Lost in Translation: Translation Mechanism in Production of

3/2006K

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I MÄ

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Lost in Translation: Translation Mechanism

s in Production of Cocksfoot M

ottle Virus Proteins

Recent Publications in this Series:

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Helsinki 2006 ISSN 1795-7079 ISBN 952-10-3016-X

3/2006

Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki

KATRI MÄKELÄINEN

Institute of Biotechnology andDepartment of Applied Biology

Department of Applied Chemistry and MicrobiologyFaculty of Agriculture and Forestry, andViikki Graduate School in Biosciences

University of Helsinki

Lost in Translation: Translation Mechanisms inProduction of Cocksfoot Mottle Virus Proteins

3/2006K

ATR

I MÄ

KELÄ

INEN

Lost in Translation: Translation Mechanism

s in Production of Cocksfoot M

ottle Virus Proteins

Lost in Translation:Translation Mechanisms in Production of

Cocksfoot Mottle Virus Proteins

Katri Mäkeläinen

Institute of Biotechnology andDepartment of Applied Biology

Department of Applied Chemistry and MicrobiologyFaculty of Agriculture and Forestry, andViikki Graduate School in Biosciences

University of Helsinki

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in the auditorium 1041 at Viikki Biocenter (Viikinkaari 5) on March 31th, 2006, at 12 noon.

Supervisor: Docent Kristiina Mäkinen Department of Applied Chemistry and Microbiology University of Helsinki Finland

Reviewers: Docent Maija Vihinen-Ranta Department of Biological and Environmental Science University of Jyväskylä Finland

And

Professor Carl-Henrik von Bonsdorff Department of Food and Environmental Hygiene University of Helsinki Finland

Opponent: Professor W. Allen Miller Plant Pathology Department Iowa State University USA

3/2006ISBN 952-10-3016-X (paperback)ISBN 952-10-3017-8 (PDF, online)ISSN 1795-7079 (paperback)ISSN 1795-8229 (PDF, online)Gummerus Kirjapaino OyVaajakoski 2006

To my family

ORIGINAL PUBLICATIONS

I Mäkeläinen K., Aspegren K., Wahlström E., Teeri T.H., and Mäkinen K. Comparison of the translational properties of Cocksfoot mottle virus 5’leader sequence with known translational enhancers from other plant viruses. (Manuscript)

II Mäkeläinen K., and Mäkinen K. Testing of internal initiation via dicistronic constructs is complicated by production of extraneous transcripts. (Submitted)

III Mäkinen K., Mäkeläinen K., Arshava N., Tamm T., Merits A., Truve E., Zavriev S., and Saarma M. 2000. Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus). J. Gen. Virol. 81: 2783-2789.

IV Lucchesi J., Mäkeläinen K., Merits A., Tamm T., and Mäkinen K. 2000. Regulation of –1 ribosomal frameshifting directed by Cocksfoot mottle sobemovirus genome. Eur. J. Biochem. 267: 3523-3529.

V Mäkeläinen K., and Mäkinen K. 2005. Factors affecting translation at the programmed –1 ribosomal frameshifting site of Cocksfoot mottle virus RNA in vivo. Nucleic Acids Res. 33: 2239-2247.

Also unpublished data will be presented.

CONTENTS

LIST OF ORIGINAL PUBLICATIONS

ABBREVIATIONS

ABSTRACT ................................................................................................................ 1

1. INTRODUCTION ................................................................................................... 2 1.1 TRANSLATION INITIATION ..................................................................... 3 1.1.1 Regulation of translation initiation during virus infection .......................6 1.1.2 Plant viral translational enhancers ........................................................... 7 1.1.3 Internal ribosome entry sites (IRESs) ..................................................... 9

1.1.3.1 Mechanism of IRES-mediated translation initiation ................. 10 1.1.3.2 IRES trans-acting factors (ITAFs) ............................................ 11

1.1.4 Leaky scanning ...................................................................................... 12 1.1.5 Reinitiation ............................................................................................ 12 1.1.6 SgRNAs ................................................................................................. 13

1.2 TRANSLATION ELONGATION ............................................................... 13 1.2.1 Regulation of elongation ....................................................................... 15 1.2.2 Programmed -1 ribosomal frameshifting (-1 PRF) ............................... 16

1.2.2.1 The -1 PRF signals .................................................................... 17 1.2.2.2 Mechanism of -1 PRF ............................................................... 19

1.3 TERMINATION OF TRANSLATION ...................................................... 21 1.3.1 Programmed termination codon readthrough ........................................ 22

1.4 POSTTRANSLATIONAL REGULATION OF GENE EXPRESSION .. 22 1.4.1 Polyprotein processing .......................................................................... 23 1.4.2 Viral proteases ....................................................................................... 24

2. AIMS OF THE STUDY ........................................................................................ 26

3. MATERIALS AND METHODS .......................................................................... 27

4. RESULTS AND DISCUSSION ............................................................................ 31 4.1 Translation initiation from CfMV RNA .................................................... 31 4.1.1 Comparison of protein production from CfMVε with known plant viral translational enhancers (I) ............................................................. 31 4.1.2 Identifi cation of regions important for gene expression from CfMVε in barley suspension cells (I) ............................................................. 32 4.1.3 Transient expression from in vitro transcribed mRNAs (I) ................... 34 4.1.4 Functioning of viral leader sequences in S. cerevisiae (II) ................... 34 4.1.5 Translational properties of CfMVε in vitro (I) ...................................... 35 4.1.6 Contribution of CfMV 3’UTR on translation initiation from CfMVε (I, unpublished) ................................................................................. 37

4.2 Does CfMVε promote internal initiation of translation? ......................... 38 4.2.1 Studies on internal initiation in WGE (I) .............................................. 38 4.2.2 Internal initiation in barley suspension cells (unpublished) .................. 42 4.2.3 Internal initiation in yeast (II) ............................................................... 43

4.2.3.1 Identifi cation of regions important for gene expression from internally positioned CfMVε in yeast (unpublished) ..............................48

4.2.3.2 Determination of 3’ cistron translation from dicistronic mRNAs in yeast spheroplasts (II) ......................................................... 49 4.2.4 Evaluation of the dicistronic approach in IRES studies ......................... 49

4.3 Proteolytic processing of CfMV polyprotein (III) ..................................... 50 4.3.1 N-terminal sequencing of CfMV VPg.....................................................50 4.3.2 Polyprotein processing in infected plants ............................................. 50 4.3.3 Putative processing sites of CfMV polyprotein .................................... 52

4.4 Synthesis of CfMV polyprotein ................................................................... 54 4.4.1 CfMV RNA programmed -1 ribosomal frameshifting in WGE (IV, V) 54 4.4.2 The -1 PRF in vivo (V) .......................................................................... 55 4.4.3 Regulation of -1 PRF by CfMV proteins (V) ........................................ 57

5. CONCLUDING REMARKS ............................................................................... 59 6. ACKNOWLEDGEMENTS ................................................................................. 61

7. REFERENCES ...................................................................................................... 70

REPRINTS OF ORIGINAL PUBLICATIONS

ABBREVIATIONS

aa-tRNA aminoacyl tRNAAMV Alfalfa mosaic virusA-site aa-tRNA binding siteATP adenosine triphosphateATPase adenosine triphosphatasebp base pairBYDV Barley yellow dwarf virusCaMV Caulifl ower mosaic viruscDNA complementary DNACfMV Cocksfoot mottle virusCfMVε Cocksfoot mottle virus 5’UTRCP coat proteinCrPV Cricket paralysis virusCrTMV Crucifer-infecting tobamovirusds double-stranded4E-BP eIF4E-binding proteineEF eukaryotic elongation factor eIF eukaryotic initiation factorER endoplasmic reticulumeRF eukaryotic release factorE-site exit siteGDP guanosine diphosphateGFP green fl uorescent proteingRNA genomic RNAGUS β-glucuronidaseGTP guanosine triphosphateHC-Pro helper component proteinaseHCRSV Hibiscus chlorotic ringspot virusHCV Hepatitis C virus HIV Human immunodefi ciency virusHP hairpinHsp101 heat-shock protein 101icDNA infectious complementary DNAICS intercistronic spacer/sequenceIRES internal ribosome entry siteITAF IRES trans-acting factorkDa kilodaltonLa lupus antigenlacZ β-galactosidase encoding genelacZ β-galactosidase proteinluc fi refl y luciferase encoding geneLUC fi refl y luciferase proteinm7G 7-methylguanosine Met-tRNAi initiator methionine-tRNA

mRNA messenger RNAMuLV Moloney murine leukemia virusNIaPro nuclear inclusion protein a proteinaseNMD nonsense-mediated RNA decay pathwayNt nucleotide

ODC ornithine decarboxylase ORF open reading framePaip PABP-interacting proteinPABP poly(A) binding proteinPCBP poly(rC) binding proteinPGK1 phosphoglycerate kinase 1PKR dsRNA-activated protein kinasePLRV Potato leafroll virusPoly(A) polyadenylic acidPRF programmed ribosomal frameshiftingPro proteaseP-site peptidyl-tRNA binding sitePTB polypyrimidine tract binding proteinPV PoliovirusPVA Potato virus APVX Potato virus XRdRp RNA-dependent RNA polymeraseRLU relative light unitRRL rabbit reticulocyte lysaterRNA ribosomal RNART-PCR reverse transcription polymerase chain reactionRuc Renilla luciferase encoding geneRUC Renilla luciferase proteinSeMV Sesbania mosaic virusSBMV Southern bean mosaic virussgRNA subgenomic RNAssRNA single-stranded RNATat trans-acting transcriptional activator TAV translational transactivator/viroplasminTBP TATA-binding protein3’TE 3’ translation elementTEV Tobacco etch virusTLS tRNA-like structureTMV Tobacco mosaic virusTMVΩ Tobacco mosaic virus 5’UTRtRNA transfer RNATS test sequenceT-site arriving aa-tRNA binding siteTYMV Turnip yellow mosaic virusUAS upstream activator sequenceuidA β-glucuronidase encoding geneuORF upstream open reading frameUNR upstream of NRASUTR untranslated regionVDS vector-derived intercistronic sequenceVPg viral protein, genome-linkedWGE wheat germ extractwt wild typeXRN1 5’-3’exoribonuclease 1

Amino acids may be described by one or three letter code

1

Abstract

ABSTRACT

The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5’cap and the 3’poly(A) tail that ensure effi cient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the effi cacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5’UTR (CfMVε) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVε alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVε involved scanning. Interestingly, CfMVε also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar effi cacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVε was eIF4G-dependent.

After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF

via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fi fth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very effi cient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF.

Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

2

Introduction

1. INTRODUCTION

The majority of viruses have positive-sense (+) single-stranded RNA (ssRNA) genomes. The (+)ssRNA viruses replicate in the cytoplasm of their host cells. Thus, they cannot utilize the host machinery for regulation of their gene expression at the transcriptional level and translational control is the most important tool for gene expression regulation. Cellular enzymes cannot copy RNA templates and viruses encode their own RNA-dependent RNA polymerases (RdRps) for the job. These enzymes lack proofreading capacity and viral genomes have to be small to maintain low levels of detrimental mutations. Because of the size limitations, the viruses do not have the capacity to encode translational machineries of their own. Consequently, the (+)ssRNA viruses are totally dependent on the host machinery. Eukaryotic mRNAs are usually monocistronic, capped at their 5’ends, and polyadenylated at their 3’termini. These structures both stabilize the mRNAs and synergetically affect translation effi cacy (Gallie et al., 1991, reviewed by Gallie, 1998). In contrast, viral genomes are often polycistronic and many viral mRNAs lack the 5’cap, the poly(A) tail, or both. Thus, one may believe that viral mRNAs are poorly translated. However, viruses have evolved multiple translational

strategies that make use of the advantages and limitations inherent within the cap-dependent translation of cellular mRNAs and enable effi cient translation of viral mRNAs (reviewed by Gallie, 1996, Gale et al., 2000). The small-sized genomes of the (+)ssRNA viruses limit their capacity to encode proteins by conventional strategies, but by using various translational strategies simultaneously, viruses can increase their coding capacity and multiply the steps that can be regulated. The (+)ssRNA viruses may also use additional gene expression strategies to avoid polycistronic mRNAs. Viral genomes may be divided into segments (multipartite genomes), in which the individual RNAs are monocistronic. Alternatively, viral RdRps may synthesize (+)-sense subgenomic RNAs (sgRNAs) that lack the 5’terminal open reading frames (ORFs) of the polycistronic viral mRNAs (reviewed by Bustamante and Hull, 1998).

In this thesis the main focus was on gene expression of Cocksfoot mottle virus (CfMV), which is a member of the Sobemovirus genus (Mäkinen et al., 1995a). Sobemoviruses have small (+)ssRNA genomes of approximately 4000 to 4500 nucleotides (nts) that are packed into small icosahedral particles (reviewed by Tamm and Truve, 2000a). According to their

Fig. 1. Genome organization and translation strategy of CfMV.

3

Introduction

putative RdRp sequences, sobemoviruses are closely related to poleroviruses. Sobemoviruses infect monocotyledonous and dicotyledonous plants, but usually the host range is narrow. The viruses are transmitted by insect vectors, seeds, or by mechanical inoculation. The type member is Southern bean mosaic virus (SBMV). CfMV infects some grass species, such as cocksfoot (Dactylis glomerata L.), but also cereals including wheat, oat, and barley. The CfMV RNA encodes four ORFs, of which ORFs 2A, 2B, and 3 overlap (Fig. 1) (Mäkinen et al., 1995a). The 5’end of the RNA lacks the 5’cap and the 3’terminus the poly(A) tail, suggesting that translation initiation occurs via a cap-independent mechanism. The translation initiation codon of ORF1 is in poor context, which allows the second ORF encoding the polyprotein to be produced by ribosomes that pass the fi rst AUG codon (Mäkinen et al., 1995a). In most sobemoviruses the polyprotein is encoded from a large continuous ORF (reviewed by Tamm and Truve, 2000a). In CfMV the polyprotein is arranged into two overlapping parts (ORF2A and ORF2B). The synthesis of ORF2B encoding the viral RdRp is regulated via -1 programmed ribosomal frameshifting (-1 PRF) (Mäkinen et al., 1995b). The last ORF3 encodes the coat protein (CP), which is produced from sgRNA (Mäkinen et al., 1995a).

1.1 TRANSLATION INITIATIONInitiation is usually the rate-limiting step of translation and is facilitated by soluble cytoplasmic eukaryotic initiation factors (eIFs) that prepare mRNA for the 40S subunit binding, assist in AUG selection, and promote 60S subunit binding (Fig. 2). Eukaryotic genomes are complex, and transcription is spatially and temporally set

apart from translation. As a result of all this, at least 11 eIFs are involved in eukaryotic protein synthesis initiation, whereas for prokaryotes three initiation factors (IF1, 2, and 3) are suffi cient (reviewed by Kapp and Lorsch, 2004). For example, eIFs 4A, 4B, 4E, 4G, and 4F do not have counterparts in prokaryotes, due to the differences in ribosome recruitment to the mRNAs. Although translation initiation is similar in all eukaryotes, there are differences in the eIFs of mammalian, plant, and yeast cells (Browning, 2004). Most eIFs are composed of several subunits and individual eIFs form multiple interactions with other eIFs, ribosomal subunits, and mRNA. The interactions stabilize and induce conformational changes in the eIFs and result in the colocalization of the factors and coordination of their binding and release (reviewed by Kapp and Lorsch, 2004). Whether the binding of eIFs to mRNA occurs individually or as preformed multicomponent complexes remains unclear. The exact order and mechanism of most binding and release events are also still largely unknown.

The majority of cellular mRNAs have a 5’terminal 7-methylguanosine (m7G) cap structure. This structure recruits eIFs 4E, 4A, and 4G, which together form an eIF4F complex (Grifo et al., 1983). Plants have two eIF4E isoforms: eIFiso4F (eIFs 4A, iso4G, and iso4E) promotes translation preferably from unstructured mRNAs, whereas eIF4F (eIFs 4A, 4G, and 4E) also mediates translation from structured, uncapped, and polycistronic mRNAs (Gallie and Browning, 2001). The 5’cap structure is specifi cally recognized by eIF4E (or eIFiso4E) via a network of interactions, the most important being base stacking of the positively charged m7G between two electron-rich tryptophans in eIF4E (Marcotrigiano et al., 1997).

4

Fig. 2. Schematic representation of the recruitment of eIFs to mRNA during translation initiation.

The eIF4G plays an important role in translation initiation by functioning as a scaffolding protein that binds mRNA and interacts with the poly(A) binding protein (PABP) and with several eIFs (Lamphear et al., 1995). Simultaneous interaction of eIF4G with eIF4E and eIF4A escorts eIF4A to the 5’end of mRNA. The adenosine triphosphatase (ATPase)/helicase activity of eIF4A then renders the cap-proximal region accessible to the incoming ribosomal subunit (Ray et al.,

1985). The eIF4A function is stimulated by eIF4B and eIF4F (Ray et al., 1985, Pestova and Kolupaeva, 2002). The eIF4G-PABP interaction enhances translation putatively by bringing the termini of mRNA together, which enables the recycling of ribosomes back to the 5’termini (Fig. 3) (Gallie, 1991, Gallie and Tanguay, 1994, Preiss and Hentze, 1998, reviewed by Gallie, 2002b). This interaction could thus prevent the synthesis of N-teminally truncated proteins (Preiss and Hentze, 1998). The eIF4G-

Introduction

5

PABP interaction may also function as a quality control mechanism for dissecting mRNAs, which are truncated at their 3’ends, as judged by their inability to bind PABP. Finally, the eIF4G-mediated binding of PABP to eIF4E stabilizes the cap interaction and protects it against decapping and degradation (Gallie, 1991, reviewed by Gallie, 1998).

The initiator methionine transfer RNA (Met-tRNAi) is selected from a pool of tRNAs by eIF2 bound to guanosine triphosphate (GTP). Discrimination against the tRNAs used for elongation occurs on the basis of certain specifi c bases, base pairings, and modifi cations in the Met-tRNAi (Åström et al., 1993). The Met-tRNAi and eIF2-GTP form a stable ternary complex, which together with the 40S subunit and eIFs 1, 1A, and 3 form

Fig. 3. The closed-loop model of translation. In plants the two termini are brought together via PABP-eIF4G and PABP-eIF4B interactions. In mammalian cells, circularization is mediated by PABP-interacting protein (Paip), which interacts with PABP, eIF4A and eIF4B. Adapted from Gallie, 2002b.

the 43S preinitiation complex (Benne et al., 1976, Pestova et al., 2001). In a manner similar to that of eIF4G, eIF3 also plays a scaffolding role during translation initiation. It binds to the 40S subunits and together with eIF1 and 1A enhances the binding of the ternary complex close to the ribosomal peptidyl-tRNA binding site (P-site, see Fig. 5B) (Benne and Hershey, 1978, Pestova et al., 1998, Majumdar et al., 2003). Furthermore, simultaneous interaction of eIF3 with the cap-bound eIF4G brings the 43S complex and the 5’end of mRNA together (Lamphear et al., 1995).

The initiation codon is located in a scanning process, during which the 43S complex migrates along the mRNA from the very 5’end towards the 3’end (Kozak, 1989). The scanning-competent form is

Introduction

6

achieved via binding of eIFs 1 and 1A to the 40S subunit (Pestova et al., 1998). The forthcoming secondary structures are unwound by the helicase activity present in the eIF4F complex (Ray et al., 1985, Kozak, 1989, Pestova and Kolupaeva, 2002). Scanning continues until the initiation codon is recognized at the ribosomal P-site. Correct base-pairing between the Met-tRNAi

anticodon and AUG results in a 48S complex formation and induces eIF5-catalyzed hydrolysis of eIF2-GTP (Pestova et al., 2000, Unbehaun et al., 2004). Thus, eIF5 controls the fi delity of initiation by controlling the GTP hydrolysis. The accuracy of initiation codon selection is also regulated by eIF1 and 1A, which recognize and destabilize aberrant preinitiation complexes and discriminate between poor and good initiation codon contexts (Pestova et al., 1998, Pestova and Kolupaeva, 2002, Unbehaun et al., 2004). The AUG selection fi xes the reading frame and eIF2-guanosine diphosphate (GDP) is released (Pestova et al., 2000). The remaining mRNA-bound complex is stabilized by eIF3 (Unbehaun et al., 2004). The 60S subunit joining triggers the hydrolysis of eIF5B-GTP and the release of eIF5B-GDP (Pestova et al., 2000) and is accompanied by the dissociation of eIFs 1, 1A, and 3 (Unbehaun et al., 2004). Finally, an empty ribosomal aminoacyl-tRNA (aa-tRNA) binding site (A-site) is ready to accept the fi rst incoming aa-tRNA.

A complete set of eIFs is not always required for translation initiation. For example, the 5’leaders with unstructured leaders can be translated in the absence of eIFs having helicase activity (Sonenberg et al., 1982, Jobling and Gherke, 1987, Browning et al., 1988, Pestova and Kolupaeva, 2002). Interestingly, some viral mRNAs with highly structured 5’leaders may also be translated in the absence of

certain eIFs. In these cases, the ribosomes are directly bound to sequence elements that are located near the initiation codons (reviewed by Martinez-Salas et al., 2001). These possibilities are discussed in the following sections.

1.1.1 Regulation of translation initiation during virus infection Many of the eIFs are present in low amounts and thus their function is tightly regulated via specifi c responses to external and internal stimuli (reviewed by Dever, 1999). Most of the regulation occurs via phosphorylation, which changes the affi nities of the eIFs towards other eIFs or mRNA. The eIFs 2, 2B, 3, 4A, 4B, 4G, 4E, 5, and PABP are at least phosphoproteins (Gallie et al., 1997, Kapp and Lorsch, 2004).

Mammalian cells respond to viral infections in a manner similar to other stressful conditions by shutting off their protein synthesis (reviewed by Gale et al., 2000). By this means cells try to prevent virus multiplication. The down-modulation of protein synthesis occurs largely via the regulation of eIF2 phosphorylation. Viral double-stranded (ds) replication intermediates activate dsRNA-activated kinases (PKR), which phosphorylate the eIF2α subunit (Crum et al., 1988, Bilgin et al., 2003, reviewed by Gale et al., 2000). Phosphorylation stabilizes the interaction between eIF2 and eIF2B and blocks the recycling of eIF2-GDP to eIF2-GTP, which results in cessation of protein synthesis (Pavitt et al., 1998). However, many animal viruses encode RNAs or proteins that prevent PKR from activating and functioning (reviewed by Gale et al., 2000). Although not as much is known on plant viruses, Tobacco mosaic virus (TMV, genus Tobamovirus) infection induces PKR-like activity in plants (Crum et al.,

Introduction

7

1988). Furthermore, successful tobamo- and potyvirus infections are dependent on cellular protein P58IPK that inhibits PKR activity (Bilgin et al., 2003).

Cap recognition is also tightly regulated. Phosphorylation increases eIF4E’s affi nity for the cap and eIF4G (Bu et al., 1993). In mammalian cells, eIF4G interacts with a kinase that regulates the eIF4E activity (Pyronnet et al., 1999). However, plant eIF4G and eIFiso4G lack the motif for the kinase binding (Browning, 2004). In animal cells, phosphorylation of eIF4 is suppressed by binding of an eIF4E-binding protein (4E-BP) (Whalen et al., 1996), whereas eIF4E-4E-BP association is prevented by phosphorylation of 4E-BP (Lin et al., 1994, Gingras et al., 1996). The 4E-BP phosphorylation correlates with translational stimulation, whereas dephosphorylation occurs during cellular stress such as viral infection or extreme temperatures (Dever, 1999). No 4E-BP homologs have been found from plants, suggesting that plants may regulate protein synthesis by different mechanisms (Browning, 2004). Since translation of cellular mRNAs is highly cap-dependent, inhibition of cap-dependent translation is a good target for viruses that use cap-independent translation strategies. Viruses

may disrupt transcription, capping, and export of host mRNAs or affect translation initiation directly via modulation of eIFs (reviewed by Gale et al., 2000). For instance, picornavirus infections render 4E-BP to the hypophosphorylated state (Gingras et al., 1996). Some picornaviruses impair cap recognition by modulating eIF4F function via proteolytic cleavage of eIF4G and PABP (Sonenberg et al., 1982, Lamphear et al., 1995, Kerekatte et al., 1999).

1.1.2 Plant viral translational enhancers Several characteristics of the 5’untranslated regions (5’UTRs) affect their capacity to mediate effi cient translation (Fig. 4). Usually the 5’UTRs of cellular mRNAs as well as many plant viral leaders are relatively AU-rich and lack strong secondary structures (Sonenberg et al., 1982, Gallie, 1996, Kozak, 2003). This is benefi cial, since simple 5’UTRs may be translated with an incomplete set of eIFs, because there is no need to unwind secondary structures (Sonenberg et al., 1982, Gallie and Browning, 2001, Pestova and Kolupaeva, 2002). Viral 5’UTRs may also be long and highly structured, due to their involvement in the regulation of

Fig. 4. Factors affecting the effi cacy of translation.

Introduction

8

mRNA stability and localization as well as virus replication and assembly. However, highly structured 5’leaders impede scanning of the ribosomal subunits (Kozak, 1989). Therefore, initiation at these mRNAs requires the participation of all eIFs (Kozak 1989, Pestova and Kolupaeva, 2002), unless scanning is replaced by the use of alternative initiation mechanisms such as internal ribosomal entry.

Certain plant viruses, such as alfamo-, tobamo-, and potexviruses, have capped RNAs. In contrast, in some plant viruses a viral genome-linked protein (VPg) replaces the 5’cap. This group of viruses includes polero-, sobemo-, and picornavirus-like viruses. There are also viruses, such as luteo- and tombusviruses, which do not have specifi c 5’terminal structures. In addition to the poly(A) tails found for instance in the 3’end of picornavirus-like viruses, the genomes of the (+)ssRNA viruses may terminate at tRNA-like structures (TLSs) as in tobamo- and tymoviruses or at heteropolymeric sequences not forming TLSs as in sobemo- and luteoviruses (reviewed by Dreher et al., 1999). Although many (+)ssRNA viruses lack either the 5’cap, the poly(A) tail, or both, viral RNAs are effi ciently translated, indicating that the viral replacements can functionally complement the 5’cap and the 3’poly(A) tail. In fact, many viral UTRs enhance translation initiation by attracting ribosomal subunits, eIFs, or some other host proteins aiding in translation initiation (reviewed by Gallie, 1996). Translational enhancers have been identifi ed from both termini of several genera of the (+)ssRNA plant viruses. The heterogeneity in these UTRs indicates that translation enhancement is accomplished in several ways. Depending on the enhancer, the functional range can be narrow or broad (Gallie et al., 1987). However, not all

viral leaders function as translational enhancers and effi cient translation of viral mRNAs may be achieved via production of large amounts of viral mRNAs, which outcompete the cellular mRNAs for translation machinery.

The 5’UTR of TMV (TMVΩ; genus Tobamovirus) is one of the best-studied examples of translational enhancers. It enhances translation in prokaryotes and eukaryotes, albeit the mechanism probably differs (Gallie et al., 1987, Sleat et al., 1988, Tanguay and Gallie, 1996, Wells et al., 1998, Gallie, 2002a). The (+)ssRNA of TMV is capped but the enhancement function of the 68-nt-long 5’UTR is cap-independent (Sleat et al., 1988). In plants, translation initiation is stimulated via binding of a host heat-shock protein, Hsp101, to the poly(CAA) sequence of TMVΩ (Tanguay and Gallie, 1996). Hsp101 further interacts with eIF4G and eIF3, which stimulates the recruitment of 40S subunits to the RNA (Wells et al., 1998, Gallie, 2002a). The TMV 3’UTR contains a TLS and an upstream pseudoknot region. The TMV 3’UTR boosts the translational enhancement conferred by the Ω element (Gallie, 2002a). Hsp101 also interacts with the 3’UTR (Tanguay and Gallie, 1996). Thus, further stimulation of translation via the 3’UTR probably results from the circularization of TMV RNA and recycling of translational components from the 3’termini to the 5’end in a manner similar to that proposed for the capped and polyadenylated cellular mRNAs (reviewed by Gallie, 1998).

The 5’UTRs from potyviruses also enhance gene expression (Nicolaisen et al., 1992, Levis and Astier-Manifacier, 1993, Gallie et al., 1995). The potyviruses belong to the Picornavirus supergroup. As in other group members, potyviral (+)ssRNA genomes are polyadenylated

Introduction

9

and linked to VPg at their 5’ends. The mechanism of translational enhancement conferred by the Tobacco etch virus (TEV) 5’UTR (143 nt) is the best studied. The 5’UTR folds into two pseudoknots, of which the 5’proximal pseudoknot is crucial for cap-independent translation initiation (Zeenko and Gallie, 2005). Capping does not improve translation from the TEV 5’leader, showing that the 5’UTR function overlaps with that of the 5’cap (Gallie et al., 1995). However, enhancement is strongly stimulated by the polyadenylated 3’terminus. The TEV 5’UTR effi ciently recruits eIF4G to the mRNA, which gives the virus a competitive advantage (Gallie, 2001). PABP boosts TEV RNA translation further, putatively via the contact formed between the 5’and 3’termini as a result of eIF4G-PABP interaction (Gallie, 2001). The potyviral 5’leaders also function at the internal positions of polycistronic mRNAs, providing further evidence for the occurrence of cap-independent translation initiation from these viral 5’UTRs (Levis and Astier-Manifacier 1993, Niepel and Gallie, 1999a, Gallie 2001, Akbergenov et al., 2004). Interestingly, potyviral VPgs interact with eIF4E, eIFiso4E, and PABP, suggesting that VPg may participitate in translation initiation (Wittmann et al., 1997, Léonard et al., 2004).

The sgRNA (RNA4) encoding the CP of Alfalfa mosaic virus (AMV; genus Alfalfamovirus) is effi ciently translated under conditions in which cap-dependent translation initiation is impaired (Sonenberg et al., 1982). This suggested that translation from this simple AU-rich leader could occur with an incomplete set of eIFs. Consistent with this hypothesis, the AMV RNA4 leader decreases the amounts of eIFs 3, 4A, 4E, and 4G needed for translation initiation (Browning et al., 1988). Recently, it was found that the

binding of AMV CP to the viral 3’UTR stimulates translation (Neeleman et al., 2001). The AMV CP interacts with eIF4G and eIFiso4G and thus appears to mimic the action of PABP in circularization of mRNA (Krab et al., 2005).

In many viruses the translation regulatory element is located in the 3’UTR instead of 5’UTR. This arrangement prevents translation initiation on mRNAs having truncated 3’UTRs. The cap-independent translation element (3’TE) of Barley yellow dwarf virus (BYDV; genus Luteovirus) functionally mimics the cap structure. The 3’TE effi ciently competes for the eIF4F complex, which is putatively delivered to the uncapped 5’leader via the kissing loop structure formed between the complementary bases of the stem-loops present in the UTRs (Wang et al., 1997, Guo et al., 2001). The 5’leader is then scanned by the ribosomal subunits in a conventional manner (Wang et al., 1997, Guo et al., 2001). The TLS in the 3’UTR of Turnip yellow mosaic virus (TYMV; genus Tymovirus) stimulates translation via a totally different but still unknown mechanism that however appears to be cap-dependent and to involve specifi c recognition of the aminoacylated TLS by a eukaryotic elongation factor 1A (eEF1A) (Matsuda and Dreher, 2004).

1.1.3 Internal ribosome entry sites (IRESs)Prokaryotic ribosomes are bound to mRNAs via base-pairing of Shine-Dalgarno sequences with 16S ribosomal RNA (rRNA). Sequences complementary to the rRNA are also frequently encountered in eukaroytic and viral mRNA 5’UTRs (Smirnyagina et al., 1991, Nicolaisen et al., 1992, Mauro and Edelman 1997, Wang et al., 1997, Niepel and Gallie, 1999, Koh et al., 2003, Akbergenov et al., 2004), which

Introduction

10

suggests that direct contact of the mRNA and the translational apparatus could be used to induce initiation (Mauro and Edelman, 1997, Akbergenov et al., 2004). Some evidence supports this hypothesis, e.g. the 40S subunits have high affi nity for mRNAs, which are complementary to the exposed regions of 18S rRNA (Akbergenov et al., 2004). Furthermore, mutations in these complementary regions reduce translation effi ciencies of the particular mRNAs (Zeenko and Gallie, 2005).

Among the DNA viruses, such as Caulifl ower mosaic virus (CaMV, genus Caulimovirus), stable secondary structures may be bypassed by `jumping´ of the migrating ribosomes over the complex regions (Ryabova and Hohn, 2000). However, (+)ssRNA viruses utilize internal binding of the 40S subunits to regions called internal ribosome entry sites (IRESs). Internal initiation was fi rst described in the picornaviruses, but IRESs were later found from several other virus groups as well as from some cellular mRNAs (reviewed by Martinez-Salas et al., 2001). Among the animal viruses, IRES-mediated translation initiation is characteristic of viruses containing complex and long 5’leaders. Thus, using internal initiation in addition to avoiding the need to scan the 5’UTRs, viruses circumvent the inhibitory effect of the upstream ORFs (uORFs) often present in long leaders. However, the IRESs characterized from plant viruses are rather simple (Ivanov et al., 1997, Koh et al., 2001, Jaag et al., 2003). The 148 nt IRES element of Crucifer-infecting tobamovirus (CrTMV; genus Tobamovirus) contains two putative stem-loops but the element important for IRES activity resides in the unstructured GA-rich region (Ivanov et al., 1997, Dorokhov et al., 2002). A similar purine-rich region is important for IRES-mediated translation initiation in Potato

leafroll virus (PLRV, genus Polerovirus) (Jaag et al., 2003). Although the 5’leader of TEV is more complex, i.e. contains two pseudoknot domains, only the fi rst pseudoknot and a region complementary to 18S rRNA in it, are important for IRES activity (Zeenko and Gallie, 2005). In a manner similar to that for cap-dependent translation initiation, IRES-mediated translation initiation may be stimulated via interaction of the IRES with the 3’terminus (Svitkin et al., 2001, Koh et al., 2003, reviewed by Martinez-Salas et al., 2001).

1.1.3.1 Mechanism of IRES-mediated translation initiation During IRES-mediated translation initiation, ribosomes attach directly to the initiation codon or a short distance upstream of it. Since cap recognition or scanning is not required, certain eIFs may be unnecessary for initiation. This improves the competitiveness of the viral mRNAs against cellular mRNA translation. In general, IRESs do not share conserved structural requirements, which suggests that the mechanisms of ribosome recruitment vary. The requirements for IRES-mediated initiation have been studied mainly in reconstituted in vitro assays (Pestova et al., 1996, 2001, Martinez-Salas et al., 2001), as illustrated in the following examples.

Picornaviral translation initiation is dependent on all eIFs, except the cap-recognizing factor eIF4E (Pestova et al., 1996). Thus, translation of viral mRNAs is not affected by regulation of eIF4E or 4E-BP activities (Pestova et al., 1996). Furthermore, cap independency allows picornaviruses to disrupt translation of cellular mRNAs by impairing cap recognition using proteolytic cleavage of eIF4G. Cleavage separates the C-terminal

Introduction

11

binding domains of eIF3 and eIF4A from the N-terminal binding domains of eIF4E and PABP (Lamphear et al., 1995). The C-terminal fragment binds to the IRES, recruits other eIFs, and enables translation initiation from the picornaviral RNAs in the absence of eIF4E (Lamphear et al., 1995, Pestova et al., 1996).

The binding of Hepatitis C virus (HCV, genus Hepacivirus) IRES to the 40S subunits occurs in the absence of eIFs. The binding causes conformational changes in the 40S subunit that place the initiation codon directly at the ribosomal P-site (Spahn et al., 2001). Subsequent 48S complex formation occurs after correct codon-anticodon recognition between the Met-tRNAi-eIF2-GTP ternary complex and AUG (Pestova et al., 1998). Finally, eIF3 is recruited to assist in the 60S subunit joining (Pestova et al., 1998).

Cricket paralysis virus (CrPV, genus Cricket paralysis -like viruses) promotes internal initiation in the absence of any eIFs, Met-tRNAi, or GTP hydrolysis (Wilson et al., 2000). Translation initiates by incorporation of the Ala-tRNA into the ribosomal A-site while the P-site remains empty. Thus, translation appears to initiate directly from the elongation phase (Wilson et al., 2000). Structural data show that the CrPV IRES forms specifi c intermolecular contacts with both ribosomal subunits and introduces conformational changes in them (Spahn et al., 2004). The conformation of the IRES also changes during the initiation process. The ability of CrPV IRES to modify the conformation of the ribosome while its conformation is also changed is a characteristic similar to protein translation factors. Therefore, CrPV IRES appears to function as an RNA-based translation initiation factor.

1.1.3.2 IRES trans-acting factors (ITAFs)Several IRESs interact with proteins that do not have previously identifi ed roles in translation initiation. For instance, picornaviral IRESs interact with polypyrimidine tract binding protein (PTB), proliferation-associated factor ITAF45, lupus antigen (La), poly(rC) binding protein (PCBP), and upstream of NRAS (UNR) (reviewed by Martinez-Salas et al., 2001). In a survey of HCV IRES-interacting proteins, approximately 90 proteins were shown to bind to this IRES, of which ~20 were specifi c for the HCV sequence (Lu et al., 2004). In addition to translation-related proteins, these proteins included RNA binding proteins, cytoskeletal proteins, as well as proteins involved in signal transduction, apoptosis, cell differentiation, and cell cycle regulation. The most popular model for ITAF function is that they act as chaperones that direct and stabilize folding of the IRESs, thus enabling the subsequent recruitment of ribosomal subunits. Consequently, variation in the ITAF content could explain the cell-type-specifi c restriction of certain viruses (reviewed by Martinez-Salas et al., 2001). However, concurrent agreement of the role played by ITAFs is lacking, since many ITAFs are nuclear proteins, whereas viral RNAs are located in the cytoplasm (Kozak, 2001, 2003). In fact, many of the ITAFs have previously characterized roles in regulation of RNA stability, splicing, or transcription. Thus, rather than being involved in translation directly, ITAFs could be involved in generating templates for the translational apparatus (Kozak, 2003). In fact, several sequences initially identifi ed as IRESs were later shown to contain cryptic promoters or splicing sites (Han and Zhang, 2002, Hecht et al., 2002,

Introduction

12

Van Eden et al., 2004, Verge et al., 2004). Thus, the involvement of the ITAFs in IRES-mediated translation initiation is confusing.

1.1.4 Leaky scanningSince the 5’UTRs are scanned from the very beginning of the 5’ends, usually the fi rst initiation codon is used for translation initiation (Kozak, 1989). However, the sequence surrounding the AUG determines the effi cacy of translation initiation. The eIF1 participates in the discrimination between favourable and poor context initiation codons (Pestova and Kolupaeva, 2002). The optimal sequence contexts for AUG recognition vary among eukaryotic species and cell types, but the purine at the –3 position (with the A of AUG as 0) is highly conserved (Kozak, 1989, Lukaszewich et al., 2000). In mammalian cells the –3 purine residue stimulates effi cient translation initiation even if the rest of the consensus sequence is imperfect (Kozak, 1989), whereas in plant cells the –1 and –2 position residues are also important (Lukaszewich et al., 2000). In some rare cases, translation may initiate from a nonconventional ACG or CUG codons, but then the rest of the codon context must be optimal (Kozak, 1989, 2002). Initiation at non-AUG codons is often observed among mRNAs whose 5’UTRs have high GC contents and strong secondary structures. Slow scanning may increase the time the mismatched codons can base-pair with Met-tRNAi (Kozak, 2002).

Since translation initiation is usually restricted to the fi rst AUG, downstream ORFs are usually silent. However, leaky scanning through nonoptimal AUGs provides viruses one mechanism for translating their polycistronic mRNAs. AUGs may also be bypassed, if they

are too closely located at the 5’end of the mRNAs (Kozak, 1989, Pestova and Kolupaeva, 2002). Actually, very few (+)ssRNA viral mRNAs have the fi rst AUG in optimal context. Leaky scanning may also be used to produce two isoforms of a single protein (reviewed by Kozak, 2002). The effi cacy of initiation on the downstream AUG again is dependent on the sequence context. AUG-bypassing results in the down-regulated expression of the fi rst cistron. On the other hand, translation initiation from a good-context AUG reduces the protein yield produced from the downstream AUGs. Thus, leaky scanning can be used to regulate protein production. However, only two or rarely three sequential proteins are usually produced via leaky scanning (Kozak, 2002). In CfMV, the initiation codon context of ORF1 contains a pyrimidine at the –3 position and lacks the G residue at position +4 (Mäkinen et al., 1995a). The initiation codon context of ORF2A is in better context, because it contains the purine at the –3 position (Mäkinen et al., 1995a). Therefore, leaky scanning down-regulates ORF1 expression and enables the trailing viral polyprotein to be encoded from the same mRNA.

1.1.5 ReinitiationThe uORFs usually limit translation of the downstream ORFs by making it dependent on leaky scanning or reinitiation (reviewed by Kozak, 2002). In contrast to leaky scanning, both the uORF and the following ORF become translated in reinitiation during the same round of protein synthesis. The factor requirements for reinitiation are not yet fully known but re-recruitment of the eIF2-GTP-Met-tRNAi ternary complex appears to be a necessity. It is hypothesized that translation of short uORFs would not lead to dissociation of

Introduction

13

all eIFs and that the remaining eIFs could then enable scanning and reinitiation (Kozak, 1987). This is supported by the fact that reinitiation effi ciency usually decreases as the uORFs become larger. However, recent data indicate that the duration of the elongation phase rather than the length of the translation product determines the feasibility of reinitiation (Kozak, 2001). On the other hand, the effi ciency of reinitiation improves as the distance to the uORF increases, probably because there is more time to bind Met-tRNAi (Kozak, 1987).

Only about 10% of the eukaryotic mRNAs have uORFs, whereas among viral and prokaryotic mRNAs they are more common (Ryabova and Hohn, 2000). Although ineffi cient reinitiation can be benefi cial in expression of toxic proteins (reviewed by Kozak, 2002), some viruses have evolved to overcome the ineffi ciency by encoding proteins that stimulate reinitiation. CaMV encodes the translational transactivator protein (TAV), which interacts with eIF3 and the 60S subunit (Park et al., 2001). These interactions keep the eIF3 associated with the elongating ribosomes. After termination eIF3 can re-recruit the ternary complex, resume scanning, and reinitiate at the downstream AUG. 1.1.6 SgRNAsSeveral (+)ssRNA viruses have 3’proximal genes that are not expressed from the genomic RNA (gRNA), since leaky scanning or reinitiation at the 3’proximal part of the genome would be too ineffi cient processes to produce the proteins in adequate amounts. These silent 3’cistrons can be made accessible to the translational apparatus by synthesizing sgRNAs in which the translation initiation codon is brought closer to the 5’end via

truncations made to the 5’terminal part during their synthesis by viral RdRPs (reviewed by Miller and Koev, 2000, Kozak, 2002). SgRNAs are subjected to the same translational control as any mRNAs. The sgRNAs may encode overlapping ORFs and viruses may encode several sgRNAs. A large percentage of (+)ssRNA viruses synthesize sgRNAs (Miller and Koev, 2000). Usually the sgRNAs encode structural and movement proteins, e.g. the CfMV CP is putatively encoded from a 1.2-kb sgRNA that is produced from the 3’proximal part of the genome (Mäkinen et al., 1995a). BYDV produces three sgRNAs: sgRNA1 encodes the structural proteins, whereas sgRNA2 and 3 do not encode any proteins. Both the gRNA and the sgRNA2 contain the 3’TE element, thus, the sgRNA2 also sequesters eIF4F and eIF4iso4F via the 3’TE. This inhibits translation of the gRNA and renders it available for replication and encapsidation (Shen and Miller, 2004). Thus, sgRNAs may also be utilized as riboregulators to regulate translation.

1.2 TRANSLATION ELONGATIONThe elongation step is highly conserved across the three kingdoms of life (reviewed by Kapp and Lorsch, 2004). During elongation the initiator Met-tRNAi at the P-site of the ribosome is elongated to a polypeptide via subsequent addition of aa-tRNAs (Fig. 5). The incoming aa-tRNAs are bound to the A-site. The A- and the P-site tRNAs interact with both subunits of the ribosomes (Moazed and Noller, 1989a). The anticodon binding sites are located in the small subunit, whereas the aa-tRNA acceptor ends (tRNA 3’ends) are located close to each other within the large ribosomal subunit. The used acylated tRNAs bound at the exit site (E-site) interact only with the large subunit via the

Introduction

14

acceptor end. The binding and movement of the tRNAs between the A- and P-sites in the small subunit are uncoupled from those occurring between the A, P, and E-sites of the large ribosomal subunit (Moazed and Noller, 1989b, reviewed by Wilson and Noller, 1998). Therefore, elongation generates hybrid states, in which the aminoacyl acceptor ends and the anticodon loops are differentially positioned between the small and large subunit A, P, or E-sites.

The aa-tRNAs are transported as a complex with GTP and eEF1A. The

initial binding is rapid and reversible and does not involve codon recognition. The aa-tRNA is initially placed in an A/T hybrid state; the anticodon loop is bound to the A-site in the small subunit and the 3’end is bound to the large subunit T-site via eEF1A. Interaction of the 3’end CCA sequence of aa-tRNA with eEF1A prevents premature peptide bond synthesis by impeding the access of the 3’terminus of the aa-tRNA to the peptidyl transferase center of the large subunit (Moazed and Noller, 1989a). Cognate tRNA binding induces conformational changes in the

Fig. 5. A) The cloverleaf structure of tRNA. The activated amino acids are attached to the 3’-OH end of tRNA. B) Hybrid state model for the elongation cycle. 1) Initiator or peptidyl-tRNA is placed in the P-site (P/P-state). 2) The anticodon of the new incoming aa-tRNA interacts with the 40S subunit, whereas the acceptor end bound to eEF1A interacts with the 60S subunit at the T-site (A/T-state). 3) GTP hydrolysis releases eEF1A-GDP and the acceptor end also enters the A-site (A/A-state). 4) Following peptide bond formation, both tRNAs are in hybrid states. 5) Movement of the anticodon ends relative to the 40S subunit and as a result the peptidyl-tRNA is moved into the P/P-state and the deacylated tRNA into the E-state. Adapted from Wilson and and Noller, 1998.

Introduction

15

small ribosomal subunit, which facilitate close contact and subsequent base-pairing of the fi rst two bases of the codon and anticodon (Ogle et al., 2001). During this interaction the ribosome senses whether the base-pairs have Watson-Crick geometry and discrimination against near-cognate tRNAs occurs. In contrast, the binding site of the third wobble position base remains relatively open and thus the geometry of the base-pairing is not as closely monitored. This explains why certain noncanonical bases may occupy the wobble position (Ogle et al., 2001). Irreversible tRNA selection occurs when a conformational change activates the eEF1A-catalyzed hydrolysis of GTP. As a result, eEF1A-GDP is released and the freed 3’end of the aa-tRNA can enter the large subunit A-site (A/A hybrid state). The acceptor ends of the tRNAs become closely arranged with each other and with the peptidyl transferase center of the large ribosomal subunit, which catalyzes the peptide bond formation (Nissen et al., 2000). Peptide bond formation is entirely catalyzed by rRNA. In this reaction a conserved adenosine residue at the catalytic site attracts a proton from the α-amino group of the amino acid moiety of the A-site aa-tRNA. This is facilitated by the active site environment, which increases the pKa of the functional adenosine residue and thus its capacity to attract protons (Nissen et al., 2000). Next, the nucleophilic α-amino group attacks the electrophilic carbonyl carbon of the ester bond linking the peptide moiety to the peptidyl-tRNA bound to the P-site. The resulting intermediate goes through rearrangements that fi nally yield a discharged tRNA bound to the P-site and a one-amino acid-longer peptidyl-tRNA bound to the A-site.

Next, the translocation step vacates the A-site for binding the following aa-

tRNA (reviewed by Joseph, 2003). During translocation the interactions between the tRNA-mRNA complex and the ribosome are broken and renewed at a new position three bases downstream towards the mRNA 3’end. This is probably achieved most accurately by performing the translocation in a stepwise manner (Moazed and Noller, 1989b). The fi rst hybrid state develops spontaneously after the peptide bond formation. The deacetylated tRNA anticodon remains at the P-site, but the acceptor stem is moved to the E-site (P/E hybrid state). Simultaneously the peptidyl-tRNA´s anticodon is retained at the A-site, whereas the acceptor stem carrying the growing peptide is moved to the P-site (A/P hybrid state) (Moazed and Noller, 1989b). The next step is catalyzed by the hydrolysis of eEF2-GTP (Rodnina et al., 1997). Movement of the anticodon ends together with the mRNA relative to the small subunit places the peptidyl-tRNA in the P/P-state and the deacylated tRNA at the E-site (Moazed and Noller, 1989b). The translocation machinery acts directly on the tRNAs and the movement of the mRNA is driven by its association with the tRNAs. The ribosomes undergo signifi cant conformational changes during translocation (Joseph, 2003). The movement of the ribosome by three nucleotides in the 3’direction also places the next codon at the A-site. Finally, the deacetylated tRNA leaves the ribosome via the E-site, and the eEF1A-GDP is recycled back to eEF1A-GTP by eEF1B. The aa-tRNA binding, transpeptidation, and translocation are repeated as many times as there are sense codons in the mRNA.

1.2.1 Regulation of elongationElongation is the fastest step in translation and is not generally considered to play a

Introduction

16

major role in the regulation of translation. However, elongation consumes a large amount of energy, since at least four high-energy bonds are used per each amino acid addition (Browne and Proud, 2002). Therefore, it is advantageous for the cells to be able to reduce the rate of protein synthesis during increased energy demand or decreased energy supply so that the energy can be used for more important processes. The benefi t from inhibition at the elongation phase is that the mRNAs remain polysome-associated (Browne and Proud, 2002). This enables rapid resumption of translation after the shortage in energy is overcome.

All eEFs are phosphoproteins and targets for various kinases and phos-phatases. However, how phosphorylation modulates eEF activities and thus protein synthesis is not yet well understood (reviewed by Browne and Proud, 2002). Phosphorylation of eEFs 1A and B by certain kinases appears to enhance their activity, whereas the reverse occurs for the phosphorylated eEF2. Human immunodefi ciency virus 1 (HIV-1; genus Lentivirus) encodes a trans-acting transcriptional activator (Tat) protein that interacts with eEF1B. This interaction reduces the translation of cellular mRNAs but not viral mRNAs (Xiao et al., 1998).

Several characteristics of the mRNAs affect the rate of elongation (Fig. 4). As a result, ribosomes move along the mRNA at non-uniform rates and become distributed unevenly along the mRNA (Wolin and Walter, 1988). Occasionally the elongating ribosomes stall and the trailing ribosomes stack behind the leading ribosome (Wolin and Walter, 1988, Tu et al., 1992, Somogyi et al., 1993). Coordinated pauses provide a correct time frame for many recoding events, cotranslational folding of proteins, cotranslational assembly of protein

or protein-membrane complexes, and cotranslational transmembrane transport. Translational pauses may be induced via codons encoding rare aa-tRNAs as well as via strong secondary RNA structures (Tu et al., 1992, Somogyi et al., 1993). Interaction of the nascent polypeptide with itself or with another protein can also inhibit elongation. For example, translation of proteins that are transported to the endoplasmic reticulum (ER) ceases after the leader peptide is synthesized and recognized by the signal recognition particle (Wolin and Walter, 1988). Translation commences after the complex is targeted to the ER.

1.2.2 Programmed -1 ribosomal frameshifting (-1 PRF)PRF provides viruses an additional mechanism for increasing the diversity of proteins produced from their small genomes. However, natural frameshift errors occur rarely, thus, certain cis-acting signals have been built into the mRNAs, which increase the frameshift frequency signifi cantly. Depending on the signals, some of the elongating ribosomes slip either backwards (-1) or forwards (+1), after which translation continues in the new overlapping ORF (Fig. 6). The nonshifted ribosomes continue translation in the original ORF (0-frame) (reviewed by Farabaugh, 1996). Both products are synthesized until the termination codons are encountered. As an outcome, two proteins are manufactured that share identical N-termini but differ at their C-terminal parts, beginning at the frameshift site.

Usually, transframe products represent the minority. Therefore, PRF is often used for production of viral RdRps, which are needed in small amounts. The +1 frameshift is less common among

Introduction

17

(+)ssRNA viruses, but widespread among bacterial, yeast, and mammalian genes (reviewed by Farabough, 1996). The -1 PRF was fi rst identifi ed among retroviruses (Jacks et al., 1985, 1987, 1988a), but since then -1 PRF sites have been found in dsRNA and (+)ssRNA viruses, prokaryotic genes, prokaryotic and eukaryotic mobile elements, bacteriophage genomes, and in some cellular mRNAs (Hammell et al., 1999, Baranov et al., 2003). Retro- and totiviruses use -1 PRF for regulating the ratio between their structural proteins (Gag) produced from the 0-frame and the enzymatic proteins (Pol) produced as a transframe Gag-Pol fusion via -1 PRF (Jacks et al., 1988a, 1988b, Dinman et al., 1991). Among the (+)ss RNA viruses, -1 PRF determines the ratio between viral RdRps and other nonstructural proteins such as viral proteases. In some viruses such as CfMV and PLRV, the -1 PRF also affects the amount of VPg synthesized (Mäkinen et al., 1995b, Prüfer et al., 1999). Since the frequency of -1 PRF determines the amount of RdRp produced, it is an important determinant of viral viability (Dinman and Wickner 1992, Hung et al.,

Fig. 6. Two cis-acting signals, a slippery heptamer and downstream secondary structure, program -1 PRF. The elongating ribosomes are stalled over the heptamer, since the downstream secondary structure cannot enter the ribosomal mRNA tunnel. During unwinding of the structure, a fraction of the ribosomes slips one nt backwards and continues translation from the overlapping -1 reading frame. Alternatively, the ribosomes may continue translation in the original reading frame.

1998, Barry and Miller 2002). In general, viral propagation occurs in a narrow -1 PRF effi ciency window. Therefore, compounds that infl uence -1 PRF frequencies may be of use in antiviral therapy (reviewed by Dinman et al., 1998).

1.2.2.1 The -1 PRF signals The fi rst cis-acting signal directing -1 PRF is the slippery site at which the actual shift in frames occurs. To prevent premature termination during slippage, the slippery site is composed of nucleotides that allow the nonwobble bases of the slipped tRNA anticodons to rebase-pair with the new -1 frame codons. These requirements lead to the fact that usually the signal is a heptanucleotide with the sequence X-XXY-YYZ in the 0-frame and thus XXX-YYY-Z in the new -1 frame. In addition to enabling the slippage to occur, the heptamer sequence affects the frequency of -1 PRF. X can be any nucleotide (Dinman et al., 1991), but it acts as a multiplicative factor of the -1 PRF frequency. The bases U and G program the highest and C the lowest -1 PRF effi ciencies in eukaryotes (Bekaert et al., 2003). Usually A or U are

Introduction

18

found at the Y position, probably because the higher amount of energy would be needed to unpair tRNAs paired to the CCZ or GGZ codons (Dinman al., 1991). Z also varies, the infl uence of Z on -1 PRF is dependent on the identity of X and Y (Dinman et al., 1991, Marczinke et al., 2000). Usually C at this position gives the highest, whereas G gives the lowest -1 PRF in eukaryotes (Marczinke et al., 2000). The tRNA modifi cations affect translation effi cacy and fi delity. Therefore, it was also speculated that alterations in the modifi cation status of tRNAs could modulate their shiftiness by affecting the bulkiness of the anticodon loop and the stability of the anticodon-codon interaction (Urbonavicius et al., 2001). However, changes in the modifi cation state of tRNAs encoding the heptamers affect -1 PRF effi ciency by at most 2-fold (Brierley et al., 1997, Marczinke et al., 2000, Urbonavicius et al., 2003), and the effect is highly dependent on the neighboring A- and P-site codons and on the type of modifi cation (Carlson et al., 2001, Urbonavicius et al., 2003).

The second signal programming -1 PRF is a downstream secondary structure that may be a hairpin (HP) (Mäkinen et al., 1995b, Dulude et al., 2002), but more often a complex pseudoknot is involved (Kim et al., 1999, Su et al., 1999, Paul et al., 2001) (Fig. 7). The secondary structure acts to pause the ribosomes over the heptamer so that the XXY and YYZ codons are placed at the P- and A-sites of the ribosome (Tu et al., 1992, Somogyi et al., 1993, Lopinski et al., 2000, Kontos et al., 2001). The shift in frames occurs during the time the secondary structure is unwound. However, pausing as such does not ensure effi cient -1 PRF (Somogyi et al., 1993, Lopinski et al., 2000, Kontos et al., 2001). Usually pseudoknots drive

more effi cient -1 PRF (Somogyi et al., 1993, Marczinke et al., 2000, Kontos et al., 2001), possibly because they may resist the unwinding better than stem-loops (Dinman, 1995, Plant and Dinman, 2005). The pseudoknots are formed when the loop of the fi rst stem base-pairs with the downstream sequences, which thus forms the second stem (Fig. 7). Thus, unwinding at the basal part of the fi rst stem of the pseudoknot causes the downstream structure to induce supercoiling in the remainder of the fi rst stem, because it cannot rotate freely due to the anchoring of the loop regions with the downstream sequences (Dinman, 1995). This results in inhibition of the unwinding. The unwinding of the pseudoknots is hardened further by nonstandard base-pairings, tertiary and quadruple interactions, triple helixes, as well as the presence of coordinated cations that stabilize these structures (Su et al., 1999). In stem-loops the loop regions are free to rotate during the unwinding process and only the basal base pairs resist unwinding, thus, the unwinding of stem-loops is easier. Mutational studies have shown that certain sequences, which are not important for secondary structure formation, are necessary for -1 PRF. These regions may be involved in modifying the contacts with the ribosomes or the putative trans-acting proteins (Shen et al., 1995, Kim et al., 1999).

For successful -1 PRF to occur, the translational apparatus must pause correctly over the heptamer. Therefore, correct spacing between the cis-acting signals is critical (Tu et al., 1992, Somogyi et al., 1993, Lopinski et al., 2000). The length of the mRNA protected by the ribosome and the length of the spacer indicates that the intact pseudoknot cannot enter the mRNA tunnel of the ribosome (Plant et al., 2003). Thus, the spacer becomes stretched before

Introduction

19

Fig. 7. Schematic presentation of stem-loop and pseudoknot structures. In addition to the loop regions, stem structures may contain bulges that contain single unpaired bases. In pseudoknots, loop regions are base-paired to fl anking regions via standard and non standard base-pairings. Sometimes the upper stem is bent with respect to the fi rst stem and the stems may also be rotated with respect to each other (indicated by arrows).

the pseudoknot is unwound. Therefore, the spacer may account for the -1 PRF effi ciency via its capacity to be stretched (Kim et al., 2001, Bekaert et al., 2003). Alternatively, the effect of the spacer nucleotides may rely on their interactions with the translational machinery, on the stabilities of the anticodon-codon interactions in the spacer sequence, or on the availability of the corresponding tRNAs (Bekaert et al., 2003).

Sequences up- and downstream of the slippery heptamer also affect -1 PRF (Honda et al., 1996, Kim et al., 2001, Barry and Miller, 2002, Dinman et al., 2002). For the upstream sequences this seems plausible because they are in contact with the elements that compose the ribosomal mRNA channel (Kim et al., 2001). In fact, the two bases upstream of the heptamer affect the -1 PRF frequencies, which suggests that the identity of the E-site codon is important (Kollmus et al., 1994, Bekaert and Rousset, 2005). The E-site-tRNA interaction may affect -1 PRF by infl uencing the stability of the P-site-anticodon interaction directly or indirectly. In some cases, the essential parts of the frameshift signals are located far from the

cis-acting signals. In BYDV, -1 PRF is regulated via interactions that occur across several kilobases between the cis-acting secondary structure and the 3’UTR (Paul et al., 2001, Barry and Miller 2002).

No viral proteins have been shown to affect -1 PRF. Due to the ease of genetic manipulation of Saccharomyces cerevisiae, it has been the main target in studies focused on the search for host factors involved in the regulation of -1 PRF (reviewed by Dinman et al., 1998). To date, no candidates having direct effect on the -1 PRF effi ciency have been identifi ed.

1.2.2.2 Mechanism of -1 PRFThe fi rst clues for the mechanism of -1 PRF came from the sequencing of transframe proteins produced from retroviral RNAs (Jacks et al., 1988a, 1988b). According to these data, a simultaneous slippage model was described which proposed that the P-site peptidyl-tRNA and the A-site aa-tRNA occupying the slippery heptamer shift frames simultaneously (Jacks et al., 1988b). Later the model included refi nements based on genetic, biochemical, molecular, and pharmacological studies

Introduction

20

(reviewed by Harger et al., 2002). These studies have revealed that the effi cacy of -1 PRF is dictated by the kinetics of the elongation steps, which affects the time during which the ribosomes occupy the -1 PRF signals. Changes in the tRNA anticodons, rRNAs, or ribosomal proteins may also affect the -1 PRF frequencies via their effects on the stabilities of the tRNA-ribosome interactions. Furthermore, defects in the translational apparatus for detecting and correcting errors account for the occurrence of -1 PRF.

The impacts of the mutations and antibiotics that affect the kinetics of each specifi c substep in elongation on the occurrence of -1 PRF have been especially important for polishing the mechanism of -1 PRF. These studies revealed that eEF1A mutations that slow down the aa-tRNA selection increase the likelihood of -1 PRF, probably by lengthening the time the ribosomes spend on the -1 PRF signals (Harger et al., 2002). Antibiotics and mutations in the ribosomal proteins resulting in reduced peptidyl transfer rates and increased residence times at -1 PRF sites also promote -1 PRF (Brunelle et al., 1999, Meskauskas et al., 2003). However, since inhibition of translocation has no effect on -1 PRF, the postpeptidyl transfer ribosomes must be incapable of slipping (Brunelle et al., 1999, reviewed by Harger et al., 2002). Thus, the conclusion from these studies is that the slippage must occur prior to the peptidyl transfer reaction. Furthermore, since peptide bond formation occurs spontaneously shortly after the A-site becomes occupied, the slippage most probably occurs before or immediately after the transition of the aa-tRNA from the A/T hybrid state to the A/A state (Brunelle et al., 1999, Plant et al., 2003)(see Fig. 5B).

High translation initiation rates also

affect -1 PRF (Paul et al., 2001, Barry and Miller, 2002). High initiation rates increase the ribosomal loads on the mRNAs and thus also the amount of ribosomes, which become stacked behind the ribosome unwinding the secondary structure. As a result, more ribosomes could pass the slippery site without pausing and the frequency of -1 PRF would be lowered (Barry and Miller, 2002).

Current data indicate that only a small portion of the mRNA, rather than the whole ribosome, is involved in the slippage (Plant et al., 2003). During aa-tRNA accommodation, the transition from the A/T hybrid state to the A-site moves the anticodon loop 9 Å in the 5’direction. Normally this movement would be accompanied by the mRNA movement, but the cis-acting secondary structure resists the movement because it cannot enter the mRNA tunnel of the ribosome. Thus, tension is generated in the spacer region (Plant et al., 2003). This local tension can be relieved via uncoupling of the A- and P-site anticodon-codon interactions and subsequent re-pairing in the -1 frame. The energy released by the eIF1A-catalyzed hydrolysis of GTP is theoretically enough to cover the energetic costs of the slippage (Plant et al., 2003). Tension may also be relieved by aborted translation or by unwinding of the structure followed by the movement of the slippery heptamer distal region of mRNA by one base forward, after which translation can continue in the original 0-frame (Lopinski et al., 2000, Plant et al., 2003).

More careful analysis of the amino acid sequencing data revealed that a less frequent mechanism of -1 PRF occurs in parallel with the simultaneous slippage mechanism (Jacks et al., 1988a, Yelverton et al., 1994, Horsfi eld et al., 1995). In this case, the A-site amino acid is determined

Introduction

21

by the -1 frame, which suggests that the P-site peptidyl-tRNA slips before the aa-tRNA incorporation. The occurrence of single peptidyl-tRNA slippage is induced in situations where the decoding of the 0-frame A-site occurs slowly (Yelverton et al., 1994). Slow decoding occurs when the A-site decodes a termination codon or when the decoding amino acid is in short supply. Slow recognition appears to result in an additional ribosomal pause, during which the peptidyl-tRNA slips in the -1 direction.

The rules concerning -1 PRF are conserved not only among the lower and higher eukaryotes (Stahl et al., 1995), but also between the eukaryotes and prokaryotes (Yelverton et al., 1994, Brierley et al., 1997, Brunelle et al., 1999, Napthine et al., 2003). The tRNAs encoding the heptamers determine which -1 PRF mechanism is most frequently used and the same mechanism is utilized in all organisms (Napthine et al., 2003). However, the -1 PRF frequencies at the same heptamers vary signifi cantly in prokaryotic and eukaryotic hosts, indicating that the translation apparatuses are not exactly identical (Brierley et al., 1997, Napthine et al., 2003). For example, the identity of the last nt of the heptamer has nearly opposite effects on the -1 PRF frequencies in Escherichia coli and in eukaryotic cells (Brierley et al., 1997). Furthermore, E. coli ribosomes usually promote equal -1 PRF effi ciencies in the presence of stem-loops and pseudoknots (Brierley et al., 1997).

1.3 TERMINATION OF TRANSLATIONEach polypeptide is elongated until a termination codon UGA, UAA, or UAG is placed at the A-site of the ribosome. These codons do not possess cognate aa-

tRNAs. Instead, termination codons are recognized by eukaryotic release factor 1 (eRF1), which has the omnipotent capacity to decode all three termination codons (Frolova et al., 1994, Drugeon et al., 1997, reviewed by Kapp and Lorsch, 2004). The eRF1 acts at the A-site and mimics aa-tRNAs structurally (Song et al., 2000). A second type of release factor, eRF3, also takes part in translation termination. It plays a postulated role in aiding eRF1 in termination codon recognition and subsequent release of eRF1 (Salas-Marco and Bedwell, 2004). Binding of eRF1 facilitates the entry of a water molecule into the active site of the ribosome and induces hydrolysis of the ester bond linking the polypeptide chain and the P-site tRNA (Song et al., 2000). The reaction is the same as during elongation, except that the nucleophilic group is a water molecule instead of the amino group of aa-tRNA. Thus, both reactions are catalyzed by the peptidyl transferase center of the ribosomal large subunit (Song et al., 2000). After hydrolysis, the peptidyl-tRNA is released. As the fi nal step, all ligands including the eRFs are released from the ribosome and translation is terminated (Kapp and Lorsch, 2004).

The effi cacy of termination is regulated by the termination codon and its sequence context. The most important determinant of termination effi cacy is the nucleotide following the stop codon; termination is favored if it is a purine (C or U) (McCaughan et al., 1995). The suggestion that stop signals are actually composed of at least four nucleotides is supported by the fi nding that eRF1 also interacts with bases downstream from the stop codons (reviewed by Bertram et al., 2001). However, the capacity of the tetranucleotides to promote peptide release varies signifi cantly (McCaughan

Introduction

22

et al., 1995). Highly expressed genes appear to contain the most effi cient termination signals recognized with high affi nity. In contrast, genes expressed at low rates accommodate a diversity of stop signals that are decoded with lower effi ciency due to their lower affi nity for eRF1 (McCaughan et al., 1995). However, the incidence of certain recoding events, such as stop codon readthrough or selenocysteine incorporation, increases when a weak stop codon is coupled to certain cis-acting signals in the mRNAs.

1.3.1 Programmed termination codon readthroughIn programmed termination codon readthrough, the stop codons are programmed to be read as sense codons. Instead of eRF1, the stop codons are recognized by tRNAs that have been reassigned via mutations to decode stop codons as sense codons. Alternatively, wild-type (wt) tRNAs may wobble base-pair at the third position. These tRNAs compete against eRF1 for the same binding site. Therefore, the concentration of the tRNAs and their affi nity for the termination codon and the A-site environment relative to that of eRF1 determines the readthrough effi cacy (Drugeon et al., 1997). In viral readthroughs, suppressor tRNAs usually outcompete the release factor complexes with 1-10% effi ciency.

Programmed termination codon readthrough is used as a gene expression strategy among many plant, animal, and bacterial viruses (reviewed by Bertram et al., 2001). However, `leaky´ termination has been observed only at the UAG or UGA stop codons. Readthrough ends up in the production of protein fusions with extended C-termini. Among the (+)ssRNA viruses, this mechanism is usually used to produce RdRps or extended forms of

CPs (Skuzeski et al., 1991, Wills et al., 1991, Li et al., 1993, Brown et al., 1996). TMV RdRp is produced via translational readthrough of UAG, but the downstream nucleotides CARYYA (where R = A/G, Y = C/T) are also detrimental to successful readthrough (Skuzeski et al., 1991). In this context, the relatively frequent recoding effi cacy at UGAC can be at least partly explained by its ineffi cient recognition by the eRF1 (McCaughan et al., 1995). In (+)ssRNA alphaviruses the signal is the simplest possible, UGAC (Li et al., 1993), whereas in Moloney murine leukemia virus (MuLV; genus Gammaretrovirus) signals directing leaky termination consist of a stop codon, a spacer sequence, and a downstream pseudoknot structure (Wills et al., 1991). The leakiness of termination can also be regulated via long-term interactions. In BYDV two regions following the stop codon are involved: a C-rich region closely after the termination codon and a distal element nearly 700 nt downstream. These elements form putative kissing-loop interactions (Brown et al., 1996).

1.4 POSTTRANSLATIONAL REGULATION OF GENE EXPRESSIONTranslation products may undergo different types of posttranslational modifi cations after or during their synthesis. These modifi cations affect the stabilities and activities of the corresponding proteins. Well-known examples of the biological consequences of protein modifi cations include phosphorylation for signal transduction, ubiquitination for proteolysis, attachment of fatty acids for membrane anchoring, and glycosylation for protein half-life regulation, targeting, and cell–cell interactions. However, in the following sections only proteolytic processing of

Introduction

23

viral polyproteins is reviewed, since other posttranslational modifi cations were not the focus of this thesis.

1.4.1 Polyprotein processingMany (+)ssRNA viruses encode large polyproteins from which the functional proteins are released via proteolytic processing. For this purpose, viruses encode proteases that catalyze the hydrolysis of specifi c peptide bonds located between two specifi c amino acids. Proteolytic processing may commence during translation and proceeds via several intermediate products to the fi nal end products that are present in equimolar amounts (Merits et al., 2002). Polyprotein processing can be the principal mode of gene expression or it can be used in conjunction with other gene expression strategies (Gorbalenya et al., 1988, reviewed by Spall et al., 1997). For instance, sobemoviral gene expression employs polyprotein processing in concert with leaky scanning, -1 PRF, and sgRNA production (Tamm and Truve, 2000a). In contrast, members of the picornavirus-like superfamily rely solely on the production of a single large polyprotein for their gene expression (reviewed by Spall et al., 1997). In Potato virus A (PVA; genus Potyvirus), a plant virus member of the group, the polyprotein is processed into as many as 10 mature proteins by three viral proteases (reviewed by Riechmann et al., 1992). Two of these, helper component proteinase (HC-Pro) and P1, cleave only their respective C-termini (Verchot et al., 1991). The third protease, nuclear inclusion protein a proteinase (NIaPro), resembles the picornaviral 3C-like proteases and processes the remainder of the polyprotein (Gorbalenya et al., 1989).

Viral proteases may be multifunctional proteins. For instance, potyviral HC-Pro also functions in suppression of gene

silencing, aphid-mediated transmission, and viral cell-to-cell movement (reviewed by Rajamäki et al., 2004). The intermediate processing products may also have important functions that differ from those of the fi nal end products, resulting in further increases in the coding capacity of viral genomes. For instance, in alphaviruses, the partially processed nonstructural polyproteins P123 and P4 catalyze the minus-strand synthesis, whereas completely cleaved proteins are needed for the plus-strand synthesis (Shirako and Strauss, 1994). Thus, it is important that viruses can regulate the occurrence of intermediate products. This can be achieved by regulating the timing of cleavages and modifying the effi cacy of the processing, but the stabilities of the intermediates also play a role (Merits et al., 2002). Potyviral NIaPro recognizes a seven-amino acid stretch and cleavage occurs between Gln (Q) and Ser (S), Gly (G), or Ala (A), which are the last two amino acids of the recognized sequence (Shukla et al, 1994). The recognition sequence is composed of residues that are highly conserved and required for effi cient cleavage, whereas few residues vary and adjust the cleavage effi ciency. Thus, some of the cleavages are rapid, whereas other sites are processed more slowly (Merits et al., 2002). However, the effi cacy of processing is also affected by other interactions between the enzyme and substrate, the most important being the accessibility of the potential cleavage site by the protease. Sometimes proteases may be regulated by cofactors, such as membranes, RNA, metal cations, or polypeptide cofactors. The protease of Sesbania mosaic virus (SeMV: genus Sobemovirus) is active if viral VPg is uncleaved from the Pro-VPg precursor. (Satheshkumar et al., 2005). Similarly,

Introduction

24

in Poliovirus (PV: genus Enterovirus) effi cient processing by 3C protease occurs only before the viral RdRp (3D) is released from the intermediate (Jore et al., 1988).

Some viruses exploit the spectra of host proteases, which are mostly utilized in the maturation of viral envelope proteins (Dougherty and Semler, 1993). Enveloped viruses may target their proteins to the host secretion route by utilizing signal peptides, which are then removed by cellular target signal peptidases. Along the secretion route, viral proteins are further processed and modifi ed by the cellular enzymes present in different compartments of the secretion route (ER and Golgi).

1.4.2 Viral proteasesViral proteases are structurally and functionally related to cellular serine, cysteine, aspartic, or metalloproteinases (Gorbalenya et al., 1988). Proteases usually contain two globular domains, cellular proteases are composed of two separately encoded protein domains, whereas viral proteases are dimers of two identical proteins (Dougherty and Semler, 1993). The amino acids involved in the catalysis are located in the crevice formed between these domains. The spacing and arrangement of the catalytic amino acids is highly conserved and used to classify the proteases. The specifi city of the proteases is mainly determined by the three-dimensional structure of the substrate-binding pocket, which is located near the active site (reviewed by Dougherty and Semler, 1993). However, the substrate-binding pockets are highly heterologous and may consist of a single binding site for one amino acid or involve several amino acids.

Serine and serinelike proteases are named according to the presence of a serine or cysteine residue at the active site of the

enzyme. The amino acids of the catalytic triad His (H), Asp (D), Ser (S), or Cys (C) are precisely spaced HX8DX30-31GXSG. These proteases are found for instance in sobemoviruses and the Picornavirus supergroup (Gorbalenya et al., 1988). In serinelike proteases of the Picornavirus supergroup, the Ser residue is replaced with Cys, whereas sobemoviral proteases more resemble the cellular serine proteases in having the Ser residue at the active site. On cleavage, the Ser or Cys residue acts as a nucleophile and donates an electron for the carbonyl carbon of the peptide bond to be cleaved (reviewed by Dougherty and Semler, 1993). Nucleophilic attack forms a covalent acyl-Ser complex between the enzyme and the polyprotein substrate. The active site His residue donates a proton for the departing amino group, facilitating its release. Hydroxylation of the acyl-Ser complex releases the carboxylic acid product and regenerates the active site.

Cysteine proteinases have a catalytic dyad that is composed of closely arranged interacting Cys and His residues. These proteases are also known as papainlike or thiol proteinases. One viral representative is the HC-Pro of potyviruses (reviewed by Shukla et al., 1994). The sulfhydryl group of the Cys acts as a nucleophile that attacks the carbonyl carbon of the target peptide bond. An enzyme forms a temporary covalent acyl-enzyme complex through the carbonyl carbon of the substrate and the sulfhydryl group of the active Cys residue (reviewed by Dougherty and Semler, 1993). The departing amide is protonated by the active site His, the active site is regenerated, and the cleavage product is released by hydrolysis.

Aspartic and metalloproteases operate on acid-base catalysis and do not form covalent intermediates (reviewed by Dougherty and Semler, 1993). Aspartic

Introduction

25

proteases have the Asp-Thr-(Ser)-Gly signature sequence in their active sites. The catalytic dyad is composed of two Asp residues originating from the individual members of the dimer. Aspartic proteases have not been found in (+)ssRNA viruses but they do exist in retroviruses and plant pararetroviruses (Spall et al., 1997). In metalloproteinases a divalent cation, often

Zn2+, is present at the catalytic site. The catalytic site also contains Glu and His residues, where the Glu residue probably donates the electron to the carbonyl carbon during peptide bond cleavage. Only a single characterized (+)ssRNA virus, HCV, is known to encode metalloproteinases (Spall et al., 1997).

Introduction

26

2. AIMS OF THE STUDY

1) To determine the mechanism of translation initiation of CfMV by comparing it with well-studied viral 5’UTRs.

2) To examine the regulation of -1 PRF in CfMV, which probably affects the viral viability by determining the amount of VPg and RdRp produced.

3) To examine the processing of CfMV polyprotein.

Aims of the Study

27

3. MATERIALS AND METHODS

A summary of the methods used is given. Detailed methods can be found in the original publications and references therein. Standard protocols were used for DNA manipulations.

Table 1. Methods used in the present study.Method ReferenceAmino acid sequencing IIIAntisera production IIIBacterial transformation I-VBarley suspension cell culturing I, IIElectroporation I,IIImmunoprecipitation IIIIodination IIIIn vitro transcription I, II, VIn vitro translation I, II, IV, VNorthern blotting I, IIParticle bombardment I, IIPlasmid construction I-VProtein overexpression; bacteria IIIProtein overexpression; yeast VProtoplast isolation IReporter gene expression analysis I, II, VRT-PCR VSDS-PAGE analysis I-VSite directed mutagenesis by PCR I, II, IV, VTotal RNA isolation I, II, VVirus inoculation IIIVirus purifi cation IIIWestern blotting III, VYeast cell culturing II, VYeast spheroplasting IIYeast transformation II, V

Table 2. Viruses or viral sequences studied.Virus or sequence element ReferenceAlfalfa mosaic virus (AMV, genus Alfamovirus) ICocksfoot mottle virus (CfMV, genus Sobemovirus) I-VCrucifer-infecting tobamovirus (CrTMV, genus Tobamovirus) I, IIHuman immunodefi ciency virus (HIV, genus Lentivirus) VPotato virus X (PVX, genus Potexvirus) ITobacco mosaic virus (TMV, genus Tobamovirus) I, II

Materials and Methods

28

Table 3. Reporter genes and proteins.Protein and gene Origin Activity and measurementβ-galactosidase, LacZ (lacZ)

E. coli Hydrolyzes fl uorogenic substrate (o-nitrophenyl-beta-D-galactopyranoside) to yield colored product (o-nitrophenyl).

β-glucuronidase, GUS (uidA)

E. coli Chemiluminescent or fl uoregenic substrate decomposed by the enzyme to yield light emission or coloured product.

Enhanced green fl uorescent protein, GFP (GFP)

Jellyfi sh (Aequorea victoria)

Fluoresces on irradiation with UV.

Luciferase LUC, (luc) Firefl y (Photinus pyralis)

Oxidiation of luciferin results in light production and in an inactive oxyluciferase.

Renilla luciferase, RUC (Ruc)

Sea pansy (Renilla reniformis)

Catalyzes coelenterate-luciferin (coelentrazine) oxidation to produce light.

Materials and Methods

29

Table 4. The most relevant plasmids used in the study.

Plasmid name Description, vector backbone, and expression system

Sequence studied at the 5’UTR or intercistronic position (ICS)

Reference

Monocistronic gene constructs 5’UTRpANU5-series 35S-ICS-uidA, pRT101 (Töpfer et

al., 1993), plant expressionAMV 5’UTR, CfMVε, PVXαβ, TMVΩ, reference

I

pANU6-series 35S-ICS-luc, pRT101 (Töpfer et al., 1993), plant expression

AMV 5’UTR, CfMVε, CrTMV IRES, PVXαβ, TMVΩ, reference

I

pMKJM GAL1-T7-ICS-luc, pYES2 (Invitrogen), yeast expression, in vitro transcription and translation

CfMVε, TMVΩ, CrTMV IRES, reference

II

pMKJMΔGAL ICS-luc, pYES2 (Invitrogen), GAL and T7 promoters deleted, yeast expression

CfMVε, TMVΩ, CrTMV IRES, reference

II

Dicistronic gene constructs ICSpHKJM GAL1-T7-HP-GFP-ICS-luc,

pYES2 (Invitrogen), yeast expression and in vitro transcription and translation

CfMVε, TMVΩ, CrTMV IRES, reference

II

pHKJMΔGAL HP-GFP-ICS-luc; pHKJM in which GAL and T7 promoters deleted, pYES2 (Invitrogen), yeast expression

CfMVε, TMVΩ, CrTMV IRES, reference

II

pHKJMB GAL1-T7-HP-lacZ-ICS-luc, pYES2 (Invitrogen), yeast expression and in vitro transcription and translation

CfMVε, TMVΩ, CrTMV IRES, reference

II

pKJM GAL1-T7-lacZ-ICS-luc, pYES2 (Invitrogen), yeast expression

CfMVε, TMVΩ, CrTMV IRES, reference

II

pKM T7-HP-GFP-ICS-uidA (Ivanov et al., 1996), pBluescript SK+ (Stratagene), in vitro transcription and translation

CfMVε, CrTMV IRES, reference

I

pPKM 35S-HP-GFP-ICS-luc, pRT101 (Töpfer et al., 1993), plant expression

CfMVε, CrTMV IRES, reference

I

pKAH T7-ICS-luc-(A)35, pSP73 (Promega), in vitro transcription and translation

CfMVε, TMVΩ, reference

I

Materials and Methods

30

-1 PRF -1 PRF casettes

pAC-A/B/C/AB/BA/AC/CA

SV40-lacZ-ICS-luc, pAC74 dual reporter plasmid (Stahl et al., 1995), bacterial and yeast expression. The -1 PRF induces translation to continue to the -1 frame luc gene.

A (CfMV 1602-1720), B (1386-2137), C (1551-1990), AB (1602-2137), BA (1386-1720), AC (1602-1900), CA (1551-1720)

V

pAC-Am/Bm/Cm/ABm/BAm/ACm/CAm

SV40-lacZ-ICS-luc inframe controls, in which one nt insertion upstream the -1 PRF site enables the luc gene to be translated without the -1 PRF event.

CfMV Am, Bm, Cm, ABm, BAm, ACm, CAm

V

pAC1789, pAC1790

SV40-lacZ-HIV-1-luc test (1789) and inframe control (1790) constructs that contain a 53 bp sequence from the HIV-1 -1 PRF region, (Stahl et al., 1995), bacterial and yeast expression

HIV-1 -1 PRF region

V

pACRF-A/B/C SV40-Ruc-ICS-luc, pAC74 (Stahl et al., 1995), in which the lacZ gene was replaced with Ruc gene, bacterial and yeast expression

A/Am, B/Bm, C/Cm

V

pACRF-Am/Bm/Cm

SV40-Ruc-ICS-luc inframe controls, one nt insertion upstream the -1 PRF site enables the luc gene to be translated without the -1 PRF event.

A/Am, B/Bm, C/Cm

V

pYES2/NT-A/Am, B/Bm, C/Cm

GAL1-T7-lacZ-ICS-luc, pYES2/NT (Invitrogen), N-terminal 6-His tag, yeast expression

A/Am, B/Bm, C/Cm

V

pAB-21 T7-ORF2A2B, pGEM-5Zf(-) (Promega), in vitro transcription and translation

CfMV polyprotein encoding region

IV

pFSC1 T7-uidA-CfMV(1621-2521)-uidA, pGEM3Z(-) (Promega), in vitro transcription and translation

CfMV 1621-2521 IV

Protein expressionpQE-VPg T5-VPg, pQE30 (Qiagen), N-terminal 6-

His tag, bacterial expressionCfMV 1386-1724 III

pYES-P27 GAL1-P27, pYES2/NT (Invitrogen), N-terminal 6-His tag, yeast expression

CfMV 1385-2137 V

pYES-Rep GAL1-RdRp, pYES2/NT (Invitrogen), N-terminal 6-His tag, yeast expression

CfMV 1669-3255 V

Materials and Methods

31

4. RESULTS AND DISCUSSION

4.1 Translation initiation from CfMV RNA Due to the simplicity of viral genomes, viruses serve as irreplaceable tools for the study of cellular processes such as translation (reviewed by Gale et al., 2000). Studies of translation can be performed in vivo or in vitro, both approaches having benefi ts and drawbacks. A complex set of factors affects the translation process in vivo and therefore the results may be diffi cult to interpret (Kozak, 2002). In the in vitro assays the reaction conditions are more easily controlled, but since the endogenous mRNAs are removed any added transcript is usually translated readily. Therefore, mRNAs that would not promote signifi cant expression under in vivo conditions, may be translated in vitro. However, in vitro conditions may be brought closer to the in vivo conditions by supplementing reactions with competitor RNAs or by depleting the lysates from eIFs by incubating them with cap analogues or poly(A) sequences (Gallie and Tanguay, 1994, Gallie, 2001, 2002a).

Several 5’UTRs from plant viruses can stimulate translation initiation (reviewed by Gallie, 1996). The enhancement results from the reduced requirement for certain eIFs (Browning et al., 1988, Pestova and Kolupaeva, 2002) or from the capacity of viral sequences to effi ciently recruit essential eIFs (Gallie, 2001, 2002a, Krab et al., 2005). Since CfMV multiplies to high titers in its host plants (Truve et al., 1997), we examined whether this was due to the strong translation activity of viral mRNA. Sequencing of CfMV RNA showed that it lacks the poly(A) tail (Mäkinen et al., 1995a). The 5’terminus of RNA is covalently linked to a viral protein, VPg (Fig. 2 in III). These facts suggest that

translation initiation in CfMV differs from cap-mediated translation initiation.

4.1.1 Comparison of protein production from CfMVε with known plant viral translational enhancers (I)To initiate studies on the translational properties of the 5’UTR of CfMV (CfMVε), we compared it with known enhancer sequences TMVΩ, CrTMV IRES, the 5’UTR of AMV RNA4, and Potato virus X αβ (PVXαβ, genus Potexvirus) (Fig. 1A in I). All these leaders promote effi cient translation in wheat germ extract (WGE) and in tobacco cells (Gallie et al., 1987, Jobling and Gherke, 1987, Browning et al., 1988, Gallie et al., 1989, Smirnyagina et al., 1991, Pooggin et al., 1992, Ivanov et al., 1997, Dorokhov et al., 2002, Gallie, 2002a), whereas with the exception of the CrTMV sequence (Dorokhov et al., 2002) they do not appear to function in yeast cells (Van den Heuvel and Raue, 1992, Everett and Gallie, 1992). We placed the viral sequences into plant expression vectors upstream from a reporter gene (luc or uidA) and compared the activities obtained with a reference construct, in which the 5’leader was composed of a multicloning site (Fig. 1A in I). The effect of the host on the functioning of viral leader sequences was studied by performing the analysis in tobacco protoplasts (Nicotiana tabaccum L.) (I), in barley suspension cells (Hordeum vulgare L. cv. Pokko) (I), and in yeast (S. cerevisiae) (II).

The competitive advantage of the AMV RNA4 leader arises from the fact that the 5’UTR does not contain secondary structures. As a result the 43S preinitiation complexes can reach the initiation codon in the absence of adenosine triphosphate (ATP) and factors associated with ATP

Results and Discussion

32

hydrolysis, i.e. eIFs 4A, 4B, and 4F (Pestova and Kolupaeva, 2002). Although TMVΩ is also unstructured (Sleat et al., 1988), it appears to use a different strategy. TMVΩ does not recruit eIFs directly (Tanguay and Gallie, 1994), instead it binds a host protein Hsp101 that presumably assists in the recruitment of eIF3 and eIF4F (Wells et al., 1998, Gallie, 2002a). In our experiments both sequences promoted effi cient gene expression in the tobacco cells, in close correlation with existing data. CfMVε also increased protein expression, but the 5’leaders from viruses infecting dicotyledonous plants were stimulating even higher protein yields (Fig. 2A in I). In contrast, CfMVε was the only element that positively affected gene expression in the barley suspension cells (Fig. 2B in I). PVX αβ can promote translation in barley protoplasts (Zelenina et al., 1992). In our experiments it mediated the highest expression among the rest of the 5’UTRs (Fig. 2B in I). Surprisingly, we observed no translational enhancement from TMVΩ in barley suspension cells, although it can enhance translation in suspension cultures of rice and maize, both monocots (Gallie et al., 1989). However, ΤΜVΩ cannot stimulate translation in all plant cell types, since no translational enhancement was obtained in Orychophragmus violaceus cells (Family Brassicaceae) (Akbergenov et al., 2004). The Hsp101 content varies, depending on the developmental stage and type of the cells (Young et al., 2001). Although Hsp101 is well expressed in barley seeds (Tangyay and Gallie, 1994), the expression levels in the barley suspension cells used may have been too low to provide competitive advantage for the TMVΩ−containing mRNAs. When we tested the Hsp101 expression with Western blotting, a very weak signal was observed compared withthat in WGE

known to contain high levels of Hsp101 (data not shown). The CrTMV sequence also functioned poorly in the barley cells at the 5’leader position (Fig. 2B in I), although it promotes effi cient translation at the intercistronic position in tobacco and in WGE (Dorokhov et al., 2002) (Fig. 2B in I). Together our results suggested that there may be substantial variations in the requirements for effi cient gene expression from viral 5’UTRs in barley and tobacco.

In some cell types longer 5’leaders promote higher translation yields (Niepel and Gallie, 1999a, 1999b, Gallie et al., 2001). The sequences studied varied greatly in length, with the CrTMV and PVX leaders being six times longer than the reference 5’UTR. However, we observed no correlation between the 5’leader length and the downstream reporter gene expression levels. The expression from CfMVε-containing plasmids occurred approximately at 12-times higher levels in barley cells when TMVΩ and CfMVε as equal-length sequences were compared. This suggests that CfMVε could be used to enhance heterologous gene expression in cereals.

4.1.2 Identifi cation of regions important for gene expression from CfMVε in barley suspension cells (I)CfMVε was mutagenized to identify regions that were crucial to enhanced gene expression in barley suspension cells. Mutagenesis was performed for the 35S-CfMVε-luc plasmid during PCR. Deletion of almost half of the 3’terminal part of CfMVε had no effect on the extent of LUC yield (DelII, Fig. 3B in I). This showed that the deleted region was not needed for improved expression from CfMVε-containing constructs and further proved that the enhanced expression relative to the reference leader did not result from the

Results and Discussion

33

difference in leader lengths. Introduction of a small uORF to CfMVε (Destab II, Fig. 3B in I) abolished translation initiation from the downstream luc AUG, indicating that translation initiation involved scanning. This is in agreement with studies on translation initiation from SBMV (Hacker and Sivakumaran, 1997).

We suggest that CfMVε folds into a stem-loop structure that begins at fi rst nt of the RNA (Fig. 3A in I). Destabilization of the putative structure by mutating the 5’stem sequence resulted in an almost 2-fold improvement in LUC expression relative to the wt sequence. In contrast, restabilization of the structure by complementary mutations in the 3’stem sequence reduced translation to half of the expression measured from the wt CfMVε (Destab I and Restab, Fig. 3B in I). Similar results were obtained from the in vitro translations, suggesting that the inhibition occurred at the translational level (data not shown). Thus, the 5’proximal position of the secondary structure appeared to inhibit either the initial binding of the 43S complex or subsequent scanning, or both. In carrot suspension cells, a 5’proximal stem-loop of seven bases reduced luc translation to 10% (Niepel and Gallie, 1999b). The stem-loop formed in the restabilization mutant had a slightly higher predicted free energy (–17.4 kcal/mol) than the wt structure (−12 kcal/mol). Thus, reduced translation from this mutant may have resulted from the inability of the preinitiation complexes to bind and to initiate scanning. Alternatively, the sequence of the 3’part of the stem may have contributed to the stimulated gene expression from CfMVε. However, since the entire 5’proximal sequence of CfMVε was altered in the restabilization mutant, solid conclusions cannot be made before less drastic mutations are studied. Since the destabilization mutation resulted in higher

translation yields than the wt sequence or the other 5’UTRs studied, it could be the best choice for stimulation of heterologous protein production in barley.

Computer analysis identifi ed several relatively short (7-14 nt) GC-rich segments from 18S rRNA that can base-pair with complementary sequences of mRNAs (Matveeva and Shabalina, 1993). Therefore, it was suggested that stretches in mRNAs that were complementary with the 18S rRNA could function in attaching mRNAs to the small ribosomal subunits. This compared favorably with results in which 5’UTRs having short complement-ary regions with 18S rRNA stimulated translation in yeast, plant, and mammalian cells (Zhou et al., 2003, Akbergenov et al., 2004, Chappell et al., 2004). Several translational enhancers from plant viruses also show complementarity with 18S rRNA (Akbergenov et al., 2004). CfMVε also has several 6-23-nt-long stretches with 70-100% complementarity with plant 18S rRNA (data not shown). In plants, the 5’UTRs complementary to the central region of plant 18S rRNA (nt 1105-1124 according to the rice 18S rRNA sequence) show the highest affi nity for the ribosomal 40S subunits and also stimulate the strongest protein expression (Akbergenov et al., 2004). The C-rich region immediately downstream from the CfMVε stem-loop structure can potentially base-pair with this central 18S rRNA region. However, deletion of the corresponding region from CfMVε did not affect expression from this element (DelI, Fig. 3B in I). The sequence forming the 3’part of the stem and the upstream loop region contained potential sites for 18S rRNA binding (data not shown). To determine whether these potential base-pairings play a role in gene expression from CfMV RNA, it should fi rst be shown whether the corresponding sites

Results and Discussion

34

are accessible in the plant 40S subunits. A more detailed mutation analysis at the single-nt level could also be performed.

4.1.3 Transient expression from in vitro transcribed mRNAs (I)To determine whether the observed enhancement in gene expression occurred at the transcriptional or translational level, in vitro transcribed mRNAs were delivered to tobacco protoplasts or barley suspension cells. In tobacco protoplasts, LUC yields from capped mRNAs containing CfMVε or TMVΩ were 1.6 ± 0.6- and 2.4 ± 1.1-fold higher than the expression driven from the reference mRNA, respectively. In barley suspension cells, the same mRNAs drove LUC expression only ~1.2 ± 0.4-fold over the reference (Fig. 4 in I). Thus, since neither CfMVε nor TMVΩ promoted relative LUC expression from the in vitro transcribed mRNAs to levels similar to those in plant expression plasmids, it appeared that the enhanced LUC expression from plant expression vectors did not result solely from the stimulated translation activity of these mRNAs. The TMVΩ function overlaps with that of the 5’cap (Gallie, 2002a). As a result, the extent of translation enhancement from polyadenylated mRNAs containing TMVΩ is higher from uncapped than from capped mRNAs in comparison to a corresponding reference mRNA lacking the viral 5’UTR (Sleat et al., 1988, Gallie, 2002a). However, capping of the mRNAs did not mask the detection of the TMVΩ effect in barley suspension cells, since translational stimulation was likewise not observed in the uncapped mRNAs (data not shown). The gRNA of TMV contains both the 5’cap and the translational enhancer element. So why does it need both? TMV may possibly use two parallel mechanisms for translation initiation to ensure its effi cient

multiplication. Under normal growth conditions, Hsp101 expression is usually low in nondeveloping tissues, such as adult leaves (Young et al., 2001). This could indicate that the cap-mediated binding of 43S preinitiation complex is utilized in the presence of low concentration of Hsp101. Under these circumstances, TMV would benefi t from having a simple 5’UTR, since scanning of unstructured leaders does not require eIF4F and eIF4A (Pestova and Kolupaeva, 2002). However, certain stressful conditions disrupt cap-mediated translation initiation via modifi cation of eIF activities (reviewed by Dever, 1999). TMV translation may possibly overcome some stress responses by recruiting eIFs via Hsp101. In tobacco cells, heat shock-mediated induction of Hsp101 expression results in as much as 10-fold stimulated translation from TMVΩ (Gallie, 2002a). It would be interesting to examine whether TMVΩ-mediated expression would improve after heat treatment of the barley suspension cells. Hsp101 expression appeared to be induced in the barley suspension cells used after 30-45-min heat treatments at +37 °C and +45 °C (data not shown).

4.1.4 Functioning of viral leader sequences in S. cerevisiae (II)To determine whether the plant host was needed for enhanced gene expression from the plant viral leaders studied, we also analyzed gene expression from CrTMV, TMVΩ, and CfMVε in yeast (II). The studied sequences were inserted into yeast expression plasmids between an inducible galactokinase 1 (GAL1) promoter and the luc gene (Fig. 1B in II). All viral sequences tested inhibited downstream reporter expression, whereas high levels of expression were measured from the reference construct containing

Results and Discussion

35

a polylinker 5’UTR (Table 2 in II). The yeast expression plasmids had identical extensions of 189 nt upstream from the studied sequences. Therefore, the initial binding of the cap-binding complex was assumed to occur with similar affi nity for all mRNAs. However, Northern blot analysis indicated that several transcripts were produced from all plasmids (Fig. 3B,C in II). One explanation for these additional RNAs is that they may have originated from opportunistic transcription initiation in the plasmid or in the studied sequences. For instance, the GAL promoter is known to contain several minor transcription initiation sites (Johnston and Davis, 1984). The shorter mRNAs may also be RNAs that have lost the major part of their poly(A) tails via deadenylation, which is the fi rst step in mRNA degradation. In contrast, the longer forms would represent the most recently synthesized mRNAs with intact poly(A) tails (Caponigro and Parker, 1996). Whatever the origin of these shorter transcripts, they were present in comparable amounts in all transformants including the reference. Thus, the differences in the expression levels probably did not result from the variable mRNA amounts but most likely from differences in the translation effi cacy of the mRNAs.

The 5’UTR length does not usually affect translation initiation effi cacy in yeast, but long runs involving Gs and Us are deleterious (reviewed by Romanos et al., 1992). The yeast scanning complex is also more sensitive to secondary structures present in the 5’UTRs than are complexes from higher eukaryotes (Kozak, 1986, Vega Laso et al., 1993). Furthermore, secondary structures are equally inhibitory at all positions of the 5’UTRs (Vega Laso et al., 1993). These facts may explain why many viral 5’UTRs function poorly

in yeast (Coward et al., 1992, Evstafi eva et al., 1993). CfMVε possibly contains a single stem-loop structure, whereas CrTMV IRES may have two of them (Ivanov et al., 1997). These structures may have reduced translation from the CrTMV and CfMV sequences by inhibiting scanning. In fact, a 5’UTR stem-loop structure with a free energy as low as – 4.5 kcal/mol reduces translation to 5% in yeast (Niepel and Gallie, 1999b). The predicted free energies of CrTMV IRES and CfMVε are clearly higher (Fig. 3A in I). Since translation initiation in yeast occurs almost exclusively via cap-mediated recruitment of initiation complexes (Preiss and Hentze, 1998), the structures present in CfMV and CrTMV 5’UTRs may have caused the low translatability of the corresponding mRNAs. Although TMVΩ is unstructured (Sleat et al., 1988), it inhibits translation in yeast (Everett and Gallie, 1992, Van den Heuvel and Raue, 1992). In our case, LUC expression was inhibited by ~90% (Table 2 in II). One reason for the poor functioning of TMVΩ in yeast appears to be the inability of yeast homologue Hsp104 to complement the corresponding protein of plant origin (Wells et al., 1998).

4.1.5 Translational properties of CfMVε in vitro (I)The translational properties of CfMVε, TMVΩ, and CrTMV IRES were also compared in WGE, which allowed us to examine more easily the role of eIFs in translation initiation from these 5’UTRs. The translations were programmed with in vitro transcribed luc mRNAs having a viral or polylinker 5’UTR and a 35-nt poly(A) tail. Commercial translation mixes have abundant translational capacity, whereas strong competition for eIFs and the translational apparatus prevails in living cells. Therefore, elements improving

Results and Discussion

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the competitiveness of mRNAs do not necessarily show up under the conditions recommended by the suppliers of lysates. For instance, translation initiation in vitro is not enhanced by the cap and the poly(A) tail to the same extent as in vivo, where a strong synergy between these terminal elements is observed (Gallie, 1991, Gallie and Tanguay, 1994). This compared favourably with our results, in which we measured only a 2-3-fold higher expression from capped mRNAs relative to uncapped mRNAs (data not shown). In the barley suspension cells the corresponding difference between the capped and uncapped mRNAs was ~42-fold (data not shown).

Translation lysates can be made to resemble more the in vivo conditions by using depleted lysates or high mRNA concentrations (Gallie and Browning, 2001, Gallie, 2002a). For instance, the translational enhancement conferred by TMVΩ becomes detectable in WGE only under these types of conditions (Sleat et al., 1988, Gallie, 2002a). Therefore, we fi rst determined the mRNA concentration in which translation in WGE became saturated and thus competition-dependent. This point was chosen based on reduced LUC expression, which indicated that the high mRNA amounts sequestered eIFs, thus reducing translation initiation (Fig. 5A in I). Below the saturation point (60 ng/μl), no strong translational advantage from TMVΩ or CfMVε was observed (Fig. 5B in I). However, at higher mRNA concentrations both viral sequences showed improved translation in relation to the reference mRNA, indicating that viral leaders succeeded better under conditions in which the eIFs became limiting. Enhanced translation from viral sequences was also observed in coupled transcription and translation reactions with mRNAs

containing a 189-bp extension upstream from the studied sequences. Tobamoviral sequences promoted approximately 10-times higher LUC yields than the reference 5’UTR (data not shown). CfMVε improved expression ~7-fold.

Next, the functional half-lives of the mRNAs were determined to compare the periods during which the mRNAs studied remained translationally active. This was done by measuring the duration of LUC synthesis in the translation mix. Cessation of LUC accumulation was taken as an indication of complete degradation of mRNAs programming translation. The functional half-life was then designated as the amount of time required for 50% of the mRNAs to become degraded (Gallie and Tanguay, 1994). This analysis did not show differences in the degradation rates of reference mRNA or mRNAs containing viral 5’UTRs (data not shown). Thus, the capacities of the viral leaders to promote translation in WGE did not result from their stabilizing effect on the mRNAs.

TMVΩ showed increasing stimulation of translation with rising mRNA concentrations, whereas less improvement was measured from CfMVε. This may have resulted from differences in the complexity of the 5’UTRs. The predicted stem-loop of CfMVε initiates from the fi rst nucleotide of CfMV RNA and is formed of 10 bps (Fig. 5A in I). It was previously shown that under in vitro conditions secondary structures are more inhibitory at higher mRNA concentrations, putatively due to titration of the eIF4A helicase activity (Gallie and Browning, 2001). A 7-bp 5’proximal G-C-rich stem-loop with free energy of –4.5 kcal/mol reduced translation in WGE to 60% with RNA concentrations as low as 10 ng/μl (Gallie and Browning, 2001). Another study showed that scanning through even weak

Results and Discussion

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A-U-rich secondary structures required that all eIFs participated in the process (Pestova and Kolupaeva, 2002). This suggests that translation initiation from CfMVε also requires the entire set of eIFs, including eIFs 4A and 4F that are putatively required to unwind the 5’proximal stem-loop of CfMVε. Translation from TMVΩ is improved via effi cient recruitment of eIFs 3 and 4F to the element via a protein bridge formed of Hsp101 (Wells et al., 1998, Gallie, 2002a). Since Hsp101 is an abundant protein in WGE (Gallie, 2002a), effi cient recruitment of eIFs to TMVΩ can presumably continue even under highly competitive conditions. As already discussed, some translational advantage can also arise from the simplicity of TMVΩ. Reconstitution assays performed in rabbit reticulocyte lysate (RRL) have shown that eIFs 4A, 4B, and 4E are not required for 48S complex formation in (CAA)n 5’UTR (Pestova and Kolupaeva, 2002), a sequence very similar to TMVΩ.

Translation lysates do not contain endogenous mRNAs. Thus, in vitro translations are performed in somewhat artifi cial environments in the absence of competition against cellular mRNAs (Gallie and Tanguay, 1994). To further test the competitiveness of the viral leaders studied, translations were programmed in the presence of total RNA from yeast (Fig. 5C in I). Under these conditions, CfMVε programmed translation only 1.6-fold more than the reference mRNA. This correlated well with the results obtained from the barley suspension cells. Thus, CfMVε did not succeed under the competitive conditions very well, which could be explained by the requirement of all eIFs for translation initiation from this 5’UTR. In contrast, TMVΩ competed successfully against the heterologous mRNAs and promoted translation 11-fold over the reference.

4.1.6. Contribution of CfMV 3’UTR on translation initiation from CfMVε (I, unpublished)In several cases viral 3’UTRs substitute the poly(A) tail functionally and cooperate with the viral 5’UTRs in promoting effi cient translation initiation. Hsp101 also binds to the TMV 3’UTR (Tanguay and Gallie, 1996) and thus, may bridge the interaction of the two termini of TMV. This correlates favorably with the translation effi ciency of mRNAs containing both TMV UTRs, which is higher than that of mRNAs containing TMVΩ and the poly(A) tail (Gallie, 2002a). In BYDV, effi cient translation is achieved when the 5’UTR and 3’TE base-pair (Guo et al., 2001). The 3’TE binds eIFs and this interaction may serve to deliver the eIFs to the 5’UTR (Guo et al., 2001).

The combined effect of CfMV UTRs on translation effi ciency was compared with luc mRNAs having a vector-derived 3’UTR (145 nt), CfMV 3’UTR (226 nt), or a vector derived 3’UTR combined into a poly(A) tail (145 nt + 35 nt of poly(A)). In WGE, uncapped mRNAs ending at the CfMV 3’UTR or at the poly(A) tail promoted gene expression 2-fold relative to the vector-derived 3’UTR. With capped mRNAs the highest expression was obtained from the polyadenylated mRNAs (data not shown). In barley suspension cells, polyadenylated mRNAs gave the best expression irrespective of whether the mRNAs were capped or not (data not shown). The expressions from mRNAs ending at the vector- or CfMV-derived 3’UTR were similar, but only about half the level of expression measured from mRNAs ending at the poly(A) tail.

In general, it is probably a rule rather than an exception that interaction of the UTRs is required for effi cient translation (reviewed by Gallie, 1998). However, the two termini of CfMV RNA did not

Results and Discussion

38

appear to have a synergistic effect on translation as such. This may indicate that a sequence from the coding region is needed or that a viral protein plays a role in connecting the UTRs. AMV RNAs are effi ciently translated when AMV CP binds to the 3’UTR and recruits eIF4G and eIFiso4G to the viral RNA (Neeleman et al., 2001, Krab et al., 2005). Translation of VPg-containing SBMV RNA is less susceptible to inhibition by cap analogues than are capped and uncapped SBMV RNAs (Hacker and Sivakumaran, 1997), which may suggest that this viral protein participates in translation initiation of sobemoviral RNAs. In fact, certain plant and animal virus VPgs are known to interact with eIFs, such as eIFs 3, 4E, iso4E and PABP, suggesting that VPg may participate in translation initiation of viral RNAs (Wittmann et al., 1997, Léonard et al., 2004, Goodfellow et al., 2005). We aim to conduct future tests to determine whether CfMV proteins affect translation from mRNAs containing CfMV UTRs. In the infected plants, CfMV CP is one of the most abundant proteins (Fig. 4 in III). This suggests that the sgRNA encoding CP may also contain elements that guarantee the high productivity of CP.

4.2 Does CfMVε promote internal initiation of translation?

4.2.1 Studies on internal initiation in WGE (I)Increasing amounts of data indicate that several 5’UTRs can also promote translation initiation from intercistronic positions (Levis and Astier-Manifacier, 1993, Niepel and Gallie, 1999a, Ivanov et al., 1997, Koh et al., 2003). Since CfMV RNA is covalently linked to a viral protein and not to the cap structure (III), we examined whether CfMVε was capable of

mediating internal initiation of translation, which is presumably cap-independent. The leader of the sgRNA encoding CrTMV CP is a strong IRES in several cell types (Dorokhov et al., 2002) and was used as a positive control. The key element in CrTMV IRES is a polypurine-rich region (Dorokhov et al., 2002). Interestingly, CfMVε also contains a GA-rich region. In fact, the only remarkable homology at the 5’UTRs of sobemoviruses is the GAAA sequence that is located in the loop of the putative 5’stem structure (Mäkinen et al., 1995a, Ryabov et al., 1996). As already mentioned, CfMVε also contains several regions complementary with the 18S rRNA. Recently it was shown that complementary interaction with the leader and 18S rRNA may enable cap-independent binding of 43S preinitiation complexes into the intercistronic spacers (ICSs) of dicistronic mRNAs (Chappell et al., 2000, Akbergenov et al., 2004).

The in vitro translations were programmed with capped and polyadenylated dicistronic mRNAs, in which the test sequences (TSs) were placed between GFP and luc genes (Fig. 1B in I). Initially, we planned to use ΤΜVΩ as a negative control, because it was previously shown that it cannot promote translation from the internal position in O. violaceus cells (Akbergenov et al., 2004). Supporting data come from the RRL system, in which a stable secondary structure placed upstream from a (CAA)n leader prevented the 48S complex formation, indicating that the (CAA)n sequence cannot mediate internal binding of 40S subunits (Pestova and Kolupaeva, 2002). In addition to viral sequences a reference control was included, in which the reference multicloning site served as the ICS. Expression of the 3’proximal LUC cistron was taken as an indication of

Results and Discussion

39

internal translation initiation. To diminish the likelihood that the 3’cistron expression arose from reinitiation or leaky scanning, translation of the 5’cistron was prevented by a 5’proximal stable HP.

In a manner similar to that of the results obtained with monocistronic mRNAs (I), low mRNA amounts showed no signifi cant variation at the level of 3’LUC production between viral and reference ICSs (Fig. 6A in I). However, the IRES activity may become detectable only under competitive in vitro conditions, similar to the results obtained with translational enhancers (Gallie, 2001). Increased mRNA amounts revealed stimulated expression from mRNAs containing viral TSs. Surprisingly, TMVΩ promoted the highest 3’cistron expression. The degradation pattern of

the dicistronic mRNAs was determined to verify that translation templates were not cleaved to monocistronic mRNAs encoding functional LUC protein. However, no such degradation products were detected from any of the mRNAs (Fig. 6B in I). Furthermore, the physical stabilities of the individual mRNAs were comparable. The functional half-lives of the dicistronic mRNAs showed that the reference mRNA remained translationally active for the longest period of time: the approximate t1/2 was 80 min (Fig. 8). The dicistronic mRNAs containing CfMVε and TMVΩ were degraded slightly more rapidly (t1/2 ~68 min), whereas the mRNA containing the CrTMV sequence had the shortest half-life (~58 min). The expression data from the same experiment

Fig. 8. Functional half-lives of dicistronic mRNAs were determined by incubating them within the WGE translation mix, which lacked the ribonuclease inhibitor. The duration of LUC expression was followed until the mRNAs became degraded and LUC accumulation ceased. This can be observed as a plateau in the curve. The functional half-life was then designated as the amount of time required for 50% of the mRNAs to become degraded. The fi nal concentration of RNA in the mix was 35 ng/μl. RLU, relative light unit.

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Results and Discussion

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showed that the enhancement of LUC expression relative to the reference mRNA remained constant from the fi rst rounds of translation until the end. Thus, the viral elements did not stabilize the dicistronic mRNAs and the stimulated expression from mRNAs containing viral sequences did not result from the stability differences or opportunistic expression from monocistronic mRNAs. In conclusion, all viral sequences promoted internal initiation in WGE.

WGE contains two isoforms of the cap-binding complex. The more abundant complex eIFiso4F (eIFiso4E, eIFiso4G) promotes translation preferentially from unstructured mRNAs, whereas eIF4F (eIF4E, eIF4G) also promotes translation from mRNAs that contain multiple cistrons, structured leaders, or uncapped mRNAs (Gallie and Browning, 2001). Interestingly, translation initiation from TMVΩ is eIF4G- but not eIFiso4G-dependent (Gallie, 2002a). WGE contains

abundant Hsp101, which is putatively used to recruit eIF4G into TMVΩ (Wells et al., 1998, Gallie, 2002a). Thus, the internally positioned TMVΩ may also be capable of recruiting eIF4G, which then further recruits the eIFs needed for translation initiation. Hsp101 binds to the CAA repeat of TMVΩ (Tanguay and Gallie, 1994). IRESs from Hibiscus chlorotic ringspot virus (HCRSV: genus Carmovirus) and TEV share the CA-richness with TMVΩ. The CA region is crucial for the HCRSV IRES function and as an unstructured region it was proposed to serve as a landing pad for the ribosomes (Koh et al., 2003). Both IRESs function in WGE (Gallie, 2001, Koh et al., 2003).

To verify that the observed 3’cistron expression also occurred in other gene combinations, the 3’luc gene was replaced with uidA. Alternatively, the 5’GFP was switched to a lacZ gene. Neither change prevented the 3’cistron expression (Fig. 9). However, we observed slightly lower

Fig. 9. Translation of the 3’proximal gene of dicistronic construcs occurs in various 5’reporter contexts. A) Autoradiogram from coupled in vitro transcription and translation reaction in WGE preformed in the presence of 35S-methionine. Linearized dicistronic pSK: T7-HP-GFP-ICS-uidA plasmids were used as templates. The CfMVε (lane 1), CrTMV IRES (lane 2), or CfMV -1 PRF signal (lane 3) was inserted between the reporter genes. B) Relative expression of 3’LUC from coupled in vitro transcription and translation reactions programmed with linearized dicistronic pKJM plasmids (T7-lacZ-ICS-luc). LUC expression from the reference was set to 1.

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Results and Discussion

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relative 3’LUC expression from constructs containing the 5’lacZ gene instead of the GFP gene, similar to earlier observations on the impact of the 5’gene on the effi cacy of downstream cistron expression (Ivanov et al., 1997, Chappell et al., 2000, Hennecke et al., 2001).

To test the competitiveness of internal initiation against cap-mediated translation, dicistronic mRNAs were translated in the presence of total RNA from yeast (Fig. 7A in I). Under these conditions the relative 3’cistron expression from tobamoviral sequences occurred at levels similar to those observed in the absence of total RNA. The relative enhancement from TMVΩ and CrTMV was 5.1 and 12.9, respectively. When a similar experiment was repeated in the presence of the monocistronic competitor Ruc mRNA, the relative enhancement of 3’LUC expression from TMVΩ was reduced to 2.5, whereas that of CrTMV increased up to 22.3 (Fig. 7A in I). The internal initiation effi ciency from CfMVε did not differ from that of the reference. Coupled transcription and translation reactions performed with equal amounts of monocistronic and dicistronic templates indicated that the 3’cistron expression attained 25% of the level of monocistronic expression (data not shown).

To increase the level of understanding of the eIFs required for translation initiation from the internally positioned viral sequences, dicistronic RNAs were translated in WGE, which was supplemented with cap analogue or poly(A) sequence to reduce the amount of eIFs interacting with them. Incubation of WGE with the cap analogue decreases the amount of components from the cap-binding complex i.e. eIF4E, eIFiso4E, eIF4G, and eIFiso4G (Gallie and Tanguay, 1994, Browning and Gallie, 2001). The

amount of eIF4A is also decreased (Gallie, 2002a). Incubation of WGE with poly(A) depletes the lysate mostly from PABP and eIF4G (Gallie and Browning, 2001), but the concentrations of eIFs 4A and 4B are also reduced (Gallie and Tanguay, 1994). In both cases indirect interactions may also reduce the amount of other eIFs (Gallie, 2002a). Free cap analogue reduces translation from the capped mRNAs and stimulates translation from the uncapped mRNAs (Tanguay and Gallie, 1994). If translation initiation from the internal position is cap-independent, the cap analogue should not affect the 3’cistron translation. This compared favorably with our results, in which we measured higher absolute LUC levels from translations supplemented with the cap than from translations lacking this additive (Table 5). This indicated that titration of eIF4E did not affect the 3’LUC expression. Stimulation of LUC expression was more pronounced in the case of CfMVε and TMVΩ, in which activities almost twice as high were obtained. In monocistronic mRNAs, the Hsp101-mediated translation initiation from TMVΩ is eIF4E-independent but eIF4G-dependent (Wells et al., 1998). For CrTMV and reference mRNAs the extent of stimulation was 30% and 48%, respectively. However, the induced LUC expression did not alter the relative 3’cistron expression ratios signifi cantly in comparison to the situation in which no addition was made (Fig. 7B in I). Supplementation of translation mixes with poly(A) inhibits translation of uncapped mRNAs more than the capped mRNAs (Tanguay and Gallie, 1994). Poly(A) treatment renders the lysates cap-dependent so that the synergistic effect of the cap and poly(A) tail on translation effi ciency of mRNAs is also observed under in vitro conditions (Gallie

Results and Discussion

42

and Browning, 2001). In our studies, the poly(A) addition reduced translation yields from all our dicistronic mRNAs (Table 5). This is expected, considering the scaffolding protein function of eIF4G. Depletion affected the reference mRNA most, in which expression attained only 12% of the level measured in the nonsupplemented translations. With viral sequences, translation was most reduced from mRNAs containing CfMVε (~26%). Tobamoviral sequences were less affected and translation was reduced to ~40%. Thus, the capacity of viral sequences to resist poly(A)-mediated depletion of eIFs correlated with their capacity to increase the relative 3’cistron expression (Fig. 7B in I). In general, translation initiation from ICSs appeared to be dependent on eIF4G.

4.2.2 Internal initiation in barley suspension cells (unpublished)We next tested the capacity of CrTMV IRES and CfMVε to promote internal initiation in barley suspension cells. Dicistronic HP-GFP-CrTMV/

Table 5. Effects of eIF depletion on 3’cistron expression. In vitro translations were programmed with capped and polyadenylated dicistronic HP-GFP-ICS-luc mRNAs and supplemented with 1 mM cap analogue or with poly(A) at fi nal concentration of 27 ng/μl. After a 90-min incubation, reactions were terminated on ice and the LUC activities were measured. Activities are presented as RLUs.

ICS No additive Cap Poly(A)

Reference 4428 ± 454 6555 ± 953 533 ± 155

CfMVε 9173 ± 1060 16594 ± 820 2432 ± 70

TMVΩ 38606 ± 3269 74283 ± 1549 13983 ± 1281

CrTMV IRES 38649 ± 3100 50173 ± 1787 16579 ± 1765

CfMVε−uidA cassettes from constructs used in the in vitro assays were transferred to plant expression vectors between the 35S promoter and transcription terminator. The resulting plasmids were then used to analyze the transient expression of the 3’proximal GUS in barley suspension cells. In these studies a 190-nt region from the CfMV -1 PRF site was used as a reference. Unfortunately, GUS expression was very low due to the low transfection effi ciency achieved via particle bombardment combined with the low activity of 35S promoter in barley. Expression from CrTMV was barely 2-fold above the reference (data not shown). In comparison to the GUS expression measured from a monocistronic control plasmid, expression from an internally positioned CrTMV sequence was less than 8%, while expression from CfMVε was clearly lower. The value reported for CrTMV-mediated IRES activity in transgenic tobaccos is much higher: 30% (Dorokhov et al., 2002). This suggested that either the viral sequences studied did

Results and Discussion

43

not function as effi cient IRESs in barley or that a required host factor was lacking from the barley cells. Due to the very low 3’GUS synthesis obtained with internally positioned CfMVε, further studies with these constructs were not performed in the barley cells.

4.2.3 Internal initiation in yeast (II)In contrast to barley suspension cells, high 3’cistron expression was achieved in S. cerevisiae with constructs in which the CfMVε, TMVΩ, and CrTMV IRESs were placed into the ICS of two reporters in yeast expression vectors (Fig. 1A in II). All viral sequences promoted higher 3’cistron expression relative to the expression measured in the reference, which contained the multicloning site as the ICS (Fig. 2 in II). CrTMV programmed the highest 3’cistron expression of the studied sequences, closely paralleling earlier reports on the effi cacy of CrTMV IRES in yeast (Dorokhov et al., 2002). In general, few viral IRESs are known to function in yeast cells and some are active only if the cap-dependent translation initiation is compromised (Thompson et al., 2001, Dorokhov et al., 2002, Rosenfeld and Racaniello, 2005). This may result from the fact that cap-mediated translation initiation is a very effi cient process in yeast (Preiss and Hentze, 1998). A survey of 2000 yeast genes found no IRES activity from a single yeast 5’UTR (Thompson et al., 2001). Thus, IRES-mediated translation initiation appears to be a relatively rare event in yeast. In our experiments the extent of 3’reporter expression was dependent on the sequence and translatability of the 5’cistron (Fig. 2 in II), even though one could assume that the binding of ribosomes to sequences promoting internal initiation should occur independently of the 5’proximal

ORF. Since IRES studies have been criticized for the uncertainty of whether mechanisms other than internal initiation may give rise to the observed 3’cistron expression (Kozak, 2003), we examined this possibility further.

Evaluation of the yeast expression data indicated that the actual expression levels of the 3’cistron were much lower than the expression measured from the monocistronic plasmids (Table 2 in II). Even more, the plant viral 5’UTRs functioned poorly in the monocistronic context compared with the reference leader. Thus, 3’cistron expression appeared to be rather ineffi cient. One alternative cause for the low-level 3’cistron expression was that it occurred via reinitiation or leaky scanning. However, both events are dependent on translation of the fi rst ORF. In two of the plasmid series used, pHKJM and pHKJMB, translation of the 5’proximal gene was blocked by 90% by a stable HP structure. Thus, in these cases leaky scanning or reinitiation would have been unusually effi cient processes if LUC was expressed via this means. Furthermore, the number of AUGs in the lacZ gene would have prevented leaky scanning to the luc AUG (Kozak, 1989). The fact that the 3’LUC was expressed at comparable levels from pKJM and pHKJM constructs, differing only in the translatability of the 5’lacZ gene (Table 2 in II), suggested that reinitiation or leaky scanning was an unlikely explanation for the observed expression from these constructs. Viral sequences also stimulated 3’LUC expression signifi cantly from pAGL constructs in which the GAL promoter was switched to an alcohol dehydrogenase 1 (ADH1) promoter and the 5’GFP translation was not blocked by the HP (Table 6). Since reinitiation does not usually occur after translation of full-length

Results and Discussion

44

ORFs (Kozak, 2001), it also appeared more likely that if the 3’LUC expression did not result from internal initiation, it would have originated from transcripts arising from splicing or transcription initiation from cryptic promoters.

Splicing or cryptic transcription could generate transcripts, in which the initiation codon of the 3’luc gene is brought close to the 5’end of the mRNA. These monocistronic mRNAs could then promote the LUC expression observed. The presence of shorter mRNAs encoding functional LUC protein was supported by the expression data. The 3’cistron was expressed when the GAL promoter was deleted (Table 3 in II). Since high expression was also measured from promoter-free monocistronic constructs, the 5’reporter appeared to be unnecessary for the observed LUC expression. The fact that transcripts synthesized from the promoter-free monocistronic constructs containing viral sequences stimulated LUC expression, whereas those produced from the GAL promoter inhibited translation (Table 2 in II), suggested that the 5’leaders differed in these mRNAs. Thus, it

ICS Galactose + Raffi nose Glucose

CrTMV IRES 28.8 ± 1.1 2.47 ± 0.63

TMVΩ 15.3 ± 3.2 1.66 ± 0.13

CfMVε 10.9 ± 1.0 0.86 ± 0.04

Reference 0.7 ± 0.2 0.09 ± 0.01

Table 6. The 3’LUC expression from dicistronic pAGD constructs, in which ADH1 promoter regulated transcription of GFP-ICS-luc mRNAs. The cells were grown in the presence of glucose or galactose and raffi nose. The LUC activities were normalized to μg of total protein concentration. Means (± SD) calculated from one experiment with three independent clones are shown.

appeared likely that the mRNAs produced from the promoter-free plasmids lacked the inhibitory parts of the viral sequences. Stimulated LUC expression was also observed in the dicistronic promoter-containing constructs when transcription from the GAL promoter was repressed by growth on glucose (Table 4 in II). No detectable LacZ expression occurred under the same conditions. Thus, the mRNAs programming LUC expression must have lacked a functional lacZ gene. Since no signifi cant expression occurred from the promoter-containing monocistronic luc mRNAs during repression, binding of transcriptional regulators to the GAL promoter appeared to prevent the occurrence of cryptic transcription. No cryptic promoter activity has been found in the yeast expression plasmid backbone used (Hecht et al., 2002). However, introduction of a reporter gene between the GAL promoter and the studied sequences allowed some cryptic transcription to occur, since LUC expression was observed. In this case the increased distance to the GAL promoter may have enabled the transcription initiation complex to form.

Results and Discussion

45

When the mRNAs produced during induced transcription from the GAL promoter were analyzed with Northern blot analysis, the dicistronic mRNAs were readily detected (Fig. 3 in II). However, several additional RNAs were detected with a probe recognizing the 3’luc gene but not with a probe recognizing the 5’GFP gene (Fig. 3 in II, data not shown). Comparison of the sizes of these RNAs with the mRNAs synthesized from the monocistronic plasmids revealed that the larger species most likely represented a monocistronic mRNA. Interestingly, the most abundant mRNAs produced from the dicistronic expression plasmids containing the ADH promoter upstream from the GFP-ICS-luc casettes (pAGL) also had sizes very similar to those of the

mRNAs produced from the corresponding monocistronic expression plasmids (pAL) (Fig. 10). However, larger mRNAs were also detected, which presumably represented the dicistronic mRNAs. Thus, the Northern blot analysis indicated that shorter RNAs were produced from the dicistronic plasmids, which could have programmed the LUC expression.

Surprisingly, although elevated expression was measured from the promoter-free expression plasmids, no mRNAs were detected during growth on glucose or galactose and raffi nose (Fig. 3C in II, Fig. 10). This suggested that the RNAs detected from the promoter-containing dicistronic transformants were not necessarily driving high LUC expression. Therefore, the short RNAs

Fig. 10. Transcript amounts detected in the Northern blot analysis did not correlate with the LUC expression data. Total RNA isolated from yeasts grown on different carbon sources was examined with antisense probe recognizing the 5’terminal part of the luc gene. Yeasts transformed with dicistronic pAGL (ADH-GFP-ICS-luc), pHKJM (GAL-HP-GFP-ICS-luc), and pHKJMΔGAL (HP-GFP-ICS-luc) plasmids and monocistronic pAL (ADH-ICS-luc) and pMKJM (GAL-ICS-luc) controls were studied. A total of 10 μg of total RNA was loaded into the gel. In vitro synthesized HP-GFP-ICS-luc and luc transcripts served as size markers.

Results and Discussion

46

may have represented stable degradation products that lacked the 5’cap structure and were thus poorly translated. In yeast, mRNA degradation is usually initiated by shortening of the poly(A) tail, which is followed by decapping and degradation by a 5’-to-3’ exonuclease (Caponigro and Park, 1996). Yeast mRNA encoding phosphoglycerate kinase 1 (PGK1) is destabilized if a stable HP in its 5’UTR blocks translation (Muhlrad et al., 1995). Thus, the blockage of 5’cistron translation from dicistronic mRNAs may have targeted them for degradation. Alternatively, degradation may have begun from the 5’end of the mRNAs independently of the deadenylation via a nonsense-mediated RNA decay (NMD) pathway. However, NMD is triggered by premature translation termination (reviewed by Weischenfeldt et al., 2005). In our case, this would take place at the termination codon of 5’cistron. Absence of these shorter RNA species in yeast mutants lacking the 5’-3’exoribonuclease 1 (XRN1) would reveal whether the short RNAs were true degradation products. However, it would not explain why the 3’proximal part of the mRNAs was left undegraded.

High LacZ activities were measured during induced transcription from the GAL promoter from dicistronic constructs having a translatable lacZ gene (pKJM). Thus, splicing at the 5’lacZ gene appeared highly unlikely. In addition, RT-PCR analysis of transformants harboring dicistronic HP-GFP-ICS-luc-plasmids (pHKJM) revealed no PCR products truncated at the GFP-ICS-luc junction (data not shown), suggesting that these mRNAs were also not spliced. Finally, a computer-based analysis of conserved yeast splicing sites (Lopez and Séraphin, 2000) found no intron patterns from the upstream reporter sequences or ICSs. Thus, it appeared that

the LUC expression observed originated from cryptic promoters rather than from spliced mRNAs. The CrTMV sequence also functions in inverted orientation (Toth et al., 2001), which suggests that it can function as a transcriptional promoter (Kozak, 2001).

Usually yeast promoters consist of at least three parts, which are an upstream activator sequence (UAS), a TATA box (consensus TATAA), and the initiator element (reviewed by Romanos et al., 1992). We next searched vector-derived intercistronic sequences (VDSs), studied viral and reference sequences, and 5’reporter genes for the putative binding sites of yeast transcription factors (Zhu and Zhang, 1999). Several sites were found from all studied viral sequences but not from the reference sequence (Table 7). For instance, both tobamoviral sequences contained several putative binding sites for TATA-binding protein (TBP) 50-130 nt upstream from the AUG. In yeast promoters, TATA elements are usually located 40-120 bp upstream from the transcription initiation site (Romanos et al., 1992). The largest number of putative binding sites for transcription factors was found from the lacZ gene (Tamle 7). Interestingly, the highest background expression from the reference construct was also observed from plasmids, in which the 5’reporter was lacZ (Tables 2 and 4 in II). This suggests that the combined effect of putative binding sites in viral sequences and in the upstream sequences may have accounted for the enhanced LUC expression observed from viral sequences. The variation in the expression levels observed during growth on different sugars (Tables 2 and 4 in II) may thus have resulted from differences in the activities of the various transcription factors binding to the viral sequences.

Results and Discussion

47

Sequence Transcription factorCfMVε GCN4, GCR1, MSN2TMVΩ GRF10(4), TBP(3)CrTMV IRES ABF1, ADR1, GCN4, GCR10(8), MCM1, TBP(3)Reference -VDSpKJM, pHKJMB GCN4, GRF10, REB1, SWI5, UME6 VDSpHKJM GCR1, LEU3, REB1, UME6(3)

LacZABF1, ACE2(5), ADR1(4), GAL4, GCN4(21), GCR1(24), GRF10(3), HSF1, LEU3(4), MBP1(8), MCM1(2), MSN2(3), NBF, RAP1, REB1(4), SWI4, SWI5(15)

GFP ABF1(5), ACE2, GCR1(2), LEU3, MAC1, MCM1, RAP1, SWI5(3)ABF1 (ARS binding factor): General transcriptional activatorACE2 (Activation of CUP1 Expression): Involved in regulation of histidine and adenine biosynthesis genes.ADR1 (Alcohol Dehydrogenase Regulator 1): Transcriptional activator of alcohol dehydrogenase 2 (ADH2).GAL4 (Galactose metabolism): Transcription factor in expression of galactose-induced genes.GCN4 (General Control Nondepressible): General control of nitrogen and purine metabolism.GCR1 (Glycolysis regulatory protein 1): Activator of glycolytic genes.GRF10 (General regulatory factor 10): Regulation of purine pathway genes.HSF1(Heat shock transcription factor 1): Regulation of transcription in response to heat shock. LEU3 (Leucine biosynthesis): Transcription regulator in branched chain amino acid biosynthesis pathways repressor and activator. MAC1 (Metal-binding activator): Repression of transcription of genes coding for copper transport proteins. MBP1 (MluI-box binding protein 1): G1/S-specifi c transcription. MCM1 (Minichromosome maintenance factor 1): Activator of a-specifi c genes.MSN2 (Multicopy suppressor of SNF1 mutation): Transcriptional activator for genes in multistress response. NBF (Nonamer binding factor): Transcriptional regulation of phospholipid biosynthesis genes.RAP1 (Repressor activator protein 1): Transcriptional regulation of most ribosomal protein genes. REB1 (RNA polymerase I Enhancer Binding protein 1): General transcription factor. SWI4 (Switching defi cient): G1/S-specifi c transcription.SWI5 (Switching defi cient): Transcription factor for control of cell cycle-specifi c transcription of homothallic switching endonuclease.TBP (TATA binding protein): Component of RNA polymerases I, II, and III; part of initiation factors TFIID and TFIIIBUME6 (Unscheduled Meiotic gene Expression): Negative transcriptional regulator involved in nitrogen repression and induction of meiosis.

Table 7. Yeast transcription factors putatively interacting with the used 5’reporter genes, VDSs, or the studied viral and reference sequences. The numbers in the parentheses indicate the number of sites found. VDS = vector-derived intercistronic sequence.

Results and Discussion

48

4.2.3.1 Identifi cation of regions important for gene expression from internally positioned CfMVε in yeast (unpublished)To determine which regions of CfMVε were important for gene expression from the internal position, mutated CfMVε elements were introduced into the HP-GFP-ICS-luc dicistronic plasmids and analyzed in yeast (Fig. 11). Deletion of the region spacing the 5’terminal secondary structure of CfMVε and the AUG of the luc gene reduced LUC expression

severely, suggesting that either the spacing or the deleted sequence was crucial for the observed 3’cistron expression (DelII in Fig. 8B). CfMVε contained putative binding site for MSN2 and GCR1 in the C-rich region following the stem-loop structure (Table 7). However, when this region was deleted (PyrDel in Fig. 8B), no repression in LUC expression occurred. In CrTMV, the GA-rich module directs cross-kingdom IRES activity (Dorokhov et al., 2002). Deletion or mutation of the GAAA motif from CfMVε reduced 3’LUC

Fig. 11. CfMVε was mutated to identify the regions, which were regulating the expression of 3’proximal luc gene from dicistronic constructs. A) Alignment of mutated CfMVε sequences. Alignment was performed with Multalin version 5.4.1. (Corpet, 1988). B) Relative 3’LUC expression from mutated CfMVε sequences. The measured activities were compared with wt CfMVε, which was given the value 1.

Results and Discussion

49

expression, indicating that these regions were also involved in the regulation of the 3’cistron expression from CfMVε (GAAdel and GAAmut in Fig. 8B). Destabilization of the 5’stem-loop structure did not affect LUC expression (5’Destab in Fig. 8B), suggesting that neither the stem structure nor the 5’stem sequence was crucial for LUC expression. However, nt changes in the 3’stem sequence reduced 3’LUC expression (3’Destab in Fig. 8B), suggesting that in addition to the GA-rich loop the downstream region was also needed for expression from the internal position.

4.2.3.2 Determination of 3’ cistron translation from dicistronic mRNAs in yeast spheroplasts (II)To measure the level of the 3’cistron translation from the dicistronic mRNAs, capped and polyadenylated mRNAs were electroporated to yeast spheroplasts together with a transcript encoding RUC. RUC was readily expressed, which indicated that electroporation had been successful (data not shown). However, no 3’LUC expression from the dicistronic mRNAs was detected, even though the assay is very sensitive. In contrast, LUC expression from the monocistronic luc mRNA was 170-fold over the background, which was practically zero. Thus, we conclude that expression from the dicistronic mRNAs was ineffi cient and less than 1% of that measured from the monocistronic mRNAs (1/170x100% = 0.6%, if background would be given value one). When the expression from the dicistronic plasmids was compared with that from the monocistronic reference plasmid, the 3’LUC expression from CrTMV was ~7% (7.2/106.7x100%), 3.7% from TMVΩ (3.9/106.7x100%), and 2.1% from CfMVε (2.2/106.7x100%)

(Table 2 in II). This indicated that the major expression from the dicistronic plasmids arose from templates other than the dicistronic mRNAs.

4.2.4 Evaluation of the dicistronic approach in IRES studiesCurrently, the dicistronic approach is the main experimental setup used to identify and study IRESs. However, the fact that the 3’cistron expression actually arises from the dicistronic mRNAs has often been poorly studied (reviewed by Kozak, 2001, 2003). Several follow-up studies have revealed that in many cases translation appears to originate from aberrant mRNAs derived from cryptic transcription or splicing rather than from the intercistronic position of dicistronic mRNAs (Kozak, 2001, Han and Zhang, 2002, Hecht et al., 2002, Verge et al., 2004, reviewed by Kozak, 2003, this study). IRES activities are also usually presented as relative values, in which expression is compared with a sequence that should not promote internal initiation. Since the level of background expression varies greatly, depending on the ICS and cell type (Niepel and Gallie, 1999a, Gallie et al., 2000) as well as on the sensitivity of the assay used to detect the 3’cistron expression, evaluation of the importance of the expression level is impossible unless comparisons are made against monocistronic controls. One problem in the use of dicistronic expression plasmids is also the fact that the mRNAs produced may undergo some unwanted processing events in the nucleus, which are left undetected due to the lack of suffi ciently sensitive methods. The best way to circumvent these problems is to perform the studies with in vitro-synthesized mRNAs. The synthesis of aberrant mRNAs from upstream reporter sequences may also be avoided by utilization of monocistronic

Results and Discussion

50

expression constructs. However, in this approach cap-mediated translation initiation should be prevented, which may lead to problems similar to those observed with the dicistronic plasmids.

4.3 Proteolytic processing of CfMV polyprotein (III)The CfMV polyprotein is encoded from the second ORF of CfMV RNA (Mäkinen et al., 1995a). Studies of CfMVε suggested that translation initiation from this 5’UTR involved scanning (I). However, since the fi rst initiation codon is in suboptimal context, some ribosomes most probably bypass the fi rst ORF and reach the polyprotein ORF via a leaky scanning mechanism. Sequence comparisons suggested that the proteolytic processing of sobemoviral polyproteins involves proteases that are similar to picornaviral 3C proteases and cellular serine proteases (Gorbalenya et al., 1988). In contrast to the picornaviral proteases, however, sobemoviral proteases contain a serine residue in the place of a cysteine residue at their active sites (Gorbalenya et al., 1988). Therefore, sobemoviral proteases also closely resemble cellular serine proteases and may represent the evolutionary link between cellular and viral proteases (Gorbalenya et al., 1988). In CfMV the conserved amino acids of the active site are located in the central part of the P2A polyprotein (Mäkinen et al., 1995a).

4.3.1 N-terminal sequencing of CfMV VPgCfMV RNA has a viral protein VPg covalently linked to its 5’end (Figs. 1 and 2 in III). Since VPg is packaged in the viral particles among the gRNA, it represents a functional end-cleavage product. To examine the cleavage sites used in CfMV polyprotein processing, N-terminal

sequencing of CfMV VPg extracted from viral RNA was performed. The 17-amino acid sequence obtained corresponded to amino acids 320-336 in the C-terminal part of ORF2A (Fig. 5 in III), verifying that CfMV proteins are organized in a Pro-VPg-Pol order similar to that in other sobemoviruses (van der Wilk et al., 1998) and in related poleroviruses (van der Wilk et al., 1997).

Based on sequence comparisons, Gorbalenya et al. (1988) suggested that sobemoviral proteases cleave between glutamate (E) and serine (S) or threonine (T). Studies of SBMV and SeMV proved that the theory is valid at least in these sobemoviruses (van der Wilk et al., 1998, Satheshkumar et al., 2004). However, N-terminal sequence analysis revealed that the CfMV VPg cleavage occurred between glutamate and asparagine (E319/320N) (III).

4.3.2 Polyprotein processing in infected plantsCfMV polyprotein is not processed in vitro, which indicates that some essential factors are lacking from the system (Tamm et al., 1999). Thus, we examined the processing further in CfMV-infected plants to ensure that all the putatively needed host factors were available. Barley plants were infected with CfMV and plant samples collected from infected and uninfected plants analyzed with antisera raised against P2A, P2B, CP (Tamm et al., 1999), and VPg (III). The sizes of the detected bands were then compared with the calculated sizes of the hypothesized processing products that were predicted, based on the identifi ed N-terminal E/N processing site of CfMV VPg, the size of the CfMV VPg, and the sizes of the proteases and RdRps in related viruses (Table 8).

P2A antisera simultaneously recognized several products with estimated

Results and Discussion

51

sizes of 12, 18, 19, 20, 23, 24, and 30 kDa from the same sample (Fig. 3A in III). The predicted sizes of the serine and serine-like proteases range from 17 to 35 kDa (Dougherty and Semler, 1993). Thus, one of the 18-24-kDa bands could be the CfMV protease (Pro). The ~12-kDa protein could have represented the VPg or the very N-or C-terminal part of P2A (Fig. 5 in II, Table 5). Since the 12-kDa protein was not detected by the VPg antisera, it most likely represented either the N-or the C-terminal fragment of P2A. However, discrimination between these two was not possible with the antisera used. The 24-kDa protein recognized with the P2A antisera was also recognized with the VPg antisera (Fig. 4B

Table 8. Sizes and detection of expected processing products of CfMV polyprotein. Sizes were calculated according to the amino acid sequence of the predicted products.

in III). This suggested that this protein was the C-terminal fragment of P2A, which included the CfMV VPg. The protein was named P27 according to its predicted mass. In PLRV, the C-terminal intermediate is also readily detected (Prüfer et al., 1999). Unexpectedly, the mature 12-kDa VPg was hardly ever detected with the VPg antisera in the CfMV-infected plants. This may indicate that CfMV VPg does not exist in its free form and that it is immediately linked to the viral RNA.

Infection was not synchronous and therefore it was possible that several cleavage intermediates were detected simultaneously. Some of the 18-24-kDa bands may have also represented differently

Results and Discussion

Protein Estimated size, kilodalton (kDa) Recognizing antisera

P2A2B * 103 P2A, P2B, VPgN131Pro-VPg-RdRp * 89 P2A, P2B, VPgN320VPg-RdRp * 69 P2A, P2B, VPgP2A 61 P2A, VPgP2B* 56 P2BT468RdRp* 53 P2BN-term-Pro-VPgE397 / E432 / E445 / E467 * 42.8, 46.5, 47.8,

50.4*P2A, VPg, (P2B)1

N131Pro-VPg-C-term 47 P2A, VPgN-term-ProE319 34 P2AN131Pro-VPgE397 / E432 / E445 / E467 * 29, 33, 34, 36.5* P2A, VPg, (P2B)1

N320VPg-C-term (P27) 27 P2A, VPgN131ProE319 20 P2AN-termE130 14 P2AN320VPgE397 / E432 / E445 / E467 * 8.7, 12.3, 13.7, 16.0* P2A, VPg, (P2B)1*

T398 / S433 / S446C-term 18.0, 14.4, 13.0 P2A1 Short transframe portion, unlikely detected with P2B antisera. * Transframe protein

52

modifi ed isoforms or degradation products of CfMV proteins. Amino acid sequencing of CfMV VPg failed to identify the second and fi fth amino acids. The CfMV RNA sequence (Mäkinen et al., 1995a) suggests that these amino acids should be serine (S321) and tyrosine (Y324). These amino acids can be modifi ed from their hydroxyl groups; one possible modifi cation could be uridylylation. In picornaviruses uridylylated VPg functions as a primer for viral RdRp during RNA replication (Paul et al., 1998).

Sometimes a 58- and a 62-kDa protein were detected with the P2A antisera (data not shown). These proteins could have represented the full-length P2A (61 kDa) precursor. However, in these blots the antisera also cross-reacted with some plant proteins from uninfected and infected plants. Thus, we cannot absolutely rule out that some ‘specifi c’ proteins detected in the infected plants represented host proteins whose expression was induced as a result of infection. The full-length P2A2B polyprotein (103.4 kDa) was never detected with the antisera used. Among many viruses, such as potyviruses, processing already initiates during polyprotein synthesis (Merits et al., 2002). In PLRV and in SeMV the large polyprotein intermediates also represent the minority (Prüfer et al., 1999, Satheshkumar et al., 2004). Therefore, polyprotein processing of sobemoviruses may also initiate cotranslationally. CfMV P2A2B transframe protein is synthesized via -1 PRF (Mäkinen et al., 1995b) and it attains ~10-20% of the amount of P2A (IV, V). Therefore, detection of transframe precursors with P2B antisera would be more diffi cult. In fact, only a single ~54-58-kDa protein, most likely representing the viral RdRp, was detected in the CfMV-infected plants (Fig. 3B in III). In

SeMV, the fi rst cleavage in the polyprotein occurs between the VPg and the RdRp (Satheshkumar et al., 2004). Therefore, it is possible that in CfMV the polyprotein processing also starts at the corresponding cleavage and that no other processing intermediates detectable with the P2B antisera exist.

Several of the predicted intermediates were never detected in the infected plant samples (Table 8), which may indicate that the intermediates were short-lived. Unfortunately, several of the predicted intermediates and the viral CP were similarly sized. Thus, large amounts of CfMV CP (~30 kDa) in the infected plants (Fig. 4A in III) may have masked the detection of intermediates with similar masses. However, a CfMV protein of ~33 kDa was occasionally detected with the P2A antisera (data not shown). This protein could have represented the N-terminal Pro or the Pro-VPg intermediate.

4.3.3 Putative processing sites of CfMV polyproteinFinally, we searched CfMV polyprotein for similar E/N sites used to cleave the Pro-VPg junction. Another E130/131N site was located upstream from the putative protease-encoding region (Fig. 5 in III). Processing at this site would release a protease of ~20 kDa (Table 8). Good candidate proteins of comparable sizes were detected in the infected plant material (Fig. 3A in III). Amino acid comparison between the N-terminal cleavage site of VPg and this putative site also revealed some consensus in the fl anking amino acids (VE/NSRLQPLESS, conserved amino acids in bold), strongly supporting the hypothesis that this was used to release the N-terminus of CfMV protease. Dissimilarities in the fl anking amino acids could be used to fi ne-tune the timing of the

Results and Discussion

53

processing events. In SeMV, the cleavage between protease and VPg is slow, probably because the VPg domain is needed to keep the protease active (Satheshkumar et al., 2004, 2005). These facts would suggest that in CfMV the site E319/320N between the protease and the VPg is also cleaved more slowly than the putative E130/131N site.

The C-terminus of VPg must be released with further processing events. However, whether the C-terminus of CfMV VPg is encoded entirely from the 0-frame or as part of the transframe protein is not currently known. We can hypothesize that the transframe VPg would already be in close contact with the RdRp, which most likely catalyzes the joining between the VPg and RNA. In contrast, if P27 served as a VPg donor, there would be more VPg to be linked with the RNA. However, there are no E/N sites in the 0- or in the -1 frames that would yield a 12-kDa VPg. This suggested that cleavage sites others than E/N must also be used to process the CfMV polyprotein. In SBMV and SeMV, the C-terminal processing of VPg occurs at the E/T site about 80 amino acids downstream from the N-terminal cleavage site (van der Wilk et al., 1998, Satheshkumar et al., 2004). A similarly located E397/T398 site can also be found in CfMV P2A (Mäkinen et al., 1995a). However, C-terminal cleavage at this site would produce a VPg of 8.7 kDa, whereas the virion-extracted VPg has a mass of 12 kDa. This indicates that if CfMV VPg was released from P27, it must have undergone signifi cant modifi cations. In contrast, a 12-kDa protein would be produced if the C-terminal cleavage site was close to residue 430. However, no suitable E/N or E/T sites could be found around that region in the 0- or the -1 frames. We next looked for E/S sites, which are used in some processing events of studied sobemoviruses and

poleroviruses (van der Wilk et al., 1998, Satheshkumar et al., 2005). Processing at P2A E432/433S or E445/446S would yield a 12-kDa protein, however, the fl anking amino acids shared no similarity with the other predicted E/N processing sites.

The 54-58-kDa size of the RdRp detected in CfMV-infected plants indicated that the cleavage must occur in the vicinity of the -1 PRF site. Once again, no suitable E/N sites were found in that region. The N-terminus of SBMV VPg is processed at RSQE326/327TLPPEL (van der Wilk et al., 1998). In SeMV the corresponding release occurs at RSNE325/326TLPPEL (Lokesh et al, 2001). Interestingly, a similar site RAAE467/ 468TEFPEL is located at the beginning of the CfMV ORF2B transframe region. Processing at this site would yield a replicase of 53 kDa and a transframe VPg of ~16 kDa. One of the 18-19-kDa proteins detected with the P2A antisera may possibly have represented this transframe VPg (Fig. 3A in III). However, a VPg of this size would probably be too large to be linked to viral RNA without further processing. After its initial release, the SeMV RdRp undergoes further processing at a suboptimal E/S site to yield an RdRp of ~52 kDa (Satheshkumar et al., 2004).

In conclusion, the processing sites used in CfMV differ clearly from those used in SeMV and SBMV. However, phylogenetic comparison of the N-terminal Pro-VPg region of sobemoviruses showed that CfMV is clearly distinguished from SeMV and SBMV (Lokesh et al., 2001). In fact, the CfMV Pro-VPg domain shares only 27% similarity with SeMV Pro-VPg (Lokesh et al., 2001). Similarities in the genome expression strategies between poleroviruses and sobemoviruses prompted us to adapt the model proposed for processing of PLRV polyprotein (Prüfer et al., 1999) to CfMV. Poleroviral

Results and Discussion

54

and sobemoviral polyproteins share an N-terminal transmembrane domain, which may indicate that a membranous location is required for processing (Prüfer et al., 1999, Satheshkumar et al., 2004). This could explain why no processing is observed under in vitro conditions. The membranous location is most probably also required for viral RNA replication. P27 contains motifs for RNA binding (Tamm and Truve, 2000b), and this part could ensure that viral RNA is transported along the P2A to the same cellular location with viral proteins. This would end up in the colocalization of all required components in the same compartment of the cell.

To be able to examine the timing of the cleavage events, synchronous infections should be obtained. This would require that either viral RNA or infectious complementary DNA (icDNA) would be delivered to plant protoplasts to attain suffi ciently high transfection effi ciency. This approach would also make metabolic labeling of the translation products possible, which could ease the detection of transient processing intermediates. The usage of icDNA would also allow the predicted processing sites to be mutagenized. Finally, a wider repertoire of antisera would enable more precise identifi cation of the cleavage products.

4.4 Synthesis of CfMV polyprotein

4.4.1 CfMV RNA programmed -1 ribosomal frameshifting in WGE (IV, V)The strategy CfMV uses to produce its RdRp differs from that of most other sobemoviruses. Instead of polyprotein synthesis from a continuous ORF, CfMV polyprotein is produced from two overlapping ORFs, 2A and 2B, via -1 PRF (Mäkinen et al., 1995b). ORF2B encodes

the viral RdRp and thus the effi ciency of -1 PRF determines its amount. Two signals putatively directing the event, a slippery UUUAAAC heptamer (1634-1640) and a downstream stem-loop structure (1648-1676), can be found in the N-terminal part of the ORF2A2B overlap (Mäkinen et al., 1995b, Fig. 1 in IV). Chemical probing of segment 1634-1690 from CfMV RNA has shown that a 12-bp stem with a 4-nt loop is formed 7 nt downstream from the heptamer (Tamm, 2000c). Mutational analysis verifi ed that the slippery heptamer and the downstream region forming the secondary structure were essential for the -1 PRF (Fig. 2 in IV).

Increasing evidence shows that both the nearby sequences as well as long-distance interactions may affect -1 PRF (Kollmus et al., 1994, Honda et al., 1996, Barry and Miller, 2002). In addition to the cis-acting signals, we examined whether some regions from the CfMV polyprotein region were required for effi cient -1 PRF. This was done with dual-reporter enzyme constructions, which can detect even small changes in -1 PRF frequencies (Stahl et al., 1995, Harger and Dinman, 2003). The beauty of this assay system resides in the fact that the translation products can be easily quantifi ed by measuring their enzymatic activities. Furthermore, experimental variation can be monitored as changes in the fi rst reporter activity. Thus, the fi rst reporter activity can be used to normalize the second reporter activity determining the effi cacy of the recoding event (Stahl et al., 1995). We selected three regions (A:1602-1720, B:1386-2137, and C:1551-1900) from the overlapping polyprotein region of CfMV for the study (Fig. 1 in V). The highest -1 PRF, ~36%, was measured from the longest CfMV sequence (B). In contrast, the medium-length region (C) promoted -1 PRF with ~28% effi cacy,

Results and Discussion

55

which was only slightly over the 25% -1 PRF frequency measured from the shortest region (A) (Fig. 4B in V). This indicated that in CfMV the surrounding regions of the cis-acting signals may also infl uence -1 PRF. One danger in the approach used was that the varying C-terminal fusions in the transframe proteins could differentially reduce the specifi c activity of the fi rst reporter used for normalization (Fig. 1A in V); however, this was not the case here (Fig. 4A in V). Further shortening of the A region to 70 nt (CfMV RNA 1621-1690) did not prevent -1 PRF (IV, Fig. 2A; pJCL24). Thus, although high -1 PRF frequencies are usually obtained only with pseudoknot structures (Dinman, 1995, Plant and Dinman, 2005), it appeared that in CfMV a simple stem-loop structure was capable of promoting effi cient -1 PRF, because chemical probing of the secondary structure revealed that the loop does not interact with the surrounding regions (Tamm, 2000c). However, improved -1 PRF from the B region suggested that some interactions leading to stimulated -1 PRF may have occurred between the cis-acting signals and the ORF2A2B overlap. Alternatively, longer insertions between the reporter genes may have affected the kinetics of the CfMV stem-loop folding and induced more frequent translational pauses at the heptamer and thus enhanced -1 PRF. Purifi ed CfMV RNA appears to induce higher levels of -1 PRF than the polyprotein encoding region (Fig. 3A in IV). Therefore, we cannot exclude the possibility that regions outside the CfMV polyprotein region may also affect -1 PRF. For instance, interactions between the 3’UTR and the cis-acting stem-loop in BYDV regulate the switch from replication to protein synthesis (Paul et al., 2001, Barry and Miller, 2002).

Differences in genome organization

between CfMV and most other sobemoviruses account for the fact that less RdRp is produced in CfMV than in its relatives. This could indicate that CfMV RdRp is more operative and therefore smaller amounts are needed. When CfMV replicase was changed to be encoded from the 0-frame to resemble the genome organization found in the majority of sobemoviruses (reviewed by Tamm and Truve, 2000a), the -1 PRF effi ciencies increased 4-5-fold (Fig. 2B in IV). However, the effect was not specifi c for the CfMV sequences, since an equal increase was observed when a similar shift in frames was introduced to a reporter gene construct (pJCL24Δ), when long regions from the RdRp sequence were deleted (pJCL16Δ), or when the gene rearrangements occurred after the entire P2A was synthesized (pJCL28, Table 1 in IV). Thus, the only thing these high -1 PRF effi ciency constructs appeared to have in common was the shortening of the transframe product and the simultaneous increase in length of the 0-frame product. Control experiments in which the 0-frame product was gradually shortened (pJCL17Δ, pJCL17ΔMfeI, pJCL17ΔSmaI) showed no change in the -1 PRF frequencies, indicating that the 0-frame length had no effect on the -1 PRF.

Shortening of the transframe product so that no gene rearrangements occurred, alone improved -1 PRF 3-fold (pJCL22 in Fig. 2B in IV). Thus, only part of the increase in the -1 PRF frequency of ORF rearrangement constructs could be explained by relocation of the transframe termination codon closer to the -1 PRF site. Since a similar movement of termination in the 0-frame did not affect -1 PRF much (pJCL22Δ in Fig. 2B in IV), the phenomenon appeared to be characteristic only for ribosomes shifting the frames.

Results and Discussion

56

Termination is a slower process than elongation and it induces pausing and stacking of ribosomes upstream from the termination codons (Wolin and Walter, 1988). In WGE up to 10 ribosomes were found stacked behind a termination codon and each ribosome protected an ~27-29-nt region from the mRNA (Wolin and Walter, 1988). For instance, in MuLV introduction of a stop codon 48 nt downstream from the recoding site stimulated the readthrough 5-fold (Wills et al., 1991). In our studies high -1 PRF frequencies were obtained even if the termination codon was located 462 nt downstream from the stem-loop structure (IV). This would mean that approximately 16 ribosomes should be queuing behind the termination codons if the increase in -1 PRF was due to stacking. We can hypothesize that the transframe ribosomes have a slower pace than ribosomes translating the 0-frame, due to the translational pause at the -1 PRF signals. However, a major fraction of the ribosomes passes the -1 PRF signals without pause (Lopinski et al., 2001). These ribosomes may possibly move more rapidly and thus could easily reach the trailing slow transframe ribosomes. This could induce stacking, and a translational pause at a nearby termination codon would give a ribosome occupying the -1 PRF signal an opportunity to shift frames, even though the secondary structure was opened.

4.4.2 The -1 PRF in vivo (V)The -1 PRF is highly dependent on the kinetics of both translation initiation as well as elongation (Barry and Miller, 2002, Harger et al., 2002). However, protein synthesis is signifi cantly slower under in vitro conditions than in vivo (Lopinski et al., 2000). Thus, we extended our studies to include in vivo conditions. Due to the

highly conserved nature of the elongation phase from prokaryotes to higher eukaryotes, recoding events such as -1 PRF and the termination codon readthrough used by viruses infecting mammalian and plant cells can be recapitulated in yeast (Stahl et al., 1995, Harger and Dinman, 2003, Bekaert et al., 2005). Although identical mechanisms of -1 PRF are used in eukaryotes and prokaryotes, the effi cacy of the process may differ (Garcia et al., 1993, Napthine et al., 2003). Thus, instead of using plant cells for the in vivo studies, we performed the studies in S. cerevisiae and E. coli for convenience. The A, B, and C regions from the CfMV polyprotein were analyzed with dual-reporter vectors, in which the regions tested were inserted between the lacZ and luc genes (Stahl et al., 1995, Fig. 1A in V).

As expected, the CfMV -1 PRF signals were functional both in yeast and in bacterial cells, although -1 PRF occurred at lower frequencies in the prokaryotic cells (Fig. 2 in V). In general, the XXXAAAC heptamers function ineffi ciently in prokaryotes (Garcia et al., 1993, Brierley et al., 1997, Napthine et al., 2003), even though the prokaryotic Asp-tRNA encoding the AAC triplet promotes effi cient -1 PRF in eukaryotic ribosomes. This indicates that the differences in -1 PRF frequencies between eukaryotes and prokaryotes must arise from the translational apparatus itself (Napthine et al., 2003). Thus, further analyses were performed in yeast.

Improved -1 PRF was measured for longer CfMV regions in vivo (Fig. 2 in V), comparing favorably with the in vitro assays performed with Ruc-luc dual-reporter RNAs. Previously, it was shown that under in vitro conditions a fraction of the ribosomes terminates at the slippery heptamer during the ribosomal pause

Results and Discussion

57

(Lopinski et al., 2000, Plant et al., 2003). Our protein analysis showed that similar premature termination products were also produced in vivo (Fig. 3 in V). These termination products appeared to be more abundant when the longer CfMV regions were programming -1 PRF. However, similar -1 PRF frequencies were obtained, irrespective of whether the additional CfMV sequences were placed upstream or downstream from the -1 PRF site (Fig. 2C in V). Thus, the longer -1 PRF cassettes may have affected the folding of the stem-loop and induced longer or more frequent translational pauses. Higher frequencies of ribosomal pausing would have presumably induced termination and -1 PRF at the heptamer.

The current hypothesis suggests that stem-loops are less resistant to unwinding than pseudoknots. This results in shorter pausing at the -1 PRF signals (Dinman, 1995, Plant et al., 2003, 2005). Keeping this in mind, the CfMV -1 PRF can be regarded as unusually high compared with reported effi ciencies of ~1-3% from other -1 PRF signals with stem-loop structures (Prüfer et al., 1992, Stahl et al., 1995). One reason for the high -1 PRF frequency in CfMV is the heptamer UUUAAAC, which directs effi cient -1 PRF in eukaryotic cells (Brierley et al., 1987, Napthine et al., 2003). The fi rst triplet of the heptamer plays a multiplicative role in determining the -1 PRF effi ciency, and U at this position promotes the highest -1 PRF frequencies (Bekaert et al., 2003). The Asp-tRNA encoding the AAC triplet also promotes effi cient -1 PRF and also dictates that slippage occurs via the simultaneous dual-tRNA slippage mechanism (Napthine et al., 2003). Finally, the CfMV spacer sequence between the cis-acting signals also contains bases that are often found in

spacers of -1 PRF signals promoting high levels of -1 PRF (Bekaert et al., 2003).

4.4.3. Regulation of -1 PRF by CfMV proteins (V)The correct ratio between RdRp and the 0-frame products is vital for viruses using -1 PRF (Dinman and Wickner, 1992, Hung et al., 1998, Barry and Miller, 2002). However, the need for RdRp probably varies during infection. At the initial stages the full-length gRNA is needed for production of proteins with enzymatic properties, whereas at the later stages CP encoded from the sgRNA is needed for particle formation. Thus, it could be benefi cial for the viruses to regulate the effi cacy of the -1 PRF to fi t the need for the amount of RdRp. Thus far no viral or cellular proteins are known to be directly involved in the regulation of -1 PRF. However, the +1 PRF event encoding mammalian ornithine decarboxylase (ODC) antizyme is up-regulated by increased cellular concentrations of polyamines, which are the biosynthesis products of ODC (Matsufuju et al., 1995). The antizyme binds to the enzyme and directs it to degradation. Thus, ODC concentration is reduced and polyamine synthesis becomes down-regulated. We tested whether CfMV proteins produced via -1 PRF could participate in the regulation of -1 PRF. We coexpressed CfMV RdRp or P27 (the C-terminus of P2A) together with the lacZ-luc dual-reporter vectors carrying the minimal CfMV -1 PRF region A or the corresponding inframe control, Am (Fig. 1 in V). The effect of the CfMV proteins on -1 PRF was then evaluated by monitoring the changes in reporter gene expression. Control coexpressions were performed with empty expression plasmids or with constructs in which the translation initiation codon of P27 or RdRp was deleted.

Results and Discussion

58

Neither of the proteins affected the expression of the fi rst reporter (lacZ) since there was no signifi cant difference in the β-galactosidase activities between protein coexpressions and control expression with the empty expression plasmids (Table 2 in V) or with the AUG deletion mutants (data not shown). In contrast, both proteins appeared to affect the downstream reporter expression to some extent when LUC activities were compared with those measured from the coexpressions performed in the presence of the empty expression vector. However, coexpressions with RepΔAUG showed that the RdRp expression did not affect -1 PRF specifi cally. In contrast, similar comparisons with P27 and P27ΔAUG expressions indicated a reproducible reduction in the amount of LUC produced in the presence of CfMV P27. The repressing effect of P27 was stronger on the inframe control than on the actual -1 PRF construct. This, however, resulted from the fact that since only ~15% of the elongating ribosomes translate the transframe protein in the -1 PRF test constructs, the effect observed in LUC expression will also be relatively lower and more diffi cult to detect.

The function of P27 is unknown, but it contains motifs for RNA binding. In fact, P2A binds RNA in an unspecifi c manner, putatively via this motif (Tamm and Truve, 2000b). Since the synthesis of the fi rst reporter was not affected by P27 expression, the effect of P27 was not due

to unspecifi c but to specifi c binding of P27 to a certain part of the downstream RNA. We believe that this binding occurred at the CfMV -1 PRF region, which resulted in the inhibition of downstream translation. Thus, during CfMV infection the accumulating amounts of P27 in the cells could indicate that no additional RdRp is needed, whereas subsequent processing of P27 could relieve the block. Alternatively, P27 could prevent -1 PRF, rendering the 3’proximal part of CfMV RNA free of ribosomes. This would enable the 3’proximal end to function as a template for sgRNA synthesis in the absence of collisions with ribosomes moving in the opposite direction. However, it is not yet known whether CfMV sgRNA is synthesized from the full-length (-) strand or from a shorter template. In BYDV, translation initiation and -1 PRF are regulated via direct interaction of specifi c elements in the 3’UTR with complementary regions in the 5’UTR and the stem-loop of the -1 PRF site (Barry and Miller, 2002). Initiation of the (-) strand synthesis from the 3’end of the (+) strands disrupts these base-pairings and prevents translation initiation and -1 PRF. Further rounds of replication would produce excess amounts of (+) strands, which would then outcompete the RdRp molecules and be free to form the long-distance interactions needed for translation. However, it is clear that the importance of P27 for -1 PRF and virus infection, and thus the specifi city of P27 binding, should be studied further.

Results and Discussion

59

5. CONCLUDING REMARKS

All the viral sequences studied, namely AMV 5’UTR, CfMVε, CrTMV IRES, PVXαβ, and TMVΩ, programmed higher gene expression in tobacco compared with the 5’UTR composed of a polylinker. In contrast, only CfMVε led to the production of higher protein yields in barley. Expression from constructs containing CfMVε was ∼12-fold higher than that from TMVΩ-containing constructs, even though TMVΩ functions as a translational enhancer in other monocots such as rice and maize. This suggests that the requirements for effi cient gene expression may differ in barley. Transient expression studies with luc mRNAs having the reference 5’UTR, CfMVε, or TMVΩ as the 5’leader showed comparable LUC accumulation from all mRNAs. Therefore, the capacity of viral sequences to enhance reporter expression did not result merely from promoted translation and the mechanism should be studied further. Introduction of an uORF to the CfMVε abolished downstream reporter expression, suggesting that translation initiation from CfMV RNA involves scanning. The fi nding that destabilization of the putative 5’proximal structure improved downstream gene expression further supported this observation. Competition assays performed in vitro showed that CfMVε did not compete as successfully against eIFs as CrTMV IRES and TMVΩ. This may have resulted from the fact that the 5’proximal stem-loop structure in CfMVε renders translation initiation dependent on the complete set of eIFs, whereas initiation from tobamoviral sequences may occur in the absence of some eIFs. In fact, translation initiation from TMVΩ was reported to be eIF4E-independent (Gallie, 2002a). Interestingly, all these sequences also promoted internal

translation initiation in WGE. Depletion of WGE from eIFs interacting with the cap analogue or the poly(A) sequence suggested that internal initiation was eIF4E-independent but eIF4G-dependent. Poly(A) addition reduced the 3’proximal cistron translation mostly from the reference mRNA, whereas the viral sequences recruited the eIFs interacting with the poly(A) sequence more effi ciently. However, in vivo studies revealed that the IRES activity of CfMVε and CrTMV was low, at least in barley and yeast. Furthermore, our studies showed that the combination of plant viral sequences with dicistronic reporter gene expression plasmids led to unpredictable behavior of the constructs and, thus, this approach was not applicable to yeast.

After successful binding of the preinitiation complexes to the 5’end of CfMV RNA, leaky scanning brings the preinitiation complex into the region encoding CfMV polyprotein. The CfMV RdRp is synthesized via -1 PRF as the C-terminal part of the transframe polyprotein. Thus, the occurrence of -1 PRF is extremely important for the viral viability. Interestingly, CfMV protein P27 repressed translation of proteins encoded downstream from the -1 PRF signals. Thus, P27 may play a role in regulating the amount of RdRp produced. Alternatively, P27 may function in regulating the ribosomal load at the 3’end of the RNA. Proteolytic processing releases the functional domains from the CfMV polyproteins. However, the E/N site used to process the CfMV VPg differs from the E/T or E/S sites recognized by the proteases of other sobemoviruses. The CfMV polyprotein did not contain a suffi cient number of E/N sites, which

Concluding Remarks

60

would have led to the complete processing of the polyprotein. Thus, additional cleavage sites are clearly utilized. The search for other putative cleavage sites suggested that the E/T or E/S sites could also be used in CfMV for some processing events. The usage of these sites could be verifi ed by mutating the suspected sites in the icDNA of CfMV.

Although, CfMVε did not function at the translational level, the clear improvement of gene expression in barley

relative to other viral sequences studied indicates that CfMVε could be utilized in biotechnological applications to increase heterologous protein expression in cereals. Furthermore, CfMV -1 PRF signals could be utilized to synthesize a fraction of proteins with certain C-terminal fusions. For instance, introduction of tags would enable affi nity purifi cation and immunodetection of a certain percentage of the expressed protein.

Results and Discussion

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6. ACKNOWLEDGEMENTS

This study was carried out at the Institute of Biotechnology and Department of Applied Biology, University of Helsinki, during the years 1999-2005. Financial founding provided by the Academy of Finland, the University of Helsinki, and the National Technology Agency of Finland (TEKES) is highly appreciated.

My warmest thanks go to my supervisor Kristiina Mäkinen, who gave me the opportunity to carry out this work. During our years together she has provided me constant guidance, support, and encouragement. I also appreciate the positive attitude, amazing memory, and endless fl ow of new ideas. I also want to thank for the possibility to work independently and freely.

I would like to thank Prof. Mart Saarma, the director of Institute of Biotechnology, for providing excellent facilities during the years 1999-2001. Equally, the help and support from the personnel of SBL during years 2002-2005 is highly appreciated. Prof. Mirja Salkinoja-Salonen is thanked for giving me the opportunity to carry out my PhD studies at the Department of Applied Chemistry and Microbiology. I also wish to thank the personnel of Viikki Graduate School in Biosciences for providing many interesting courses. Mikko Frilander and Tero Ahola, the follow up group of my PhD project, are warmly thanked for the annual meetings and helpful comments.

I also wish to thank reviewers docent Maija Vihinen-Ranta and professor Carl-Henrik von Bonsdorff for the time they spent reading my text as well as for the comments, which led to the improvement of the text.

The previous and the current members of the lab: Andres M., Kostya I., Pietri P., Jimmy L., Deyin G., Minna R., Eva W., Anders H., Kimmo R. and Rasa G. are thanked for creating splendid and humoristic working environment. I am indepted to Andres, who initially thought me patiently the methods of molecular biology as well as the basics of cloning. My special thanks go to my closest colleagues in the group, Pietri and Kostya. Their skilfull assistance in the case of technical problems during experiments is much appreciated. For Rasa, Anders, and Kimmo, I wish good luck with your projects, productive collaborations, and nice karonkkas in the near future. Julia P. and Anne S. from the animal virus laboratory are greatly appreciated of joined courses, lunches, and congress trips. I also wish to acknowledge all the co-authors for their contribution to our mutual papers.

Warm thanks also go to my friends, Jonna J., Jonna P., Petra, Soile, and Leena, who have actively organized events, which have forced me to switch from science to free time. Also Ninnu and Merlin are thanked for giving me an opportunity to relax and have some phycical exercise after work.

Finally, my very special thanks go to my family. My parents Tuovi and Arvo have provided me constant trust and support but also tought me to work hard. My sister Kirsi and his husband Marko are thanked for their encouragement. Most of all, I want to thank Harri for the interest, endless inspiration and support.

Helsinki, March 2006

Acknowledgements

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