Dimerization of retroviral RNA genomes: an inseparable pair

RNA dimerization is the non-covalent process by whichretroviruses carry two identical (or nearly identical)RNA genomes into the virion particle. In recent years,significant progress has been made towards elucidatingthe relationships between the conformation of RNAgenomes, RNA dimerization and retroviral replication.This review summarizes the contributions of in vitroanalysis to the identification of important retroviralRNA dimerization signals and how the RNA tertiarystructures might regulate viral replication. Our currentunderstanding of trafficking, dimerization and virionpackaging of the RNA genomes during retroviral assem-bly is presented (FIG. 1), and the impact of RNA dimer-ization on the generation of multidrug-resistantmutants and the potential of using RNA dimerization asan antiviral target is also discussed. As HIV-1 is one ofthe most intensively studied retroviruses, many exam-ples of retroviral RNA dimerization will be drawn fromHIV-1-based research.Whenever it is possible, however,examples of other retroviruses or retrotransposons willbe presented to emphasize the similarities, as well as thedifferences, of retroviral RNA dimerization. Here, wereview the current understanding of RNA dimerizationand discuss some of the implications for future study.

In vitro dimerization of retroviral RNAIdentification of the dimerization initiation site.Analysis of the Rous sarcoma virus (RSV) genome pro-vided the first indication that retroviruses contain twocopies of their RNA genome. Subsequent studies have

shown that the two copies of virion RNA genomes are dimerized in several type C retroviruses by a non-covalent interaction near the 5′ end of the genomes.This region has been designated as a dimer linkagestructure (DLS)1–4. Viral RNA genomes are likely tointeract with proteins throughout the viral replicationcycle, making it difficult to delineate the contributionsof RNA and proteins to retroviral RNA dimerization. Inthe early 1990s, it was shown that short in vitro tran-scribed RNAs that contain the DLS dimerize sponta-neously in the absence of proteins5–7. This approach hassince been instrumental in dissecting the contributionof RNA sequences in retroviral RNA dimerization. Thedimerization initiation site (DIS) of the HIV-1

Malisolate

(a recombinant of HIV-1 subtypes A, D and K) was thefirst to be identified8,9. The DIS consists of a shortsequence that is located between the primer-bindingsite and the major splice-donor site, which folds into ahairpin structure (FIG. 2a,b). Following this finding, sev-eral groups have reported that a homologous sequencefrom another HIV-1 isolate (HIV

Lai) also mediates the

in vitro dimerization of the RNA genome10–12.

The loop–loop kissing-complex model: the case of HIV-1.The presence of a self-complementary sequence in theDIS loop of all HIV-1 subtypes (FIG. 2d) suggests thatdimerization occurs through symmetrical intermolec-ular interactions between these sequences to form aKISSING-LOOP COMPLEX8–10,13 (FIG. 2c). Accordingly, anymutation that destroys the self-complementarity in the

DIMERIZATION OF RETROVIRAL RNAGENOMES: AN INSEPARABLE PAIRJean-Christophe Paillart*, Miranda Shehu-Xhilaga‡, Roland Marquet* and Johnson Mak‡,§

Many viruses carry more than one segment of nucleic acid into the virion particle, but retrovirusesare the only known group of viruses that contain two identical (or nearly identical) copies of the RNAgenome within the virion. These RNA genomes are non-covalently joined together through a processknown as genomic RNA dimerization. Uniquely, the RNA dimerization of the retroviral genome isof crucial importance for efficient retroviral replication. In this article, our current understanding ofthe relationship between retroviral genome conformation, dimerization and replication is reviewed.

KISSING-LOOP COMPLEX

Formed when two RNAhairpins interact through theircomplementary loops.

NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | JUNE 2004 | 461

*UPR 9002 du CNRS affiliée à l’Université Louis Pasteur, Institut deBiologie Moléculaire etCellulaire, 67084 StrasbourgCedex, France.‡Macfarlane Burnet Institutefor Medical Research andPublic Health, Melbourne3004, Victoria, Australia.§Department ofBiochemistry and MolecularBiology, Monash University,Clayton 3800, Victoria,Australia.Correspondence to R.M.and J.M.e-mails: [email protected];[email protected]. and M.S.-X.contributed equally to thiswork.doi:10.1038/nrmicro903

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462 | JUNE 2004 | VOLUME 2 www.nature.com/reviews/micro

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Accordingly, all mutations that maintain a self-comple-mentary sequence do not guarantee stable dimer for-mation11,14,16. Furthermore, selection of RNAs that arecapable of homo- and heterodimerization from poolsof degenerated DIS loops only yields a limited numberof combinations that resemble natural HIV-1 DISsequences17. Selection experiments indicated that, atmost, two A–U base-pairs are tolerated in the kissing-loop interaction17, and a mutant virus with a DIS loopcontaining four A–U base-pairs replicates poorly16.Selection experiments also indicated that the two cen-tral nucleotides of the self-complementary sequencehave a pivotal influence on the stability of the kissing-loop complex, which indicates that they might consti-tute the nucleation point of the RNA dimer17 — aproposal that is consistent with mutagenesis analysesof the self-complementary sequence11. Finally, extend-ing the complementarities between the two loops to more than six nucleotides does not increase the stability of the kissing-loop complex17,18.

In most HIV-1 isolates, the self-complementarysequence in the DIS loop is flanked by two 5′ and one 3′unpaired nucleotides (FIG. 2d). Deletion or substitutionof these nucleotides strongly impairs dimerization11,14.Selection of the most stable RNA dimers from pools ofdegenerated DIS loops reveals a strong pressure tomaintain unpaired adenines at the first and last posi-tions of the loop17, in agreement with the naturallyoccurring DIS loops. On the basis of chemical probingand interference data, the flanking nucleotides wereproposed to be involved in non-canonical interac-tions14,19. However, such interactions are not observedin the crystallographic structures of the DIS kissing-loop complexes20 (FIG. 3). It is conceivable that althoughthese interactions are not present in the final structure,they still have an important role in the dimerizationprocess, most likely by structuring the monomeric DISloop. This hypothesis is strengthened by the fact thatdeletion of the unpaired purines mainly affects theRNA association rate14.

Stabilization of the kissing-loop complex. The kissing-loop model of HIV-1 RNA dimerization proposes thatthe initial complex could be transformed into a morestable EXTENDED DUPLEX (FIGS 2c,3). Indeed, nuclear mag-netic resonance (NMR) structures21,22 and X-ray crystalstructures20,23 of both DIS kissing-loop complexes20,22

and extended duplexes21,23,24 have been published. Thesestudies all used 23-mer RNAs that corresponded to theapical stem loop of the DIS (FIG. 3). For these shortRNAs, there is evidence to support transition from thekissing-loop complex to the extended duplex withoutdissociation of the loop–loop interaction, rather than byformation of the duplex from denatured stem loops25–27.Retroviral RNA dimerization is facilitated by the nucle-ocapsid protein7,28–30. It has recently been shown that inthe presence of magnesium chloride the nucleocapsid-mediated transition is favoured by protonation of thefirst adenine in the loop27. Formation of an extendedduplex by a 39-mer RNA oligonucleotide that containsboth the upper and lower DIS stems (FIG. 2c) is much

DIS loop prevents RNA dimerization8,9,11,14,15, whereasthe introduction of compensatory mutations restoresthe process.

Only two of the possible 64 self-complementaryhexanucleotides are commonly found in the DIS loop,which indicates that not all self-complementarysequences promote the formation of RNA dimers.

Nucleus

Maturation

Translation

Reversetranscription

Budding

Genomic RNA

cDNAIntegration

PROVIRUS

Transcription

d

c

Gag

LDI BMH

b

NC?

a

Protein assemblyand packaging

Figure 1 | Schematic of the HIV-1 replication cycle. The figure shows the importance of tertiaryRNA genome structures in viral cDNA synthesis, the translation of viral proteins and the assemblyand maturation of viral particles. Upon viral infection, the virion core is released into the infected cell,where the dimeric RNA genome is used as a template for the synthesis of viral cDNA by reversetranscription. Studies have indicated that dimerization of the RNA genome facilitates recombinationduring synthesis of viral cDNA (see panel a; arrows represent template switching). Full-lengthgenomic RNA functions as mRNA to produce structural and enzymatic proteins and/or copies ofthe RNA genome. The dimerization initiation site is located in a region of the genome that is crucialfor viral replication. It has been proposed that the conformation of this region could have animportant role in the regulation of functions such as translation and packaging (panel b; see FIG. 4

for more details). Virion encapsidation of the RNA genome is mediated through the nucleocapsid(NC) domain of the Gag precursor protein, which is also involved in the dimerization of the viralRNA genome (see FIG. 4 for more details). Genomic viral RNA also acts as a scaffold in themultimerization of viral structural protein Gag during viral assembly (panel c). Gag is thought to re-enter the nucleus prior to virion release. Upon virion release, the virion particle undergoes amaturation process in which the precursor proteins are cleaved for the formation of mature virioncore. Changes in the virion core morphology during maturation coincide with the rearrangement(or maturation) of the dimeric viral RNA genomes (panel d). BMH, branched multiple hairpin; LDI,long-distance interaction.


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formation indicates that the tight dimer that is formed at high temperatures does not originate from the kissing-loop complex13. Indeed, no dimer is formed at a temperature that is intermediate between the optimaltemperatures for formation of the kissing loop complexand the tight dimer (37°C and 55°C, respectively)13.However, this does not preclude the possibility that a DISextended duplex can form from partially denatured RNAmolecules.A nucleocapsid-induced in vitro ‘maturation’of a retroviral RNA dimer is also observed in the case ofHarvey sarcoma virus (HaSV)29 and HIV-2 (REF. 33).

Interestingly, stabilization of the HIV-1 RNA dimercan occur independently from formation of anextended duplex9,14,15,34,35. A short sequence of ~100

slower than for the 23-mer RNAs, and is prevented bydeletion of the internal loop31.

In longer RNAs (more than 300 nucleotides), the DISkissing-loop complex is not converted into an extendedduplex in the absence of protein cofactors at physiologicaltemperatures (37°C)15. An alternative form of RNAdimer that is more stable than the kissing-loop complex isobtained when incubation is performed either in thepresence of nucleocapsid protein28 or at high tempera-tures13,32. This tight dimer (as opposed to the kissing-loop dimer, which has been referred to as the loosedimer) has often been assumed to be an extended duplex,even though there is little evidence to support this con-clusion. The biphasic temperature dependence of dimer

EXTENDED DUPLEX

A complex that results fromcomplete intermolecular base-pairing of two stem loops. It canbe obtained from the kissing-loop complex by disrupting theintramolecular stems andforming intermolecular base-pairs.

1

1

TAR Poly-A

Poly-A

PBS

PBS

DIS

DIS

615

SD

SDAUG

Psi

IRES

IRES

Dimerization

gag

TAR

gag–pol envMatrixU5R

200100 300 400 450

AGGUGCACA�

AAGCGCGCA�

AAGUGCACU�

AAGUGCACA�

GAGUGCACC

a

b

c d

U3 R

Nucleocapsid ?

CUUGCUG GUGCAC ACAGCAAG��

GAACGACA CACGUG GUCGUUC

AG

GA

Kissing-loop complex

Extended duplex

CUUGCUG GUGCAC ACAGCAAG��

GAACGACAA CACGUG GUCGUUC

CUCG

GAGC

AG

GA

260* 280

272*

294

294* 280* 260

273

5′

3′

5′

3′

3′

5′

3′

5′

Stem Stem Subtypes

A

M

B/D

C

F/H, G

O

Loop

G

G AG

CGAG

GCUG

GA G

G

CUCG

GAGC

G

G AG

CGAG

GCUG

GA G

G

Figure 2 | The HIV-1 dimerization initiation site. a | Schematic representation of the HIV-1 genomic RNA. R, repeat sequence;TAR, trans-acting responsive element; poly-A, 5′ copy of the polyadenylation signal; U5, unique at the 5′-end of the RNA genome;gag, 5′-end of the structural protein coding sequence; PBS, primer-binding site; DIS, dimerization initiation site; SD, major splice-donor site. b | Secondary structure of the 500 nucleotides at the 5′ end of HIV-1 genomic RNA. The main secondary structureelements are indicated: TAR, Poly-A, PBS, DIS, SD, Psi (main part of the RNA packaging signal), AUG (translation initiation codon ofthe gag gene). The two regions involved in the kissing-loop complex are highlighted by the yellow box. c | Schematic of the HIV-1RNA dimerization mechanism, showing the kissing-loop complex and the putative extended duplex. d | Comparison of the DISsequences of various HIV-1 groups (M, main; O, outlier) and subtypes (A, B, C, D, F, G and H).


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corresponds to an extended duplex is unknown. Invivo, deletion of the internal loop and lower stem ofthe DIS decreases the amount of the dimer that isinside the virions, but does not affect its thermal sta-bility42. This finding contrasts with the effect of theinternal loop deletion in vitro, which prevents forma-tion of the extended duplex31 and suggests that in vivomaturation does not correspond to formation of anextended duplex. In addition, transition from the kissing-loop complex to an extended duplex by full-length genomic RNA inside the virions would pose aformidable topological problem. Of note, the contro-versial idea that this transition might proceed via atransesterification mechanism, rather than by ‘melting’of the DIS stem, has recently been proposed20,43.

Is dimerization regulated by an RNA switch? Thedimerization signal of retroviruses is located in a regionthat is crucial for retroviral replication. Indeed, the 5′-UNTRANSLATED REGION (5′-UTR) of HIV comprises several functionally independent domains, the sec-ondary structures of which are crucial to their func-tions. These domains include signals for transcriptionaltransactivation (the TAR domain), splicing (the splice-donor site domain), reverse transcription (the primer-binding site domain) and genomic RNA encapsidation(FIG. 2a,b). In vitro experiments show that long-distanceinteractions involving sequences that are several hun-dred nucleotides apart could take place within the 5′-UTR33,44,45. These tertiary interactions involve eitherthe polyadenylation signal loop and a region in thematrix coding sequence44, or a U5 sequence, which isupstream of the primer-binding site, and nucleotidesencompassing the gag initiation codon33,45 It has beenproposed that these interactions could regulate the 5′ polyadenylation signal and the translation of Gag byoccluding functional cis elements, respectively (FIG. 1).Recent studies have indicated that the 5′-UTRs of HIV-1(REFS 45–49) and HIV-2 (REFS 50,51) could form alternativesecondary structures in which functional elementswould be either accessible or silenced. A conforma-tional ‘switch’ between these structures would allowtemporal regulation of the functions that are associatedwith the 5′-UTR. Depending on the RNA secondarystructure — which in HIV-1 RNA is either long-distance interaction (LDI) or branched structure withmultiple hairpins (BMH); see FIG. 4 — the DIS self-complementary sequence would be either base-pairedto the polyadenylation signal (in the case of LDI struc-tures)45, thereby preventing genomic RNA dimeriza-tion, or accessible in the DIS stem loop (in the case ofBMH structures). In this latter case, the AUG startcodon would interact with a palindromic sequence inU5 (REF. 45), possibly inhibiting protein translation. Ofnote, palindromic sequences in the TAR domain52 andthe poly-A region53 could also be directly involved inintermolecular interactions between two genomic RNAsas part of the DLS53. As the in vitro RNA conformationmight be affected by experimental conditions, it wouldbe helpful to gain direct insights into the RNA structurein infected cells and virions.

nucleotides immediately downstream of the 5′ splice-donor site increases the thermal stability of the HIV-1

Mal

RNA dimer by about 12°C. The stabilization mecha-nism is unknown, but substitution of the loop of thesmall hairpin after the gag initiation codon was shownto prevent stabilization. The amplitude of the stabiliza-tion that is provided by the sequences downstream fromthe splice-donor site depends on the self-complemen-tary sequence that is present in the DIS loop14,35, whichraises the possibility of a direct interaction betweenthese two RNA regions.

A stabilization or maturation of the genomic RNAdimer has also been described in vivo for RSV36,37,Moloney murine leukaemia virus (MoMuLV)38, HIV-1 (REF. 39) and the retrotransposon Ty1 (REF. 40). In allcases, maturation requires processing of the viral pre-cursor protein Gag. As this processing generates themature nucleocapsid protein, the IN VIVO MATURATION

could correspond to the nucleocapsid-protein-induced stabilization of the dimer that is observed invitro. However, it has been shown that, in vitro, themature nucleocapsid protein and the unprocessedGag protein have identical effects on HIV-1 RNAdimerization and on the stabilization of the HaSVRNA dimer41. Whether the mature in vivo dimer

IN VIVO MATURATION

A process that depends on theprocessing of the polyproteinGag and results in increasedthermal stability of the RNAdimer.

5′-UNTRANSLATED REGION

(5′-UTR). An RNA sequencethat is found upstream of theprotein-coding sequence.

a Kissing complex b Extended duplex

Figure 3 | X-ray crystal structures of the kissing-loop complex and extended duplex of a23-mer RNA (subtype A isolate). The two RNA strands are represented in red and green.Structures were drawn using the coordinates deposited in the Nucleic Acid Database (NDB IDUR005 and UR0016).


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For instance, two sequences are implicated in the invitro dimerization of HIV-2 RNA19,50,51,55,76. The first is aself-complementary sequence located in the 5′-end ofthe primer-binding site that acts as a loose-dimer-inducing element, whereas the second involves SL1 — astem-loop structure that is homologous to the HIV-1DIS that mediates the formation of tight dimers50,51,76.The transition of HIV-2 RNA from loose to tight dimersis mediated by disruption of a long-distance interactionby the nucleocapsid protein51,76. More recently, it has been suggested that a palindromic sequence in the HIV-2 packaging signal promotes the formation ofHIV-2 RNA dimers33.

In MoMuLV, and the related retrovirus HaSV,base pairing of the self-complementary 18-nucleotidesequence does not constitute the initial step, but contributes to formation of a stable dimer71,74,75,77. It isunderstood that the recognition step would be medi-ated either by two hairpins with the same GACGtetraloop75, which form a kissing-loop complex withonly two base pairs78, or by two separate stem-loop ele-ments that are positioned 55 bases apart in the leaderregion, each of which contributes to genomic dimer ini-tiation through independent kissing-loop interactions74.In MuSV, the genomic strands are thought to associatethrough specific G-rich elements that facilitate dimer-ization through an adjacent kissing loop72,73. In HTLV-1,a 13-nucleotide sequence, which, except for an adenineat its centre, is self-complementary, is believed to governRNA dimerization; although the precise mechanismremains unknown56–58. With other retroviruses, the situation is not so clear and work is still required to pinpoint the site of dimer initiation and the mechanismof dimerization.

RNA dimerization in retroviral replicationIs the DIS stem loop (SL1) the sole determinant for retro-viral RNA dimerization? Although it is unlikely that in vitro RNA dimerization perfectly mirrors the dimer-ization process in the virion, in vitro RNA dimerizationhas provided numerous clues to the process of retroviralRNA dimerization. It is now well established that theself-complementary stem loop of SL1 (which has beenidentified by an in vitro assay) is crucial for RETROVIRAL

REPLICATION79–86 (TABLE 1). However, RNA sequences outsideof the DIS stem loop are also important in the process ofretroviral RNA dimerization (TABLE 2).When avian retro-viral dimeric RNA was isolated under conditions thatpromote RNA nicking, it was observed that dimericRNA remains heat stable at various temperatures, whichindicates that the RNA interacts at multiple sites withinthe viral genome68,87. Using a HIV-1 vector-based system,it was noted that removal of the polymerase gene (pol)results in the formation of a more stable RNA dimer,which indicates the presence of putative RNA elementsin the retroviral pol gene that might negatively regulatethe stability of RNA dimers88. In addition, partial orcomplete deletion of the DIS stem loop does not elimi-nate the presence of detectable dimeric RNA in the virions35,42,80,88–91. The aberrant genomic RNA dimersthat are found in these mutants support the hypothesis

It has been speculated that the conformationalswitch could be induced in vivo by the nucleocapsidprotein49 (FIG. 4) and a two-step mechanism is possible.First, during translation, the ribosome would disruptlong-distance interactions to induce a conformationalchange within the 5′-UTR. Second, after translation, thenucleocapsid domain of Gag would induce importantconformational changes in the unspliced viral RNA andmodulate dimerization and encapsidation of thegenomic RNA. This conformational RNA switch couldalso explain the activation of an internal ribosome entrysite (IRES), which partially overlaps the main RNApackaging signal, during the G2/M cell-cycle phase inwhich cap-dependent translation is inhibited54.

The dimerization site of other retroviruses. In addition toHIV-1, self-complementary sequences have been shownto be involved in the dimerization of several other retro-viral RNAs. These include HIV-2 (REFS 19,50,55), HTLV-I56–58, human foamy virus59–61, bovine leukaemiavirus62,63, avian retroviruses64–68 and the murine retro-viruses HaSV, murine sarcoma virus (MuSV) andMoMuLV5,30,69–75. In avian retroviruses, the process seemsto be similar to that observed with HIV-1. Dimerizationis promoted by a hairpin containing an 8-nucleotideloop, which includes a 6-nucleotide self-complementarysequence in its centre64.As in HIV-1, mutational analysissupports the formation of a kissing-loop complex.However, for other retroviruses, it seems that a self-complementary sequence is not sufficient to promotecomplete and functional RNA dimerization.

RETROVIRAL RECOMBINATION

A process to generate new strainsof retroviruses by mixing geneticmaterials of two different strainsof parental retroviruses.

PBS

TAR

SD

DIS

AUG

Psi

gag

gag

Psi

Long-distance interaction (LDI) Branched multiple hairpin (BMH)

SD

DIS

PBS

TAR

R U5

AUG

R

Poly-A

Poly-AU5

Nucleocapsid ?

*

*

LDI BMH

No Yes

Yes No

RNA genome dimerization(and encapsidation)

Protein translation

Figure 4 | Model showing the putative conformational switch that is proposed toregulate translation and packaging of the HIV-1 genomic RNA. The long-distanceinteraction (LDI) secondary structure is proposed to be the translation-competent form, whereasa conformational change to the branched multiple hairpin (BMH) secondary structure would allowthe genome to be encapsidated through interactions between its Psi and dimerization initiationsite (DIS) domains and Gag (mainly its nucleocapsid domain). The regulatory motifs are shown indifferent colours. PBS, primer-binding site; SD, splice-donor site. Adapted with permission fromREF. 45 © (2003) American Society for Biochemistry and Molecular Biology.


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viral RNA genomes can be detected in the lysate of avianreticuloendotheliosis virus (REV)-producing cells67.

The hypothesis that the dimerization of retroviralRNA genomes occurs prior to RNA packaging is alsosupported by the fact that mutant retroviruses that aredefective in RNA dimerization are often defective inRNA packaging mechanisms79,82,85,105–107. Several attemptshave also been made to generate virions that containmonomeric RNA genomes, including: mutating eitherthe RNA dimerization sequence42 or the amino acidsequence of the viral Gag protein108,109; placing additionalDLSs into other parts of the retroviral genome110,111; oraltering the ratio and the cleavage of viral precursor pro-teins112. Three studies have shown that only monomericRNA genomes are found in these mutant mature HIV-1(REFS 110,113) and RSV108, although the amounts of viralRNA packaged in these mutants are reduced.

By inserting additional DLSs into the HIV-1genome, Sakuragi et al.110 were able to ‘trick’ these DLSsto form intramolecular RNA–RNA interactions, whichresulted in the production of virion particles containingexclusively monomeric RNA genomes110. This studyindicates that RNA–RNA interactions between twoDLSs are required for optimal packaging and that theseinteractions can be either inter- or intramolecular94,110.By maintaining the authentic DLS and performingmutational mapping on the second DLS without alter-ing the amounts of virion RNA , it was found that the RNA dimerization domain is embedded in theRNA-packaging signal, which indicates that RNAdimerization is part of the virion RNA packagingprocess in wild-type HIV-1 (REF. 94). Mutating the HIV-1matrix nuclear-export signal 113 or inserting a plasma

that the DIS palindromic sequence is the nucleationpoint for genomic RNA dimerization11,17, but these dataalso highlight the involvement of additional RNAsequences in virion RNA dimerization91–95.

Similar to genomic RNA dimerization, the place-ment of primer tRNA onto the primer-binding site ofthe viral genome is also facilitated by retroviral nucleo-capsid sequences in the context of Gag96–98. As theprimer-binding site is positioned in close proximity tothe DLS in all retroviruses, it has long been speculatedthat primer tRNA might have a role in the process ofretroviral RNA dimerization. Studies on Ty3 and Ty1retrotransposons indicate the involvement of primertRNA in the dimerization of retrotransposon RNA ele-ments99,100; however, a similar requirement for primertRNA has not been observed with retroviruses87,94,101.

Is RNA dimerization necessary for virion packaging ofthe RNA genome? With the exception of some initialreports showing that selected members of the avianretrovirus family might package RNA genomes intovirions as monomers that later dimerize during virionmaturation36,37,68,87,102,103, it has been generally found thatnewly released wild-type retroviral particles containdimeric RNA. In addition, when murine leukaemiavirus (MLV)-producing cells are treated with actino-mycin D for several hours to inhibit the de novo synthe-sis of viral mRNA104, the amount of genomic RNA thatis packaged per particle is drastically reduced. However,the RNA in these particles is still dimeric. These obser-vations indicate that the formation of an RNA dimer is aprerequisite for the virion packaging of retroviral RNAgenomes. Furthermore, it has been shown that dimeric

Table 1 | Impact of DIS stem-loop mutations on stages of the HIV-1 life cycle compared with wild-type virus

DIS Replication RNA Reverse Dimerization Thermal Processing Referencesmutation packaging transcription stability

Loop substitutions Delayed 5–6-fold n.d. n.d. n.d. n.d. 156∆257–269* reduction

Loop substitutions Delayed n.d. n.d. Decreased Identical n.d. 81

Loop substitutions 10-fold reduction 2-fold reduction n.d. Identical Identical n.d. 79

Loop substitutions 10–103-fold 5-fold reduction 3–10-fold reduction n.d. n.d. No effect 85∆243–277 reduction (2nd strand transfer)

Loop disruption n.d. n.d. n.d. Identical Identical n.d. 88

Stem and loop 10-fold reduction 5-fold reduction n.d. Decreased Identical No effect 80mutations

Stem and loop 99% reduction 2–5-fold n.d. 4-fold reduction n.d. No effect 82substitutions reduction∆248–261, ∆241–256*

Stem deletions 99% reduction 5-fold reduction n.d. n.d. n.d. p2–capsid 134∆241–256, ∆264–277a reduced

Loop substitutions 80–98% reduction 5-fold reduction n.d. n.d. n.d. n.d. 16

Stem deletions 99% reduction 5-fold reduction n.d. n.d. n.d. Reduced 83∆241–256 + ∆264–277*

Deletion of stem loop B n.d. 4-fold reduction 5-fold reduction 2-fold reduction Identical n.d. 42∆243–247, ∆271–277*

Stem loop B 2–3-fold reduction n.d. n.d. 2-fold reduction Identical n.d. 90substitutions

Stem deletions Identical in PBMC 2-fold reduction n.d. Diffuse RNA dimer Identical No effect 91

*Numbering refers to genomic RNA HxB2 isolate. n.d., not determined; PBMC, peripheral blood mononuclear cells.


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detected in the gag genes of other retroviruses115.Furthermore, a mRNA transport protein, known asStaufen, is selectively packaged into HIV-1, HIV-2 andMLV, but not into virus-like particles that lack RNAgenomes116. Either over- or underexpression of theStaufen protein can interfere with the level of virion-packaged RNA, which leads to a reduction in HIV-1infectivity116,117. It was speculated that the virion packag-ing of Staufen could be linked with HIV-1 RNA dimer-ization117, in particular because the association ofDrosophila Staufen is required for the dimerization-dependent localization of bicoid mRNA118. Furtherexperiments will be required to define the potential con-tributions of RNA and protein trafficking to the processof RNA dimerization during retroviral assembly.

Viral assembly, protein processing and RNA dimer maturation. The process of genomic RNA dimer forma-tion and stabilization in retroviruses is intricately linkedto the assembly and maturation of viral particles. In thecase of type C retroviruses, virions are first assembled atthe plasma membrane and released by ‘pinching off ’from the infected cell as immature virions (for reviews,see REFS 119,120). The immature virions are subsequentlyconverted to mature, infectious retroviruses throughviral protease-mediated protein cleavages. It has alsobeen shown that the cleavage of viral precursor proteinsin HIV-1 enhances the rate of virion release121, althoughinactivation of the viral protease does not prevent therelease of retroviruses from the infected cells36,37,122,123.

The formation of virion particles requires the multi-merization of the precursor proteins Gag, GagPol and,for selected retroviruses, GagPro. Together with thematrix and capsid domains, the interacting domains(which overlap with the basic sequences found withinthe nucleocapsid domain) drive the multimerization ofretroviral precursor proteins. In vitro assembly analyseshave shown that viral RNA might function as a scaffoldfor the multimerization of viral precursor proteins124–126,and the role of viral RNA scaffolding in virion forma-tion could be substituted by host cell RNA127. Otherresearchers have proposed that virion RNA is importantfor stabilization of the virion particle architecture128.This is consistent with early reports that virion RNApackaging-defective mutants are enriched with host-cell

membrane-targeting signal at the amino terminus RSV Gag108,109 results in the production of non-infectious virions that encapsidate only monomericRNA. In both cases, the defects in RNA dimerizationare likely to be related to the intracellular trafficking ofGag during viral assembly (see below). Although gen-eration of retroviral particles containing monomericRNA genomes is possible108,110, the formation of adimeric RNA genome is likely to be a prerequisite forthe production of infectious retroviral particles.

What is the role of trafficking in the formation ofretroviral RNA dimers? By blocking the chromosomeregion maintenance 1 (CRM1)-mediated nuclear export,Schiefele and co-workers114 demonstrated that traffick-ing of RSV Gag into and out of the nucleus is crucial forviral assembly. Insertion of a plasma membrane-target-ing signal at the N-terminus of RSV Gag prevented Gagfrom undergoing this cytoplasm–nucleus–cytoplasmtransport109,114, and this correlated with the detection ofmonomeric RNA RSV particles. An earlier study hadshown that the HIV-1 matrix sequence in Gag containsboth a nuclear-localization signal and a nuclear-exportsignal113. Similar to RSV, HIV-1 Gag also undergoescytoplasm–nucleus–cytoplasm transport in a CRM1-dependent manner113. HIV-1 with a mutation in thematrix nuclear-export signal are defective in Gag traf-ficking and yield virion particles with monomeric RNAgenomes113. At present, it is presumed that the traffick-ing redirection of RSV or HIV-1 Gag is the reason forthe monomeric RNA that is observed in these virions,although direct evidence has yet to be provided. If thetrafficking of Gag is important for RNA dimerization,then perhaps only the nuclear-localized, unspliced,retroviral RNA is destined to be packaged as thedimeric RNA genome, hence the entry of retroviral Gaginto the nucleus to recruit the appropriate RNAgenomes for retroviral assembly113,114. Alternatively, thenuclear transport of retroviral Gag could be required tomodify specific viral factors for the formation ofdimeric RNA genomes.

In addition to Gag, a cis-acting RNA-traffickingsequence, A2RE, that could interact with the host celltrans-acting trafficking factor hnRNP 2A is found in theHIV-1 gag RNA. Similar A2RE-like sequences are also

CRM1

(Chromosome regionmaintenance 1). A cellularprotein that mediates thenuclear export of numerousproteins.

Table 2 | Investigating the impact of mutations outside of the DIS hairpin on aspects of the virus life cycle

Mutation Replication RNA Reverse Dimerization Thermal Processing Referencespackaging transcription stability

Mutations in matrix None detected Reduced n.d. Mainly monomeric n.d. No effect 113(K18A and R22G)

Deletions in U5 Delayed 4–6-fold n.d. 5-fold reduction Reduced No effect 93∆99–108, ∆112–123* reduction

SL3 mutations Delayed 2-fold reduction n.d. 2-fold reduction Reduced n.d. 92

GA-rich region Delayed or dead 2-fold reduction n.d. Reduced Reduced n.d. 92(324–336)

∆306–325* Delayed 2-fold reduction n.d. 2-fold reduction Reduced No effect 95

Lower stem of n.d. 2–5-fold n.d. 2-fold reduction Identical n.d. 94the PBS domain reduction

*Numbering refers to genomic RNA HxB2 isolate. n.d., not determined; PBS, primer-binding site.


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Gag that correct both the RNA packaging and dimeriza-tion defects. However, this positive effect on virion RNAdimerization is not detected with SL3-deleted mutantswhen these mutations are presented in the context ofprotease-negative immature HIV-1, indicating that thep2 and nucleocapsid compensatory mutations areimportant during virion protein processing and coreformation 95. These data further strengthen the existingevidence on the dynamic relationship between retroviralRNA dimers and virion core formation.

RNA dimerization, reverse transcription and recombi-nation. The evolutionary conservation of RNA dimer-ization among retroviruses highlights its importance inthe replication cycle of retroviruses. When comparedwith wild-type RSV, mutant RSV particles containingmonomeric RNA genomes exhibit a 100-fold reductionin the synthesis of viral cDNA108, which supports a rolefor RNA dimerization in reverse transcription. Synthesisof the viral cDNA is initiated near the 5′ end of the viralRNA genome to generate an intermediate productknown as negative-strand strong-stop DNA. The nega-tive-strand strong-stop DNA is then translocated to the3′ end of the RNA genome to enable the synthesis of theviral cDNA to resume. The translocation process iscalled first-template switching, and might occurthrough an intermolecular135 or intramolecularpathway136,137. However, heat dissociation of virion RNAdimers suppresses the synthesis of HIV-1 cDNA101.Furthermore, HIV-1 mutants with an altered DIS havedefects in reverse transcription that are linked to a defectin strand transfer to complete the synthesis of the viralcDNA42,85. Therefore, the most likely role of RNA dimer-ization in reverse transcription is to drive the recombina-tion of retroviral genomes during the synthesis of theviral cDNA138 (FIG. 1).

Studies on MLV have shown that, in vivo, templateswitching during retroviral cDNA synthesis frequentlyoccurs at direct RNA–RNA interactions that are medi-ated by palindromic sequences139–143. It is thought thatthe dimerization of RNA templates enhances recombi-nation, and this hypothesis is supported by several invitro biochemical studies on HIV-1 (REFS 144–146).Epidemiology studies have shown that recombinationbetween different subtypes of HIV-1 occurs throughoutthe viral RNA genome147,148, which is consistent with theview that virion RNA dimers are linked at multiple sitesof the RNA genomes68,87. Identification of locations atwhich recombination during reverse transcription isfrequent might help to reveal other RNA sequences inthe retroviral genome that can support retroviral RNAdimerization.

It has been shown that deletion of the complete DISstem loop strongly decreases the HIV-1 infectious titre inboth primary cells86 and T-cell lines80,85,92. However,requirement for the DIS stem in the replication of HIV-1is cell-type dependent91. Hill and co-workers found thatHIV-1 mutants lacking the DIS stem are replicationcompetent in primary peripheral blood mononuclearcells (PBMCs) but not in a SupT1 T-cell line. Despitelong-term passage in PBMCs, restoration of the DIS

RNA. As the retroviral Gag precursor protein exhibitsRNA chaperone activity41 and dimeric retroviral RNAgenomes can be found at the earliest stage of virionrelease (rapid harvest and immature retroviruses), thedimeric viral RNA genome, rather than monomericRNA, might be a scaffold for the multimerization ofGag.

During the maturation of the virion, dimeric RNAalso undergoes a rearrangement process38,102,129. DimericRNAs that are extracted from these newly assembledparticles (which are collected 5–10 minutes after releasefrom the host cell) have a slower electrophoretic mobil-ity38 and altered density-sedimentation profiles102,129

when compared with RNAs that are extracted from thefully mature retroviral particles. The idea that virionRNA undergoes a maturation process after release isfurther supported by analysis of the genomic RNA ofprotease-inactive retrovirus mutants36–39. Using a geneticapproach to ‘freeze’ the process of virion maturation,several groups have reported that the dimeric RNAfrom immature retroviruses displays an altered elec-trophoretic mobility compared with the wild-type con-trol36–39. Furthermore, incubation of the purified viriondimeric RNA at increasing temperatures has shown thatthe RNA dimers from mature particles are more resis-tant to heat denaturation than are those from immatureparticles38,39, which shows that virion maturation leadsto a stronger association between the two copies of theRNA genome. The proteolytic processing of retroviralprecursor proteins (Gag and GagPol) during virionmaturation is a highly regulated stepwise event130.Analysis of HIV-1 mutants that are defective at variousstages of proteolytic processing shows that the release ofthe N-terminus of the nucleocapsid protein from theGag precursor protein is critical for the maturation of virion RNA dimers, whereas proteolytic cleavage ofother processing sites in the Gag protein does not affectthe formation of heat-stable RNA dimers131 — a findingthat is likely to also be true for MLV132.

Interestingly, RNA enhances the in vitro cleavage ofviral proteins133, and the HIV-1 RNA genome itself hasbeen implicated in protein processing and maturationof the virion. A HIV-1 mutant with a deletion in the DISstem loop that is defective in RNA dimerization is alsounable to complete the processing of virion proteins83.Evolutionary pressure via long-term culturing of thismutant in the MT2 T-cell line84,134 shows conservationof the deletion throughout the study and the emergenceof compensatory mutations in the gag region, includinga p2 mutation that is immediately upstream of the N-terminal nucleocapsid-cleavage site84,134. These com-pensatory mutant virions have a similar replicationcapacity as the wild-type virus84,86,134 and show a patternof fully processed virion protein84,134. These findingsreflect an intimate relationship between the DLS, theRNA-dimerization process, retroviral RNA dimers and virion protein processing. Recently, Rong and co-workers95 reported that long-term culture of a HIV-1mutant in which the SL3 RNA packaging signal hasbeen deleted yields virions containing compensatorymutations in the p2 and the nucleocapsid regions of


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structural similarity between the DIS and the eubacte-rial 16S ribosomal aminoacyl-tRNA site (A site); andthe finding that aminoglycoside antibiotics bind the DIS with high affinity and specificity154,155. In additionto inhibiting RNA dimerization and packaging,molecules that can target RNA dimerization couldpotentially prevent reverse transcriptase recombina-tion and limit the generation of multidrug-resistantHIV-1 strains. Such molecules might also reduce thenatural variability of HIV-1 and help the immune system to clear infection.

Concluding remarksThe significance of retroviral RNA dimerization lieswith its evolutionary conservation and the potential fordeveloping antiretroviral agents that target reverse tran-scriptase-mediated recombination, which is believed tobe an important mechanism for the generation ofmultidrug-resistant mutants. The findings from in vitroRNA dimerization studies have proven to be a valuableguide in defining this complex and intimate relationshipbetween retroviral RNA dimerization, genomic RNApackaging and virion maturation. However, the in vitro-defined mechanism of RNA dimerization and the con-tributions of RNA tertiary structures to viral replicationmight not accurately reflect the in vivo situation. It istherefore crucial that we gain insights into the RNAstructure in both infected cells and virions to validatethe roles of these RNA structures in viral replication.More importantly, such assessment will be vital in high-throughput screening for the design of RNAdimerization or structure inhibitors. The role of traf-ficking of both viral protein and RNA, and the potentialinvolvement of host cell factors also provide newavenues for dissecting the mechanism of retroviral RNAdimerization. Eradication of HIV-1 will require aneffective strategy to control the emergence of mul-tidrug-resistant mutants, and understanding the processof RNA dimerization might provide important clues forthe development of methods by which this problem canbe addressed.

stem or the presence of compensatory mutations is notdetected91 (TABLE 1). Further analysis of these mutants inadditional T-cell lines (such as H9 and CEM) have failedto identify any other cell type that can support the repli-cation of a DIS-stem-deleted mutant (J.M. et al.,unpublished observations). The reported discrepancy— the propagation of DIS stem mutants in primarycells compared with T-cell lines — could imply the lossof specific host cell factors during T-cell transformation.Such cellular factors might be important for reverse transcription through direct or indirect interaction withthe DIS stem during virion uncoating85, perhaps by par-ticipating in template switching (or even recombination)during the synthesis of viral cDNA.

RNA dimerization as an antiviral target?Highly active antiretroviral therapy (HAART) has shownexceptional promise in prolonging the life expectancy forHIV-1-infected patients. However, the increasing fre-quency of multidrug-resistant HIV-1 mutants highlightsthe need for new antiviral drugs. In this respect, the roleof the DIS in viral recombination is an attractive targetfor multidrug therapy for several reasons. First, reversetranscriptase-mediated recombination is thought to bean important mechanism for the generation of mul-tidrug-resistant HIV-1 mutants in cell culture149,150.Second, splenocytes from HIV-1-infected individualsrevealed up to eight integrated proviruses per infectedcell (with a mean of 3.2)151. Third, recent studies haveshown that the frequency of HIV-1 double infections isgreater than if they occurred at random152 and the dou-bly HIV-1-infected cells are more recombinogenic thanpreviously estimated153. Therefore, the ability of HIV-1 toreplicate at a low level in patients on HAART provides anideal environment for the generation of multidrug-resistant HIV-1 mutants through reverse transcriptase-mediated recombination. The first steps towardsinhibiting RNA genome dimerization by targeting theDIS have been facilitated by: the high-resolution X-raycrystal structures of the kissing complex20 and extendedduplex23 forms of the DIS; the recognition of the

HAART

(Highly Active AntiretroviralTreatment). Consists of aminimum of three drugs thatinhibit the activity of the viralenzymes reverse transcriptase orprotease. The first compound ofa third drug class, the fusioninhibitors, has recently beenapproved for treatment.

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AcknowledgementsJ.-C.P. and R.M. thank B. Ehresmann and C. Ehresmann fortheir constant interest and support, and thank the former mem-bers of the Ehresmann laboratory for their contribution to thestudy of retroviral RNA dimerization. M.S.-X. and J.M. thank S.Campbell for her assistance in some of the graphic design, plusformer and current members of the Burnet Institute for their sup-port and contribution to the study of retroviral assembly. Wethank all our reviewers for their insightful comments, and apolo-gize to those colleagues whose research has not been high-lighted owing to length constrains. The selected examplesmerely reflect our personal interest. This work was supported bythe Agence Nationale de Recherches sur le SIDA in France, andthe Australian National Health and Medical Research Council C.J. Martin Fellowship, Monash University and Pharmacia–PfizerFoundation in Australia.

Competing interests statementThe authors declare that they have no competing financial interests

Online linksDATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/Entrez/HIV-1 | HIV-2SwissProt: http://www.ca.expasy.org/sprot/Gag

FURTHER INFORMATIONJohnson Mak’s laboratory: http://www.burnet.edu.auRoland Marquet’s laboratory: http://www-ibmc.u-strasbg.fr/smbmr/UPR_9002/Pages/site_Marquet/1.Accueil_Marquet.htmlAccess to this links box is available online.

Dimerization of retroviral RNA genomes: an inseparable pair

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