TOBACCO MOSAIC VIRUS REPLICASE AND ......Tobacco mosaic virus replication 279 fully double-stranded (ds). The RI-like RNA was partially sensitive to both enzymes, showing it had the

J. Cell Sci. Suppl. 7, 277-285 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987

111

TOBACCO MOSAIC VIRUS REPLICASE AND

REPLICATIVE STRUCTURES

N E V IN Y O U N G , J U L IE F O R N E Y a n d M IL T O N Z A IT L IN Department of Plant Pathology, Cornell University, Ithaca, New York 14853, USA

S U M M A R Y

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complemen­tary RNA, initiated in vivo\ however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase.

Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity.

Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography.

The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126 000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

I N T R O D U C T I O N

The genomes of RNA viruses are replicated by enzymes (replicases) which are specifically generated by the infecting virus, and which are most probably comprised of both virus-coded and host-coded subunits (Hall e ta l. 1982). The ultimate objective in studies of replicase enzymes is to obtain the replicase in a soluble form, the activity of which is dependent on a specific template. Moreover, the enzyme should recognize only the RNA of the virus from which it has been prepared (and, perhaps, of closely related strains). Finally, the true ‘replicase’ should be capable of synthesizing full-length viral nucleic acid of virus-sense polarity from a virus-sense

278 N. Young, J. Forney and M. Zaitlintemplate. In no case have all of the objectives been achieved with any plant virus replicase. There are studies with some replicases, however, where one or two of the objectives have been met, and these enzyme preparations have been utilized to glean useful viral replication data. The best example is given by Hall and his associates in this volume. They have isolated an enzyme capable of synthesizing the RNAs of brome mosaic virus. The enzyme has an absolute requirement for template and it is reasonably fastidious in its template acceptance. It will accept BMV RNA templates of either plus (virus-sense) or minus (anti-sense) polarity, but in each case, only the specific RNA complementary to the template (minus or plus) is produced (Miller et al. 1985); i.e. the replication cycle is not completed. Another example of progress in replicase purification is with turnip yellow mosaic virus (Mouches et al. 1984). This enzyme comprises both a host and a viral-encoded protein (Candresse et al. 1986). Other useful replicase systems under active investigation are cucumber mosaic virus (Jaspars et al, 1985), cowpea mosaic virus (Dorssers et al. 1984), and alfalfa mosaic virus (Clerx & Bol, 1978).

These replicases are all RNA-dependent RNA polymerases. The early replicase purification efforts with a number of plant viruses were confounded because of the presence in plants of a host-encoded RdRp, the activity of which is enhanced by many perturbations, including virus infection (for reviews, see: Fraenkel-Conrat, 1986; Zaitlin & Hull, 1987). This virus-stimulated RNA synthesis persuaded many workers in this field to conclude that they had isolated the true viral replicase, but they most probably had a mixed preparation of the host enzyme and the viral replicase. There is good evidence, however, which indicates that the host RdRp is not the replicase (Dorssers et al. 1983), although there is still some advocacy for its involvement (Khan et al. 1986).

A N A L Y S I S OF TMV R E P L I C A T I V E S T R U C T U R E S

It has long been known that a membranous fraction of cell-free extracts of TMV- infected tobacco leaves is capable of synthesis of replicative structures resembling TM V-RF and RI (Bradley & Zaitlin, 1971). This system can synthesize viral-specific products in a template-independent manner, apparently by continuing the synthesis of partially completed replicative structures. We have analysed the products of this in vitro system by using its radiolabelled products as hybridization probes with a collection of M13 clones (Young & Zaitlin, 1986). Each M13 clone contains a different portion of either the plus or minus strand of the TMV genome (Goelet et al. 1982); thus, the amount of hybridization to a given clone indicates the level of in vitro synthesis occurring in the portion of the TMV genome complementary to the insert.

Viral-specific products of TMV replication that are synthesized in vitro consist of two types of RNA molecules, one resembling RF according to its electrophoretic migration and another, more heterogeneous set of molecules, resembling RI. We characterized these two forms and showed that the RF-like RNA was resistant to RNase A at high salt concentrations, but sensitive to RNase III, showing it to be

Tobacco mosaic virus replication 279fully double-stranded (ds). The RI-like RNA was partially sensitive to both enzymes, showing it had the expected double- and single-stranded moieties. Electroelution of RNA from gel fragments containing either RF-like or RI-like RNA provided the labelled RNAs for use as hybridization probes with the collection of M13 clones.

Isolated RI-like RNA hybridized only to clones of minus polarity, indicating only plus strand synthesis. A majority of the incorporation of label (i.e. extent of in vitro synthesis) occurred near the 5'-end of the plus strand, with substantially lower incorporation near the 3'-end and virtually none in the centre. Isolated RF-like RNA, by contrast, hybridized to M13 clones with inserts of both polarities, with roughly six times greater incorporation into the plus strand than the minus. Incorporation into the plus strand was found exclusively near the 3'-end.

These experiments indicate that synthesis of plus strands takes place on replication complexes with one or more progeny strands, while synthesis of minus strands takes place on complexes with only a single progeny RNA molecule. Incorporation near the 5'-end of the plus strand in RI-like RNA, but not in the middle of the molecule, suggests that synthesis of these molecules terminates early in vitro , while incorpor­ation near the 3'-end may have resulted from either internal initiation (as in the synthesis of 3'-coterminal subgenomic RNAs) or the finishing off of genomic plus strands which had been nearly completed at the time of isolation. Such ‘finishing-off’ is also probably the explanation of the high level of incorporation very near the 3'-end of the plus strands of RF-like RNA.

In a similar study, Watanabe & Okada (1986) analysed the in vitro replication products from an extract prepared from TMV-infected tobacco protoplasts. Although RF and RI were not separately analysed in their experiments, their results agree with those outlined here and described in detail in Young & Zaitlin (1986). Moreover, by gel analysis of hybrids formed between the labelled replicative structures and an M13 clone containing a large insert complementary to the 3'-portion of the plus strand of TMV, Watanabe & Okada were able to clearly demonstrate in vitro synthesis of the subgenomic coat protein mRNA of TMV.

P A R T I A L P U R I F I C A T I O N OF TMV R E P L I C A T I O N C O M P L E X

In spite of an early start in the TMV replicase field (Ralph & Wojcik, 1969; Bradley & Zaitlin, 1971), there has been no significant progress in solubilizing the TM V enzyme until this report. We adopted the strategy of solubilizing the complex containing TM V replicase activity and then fractionating this preparation by Sepharose 4B chromatography. The first step, solubilization of replicase from the 30 000g pellet fraction, was essential in separating the replication complex away from the host-encoded RdRp and other cellular components which reside in the same fraction. We tested several non-ionic detergents, including Triton X-100 and dodecyl-/3-D-maltoside (12-M), both successful in replicase studies of other plant viruses (Dorssersei al. 1984; Bujarskiei al. 1982). Of the detergents examined, only one, CHAPS (Hjelmeland, 1980), solubilized high levels of TMV replicase from the

280 N. Young, J. Forney and M. Zaitlin30 0 0 0 ^ pellet fraction (Fig. 1). At a concentration of 0-1 % CHAPS, approximately 14% of the replicase activity was recovered in the supernatant (as monitored by synthesis of RF and RI in vitro), while at 0-5% , far less replicase activity was recovered. Host-encoded RdRp was also partially solubilized from the pellet by 0-1 % CHAPS treatment (Pig. 1).

The next step in the purification was the fractionation of the solubilized replicase by Sepharose 4B chromatography (Dorssers et al. 1984) in the presence of 0-1 % CHAPS. Fractions taken from the column were assayed for replicase activity (Fig. 2) and protein composition (Fig. 3). Virtually all of the TMV replicase activity was

Fig. 1. Solubilization of TMV replicase by CHAPS and 12-M. The 30000^ pelletable fraction was prepared from TMV-infected leaves as described (Young & Zaitlin, 1986). This fraction was split into aliquots and 10 % solutions of either 12-M or CHAPS were added to give final concentrations of 0-1 % or 0'5 %. Detergent treatments were carried out for 30 min at 4 °C after which the samples were microfuged at 15 600 g for 20 m in. The supernatants of each sample were assayed for replicase activity as described (Young & Zaitlin, 1986) and analysed by electrophoresis in a 1-8% non-denaturing agarose gel containing Tris-acetate-EDTA buffer (Loening, 1967). Lane 1, molecular weight markers (23-1, 9-4, 6-6, 4-4, 2-3, 2-0, and 0-56kilobase pairs); Lane 2, 30000^ pellet fraction; Lane 3, 0-1% 12-M; Lane 4, 0-5% 12-M; Lane 5, 0-1% CHAPS; Lane 6, 0-5% CHAPS. The closed arrow marks the migration position of TMV-RF, while the open arrow marks the migration of host RdRp products.

Tobacco mosaic virus replication

1 2 3 4 5 6 7 8 9 10* *

• »>4 <

Fig. 2. Replicase assay of fractions from Sepharose 4B colum n. A 0-1% CH A PS- solubilized T M V replicase fraction was prepared as described in the legend of Fig. 1 and O 'lm l of th is preparation was loaded onto a Sepharose 4B colum n (20 x0 -8 cm ). F ractions (0-5 ml) were collected at a rate of 8 m ill-1 . Each fraction was assayed for replicase as described (Young & Zaitlin, 1986). Lane 1, molecular weight m arkers; Lanes 2 through 10 correspond to colum n fractions 2 through 10. M olecular weight standards are identical to those in Fig. 1. T h e closed arrow marks the m igration position of TM V - R F while the open arrow marks the m igration of host R dR p products.

found in the void volume (Fraction 4 on Fig. 2). Host-encoded RdRp activity, as inferred from the synthesis of low molecular weight RNAs, was found in several fractions, with roughly 20 % of the host RdRp copurifying with the TMV replicase- containing fraction.

The protein composition of each fraction was determined by gel analysis. Our attention was focused on the fractionation of the two TMV-encoded proteins, the 126K and 183K proteins, both of which are thought to have a role in replication (for review, see: Palukaitis & Zaitlin, 1986). These proteins co-purified with TMV replicase activity on the Sepharose column, eluting in the void volume (Fraction 4 in Fig. 3). There were, however, many other proteins eluting in this fraction, so it was impossible to conclude that the 126K or 183K proteins were responsible for the replicase activity observed in this fraction. Nevertheless, the prominence of the 126K and 183K proteins in the partially purified replicase preparation strongly supports the proposal that these proteins are components of the TMV replication complex.

282 N. Young, J. Forney and M. ZaitlinI N T R A C E L L U L A R S I T E OF R E P L I C A T I O N OF TMV R N A

During the past 30 years, TM V RNA replication has been postulated to take place at various sites within the cells of its host (Zaitlin & Boardman, 1958; Reddi, 1964; Ralph et al. 1971; Nilsson-Tillgren et al. 1974). Recent studies by van Telgen et al. (1983) and by Watanabe & Okada (1986) utilizing cell fractionation procedures, indicate a nuclear association of viral-synthesized components and could be interpreted to support replication of the RNA in the nucleus. These conclusions are based on the observations that the viral-encoded 126K protein is associated with chromatin in infected cells (van Telgen et al. 1985) and is also found in a subcellular fraction from protoplasts which contains nuclei (Watanabe & Okada, 1986), and that the replicative form of TMV RNA is produced by the ‘nuclear’ fraction (Watanabe & Okada, 1986).

These results contradict earlier findings of Ralph et al. (1971) and of Nilsson- Tillgren et al. (1974). These workers followed the distribution of ds TMV RNA and various organelles or marker enzymes in subcellular fractions of tobacco tissue. They observed ds TMV RNA associated with cytoplasmic membranes. The conflicting evidence presented by such studies may be due to discrepancies in the different methodologies used in subcellular fractionation.

We have compared various methods of fractionating tobacco tissue and mesophyll protoplasts to determine which factors were important in subcellular localization of

Fig. 3. Protein gel analysis of fractions from Sepharose 4B colum n. Sam ples from the colum n fractions described in Fig. 2 were analysed by 10% polyacrylam ide gel electrophoresis and proteins were visualized by silver staining. Lane 1, unfractionated 0 -1% C H A P S-supernatan t preparation; Lanes 2 through 10 correspond to column fractions 2 through 10. Arrows note the 126K and 183K viral coded proteins of TM V .

Tobacco mosaic virus replication 283TM V replication. The TMV-associated 126K protein, replicase activity, and ds RNA were used as markers to follow TMV replication. DNA was quantified as a guide to nuclear isolation.

These studies will be documented elsewhere in detail; they show that association of a virus-related component is not necessarily a reliable indicator of the site of synthesis or accumulation of that component. We found, for example, that depending on the isolation method used, the replicase activity and the 126K protein were distributed among all of the fractions of the homogenate tested. On the other hand, only trace amounts of ds TMV RNA were found in the nuclear fraction (which contained 85 % of the total DNA in the homogenate); most (60%) of the ds TMV RNA was found in the post-nuclear supernatant. The results of these studies make us question the relevance of the association of viral-related components in the nuclear fraction of disrupted tissue as implicating the nucleus as a site of synthesis of TMV RNA.

D I S C U S S I O N

For years, the study of TMV replicase has been hindered by the inability to analyse its properties in solution. As long as the replicase was associated with the sedimentable fraction after homogenization, it was impossible to purify the replicase away from the host-encoded RdRp or from other insoluble components of the cell. While protocols were developed for the solubilization and partial purification of other plant viral replicases (Bujarski et al. 1982; Dorssers et al. 1984; Mouches et al. 1984), application of these protocols to TMV replicase was consistently unsuccessful (Young & Zaitlin, unpublished results). In this report, however, we describe the release of TM V replicase activity into solution by treatment with low concentrations of the nondenaturing, zwitterionic detergent, CHAPS. Host-encoded RdRp activity is also solubilized by CHAPS treatment, but Sepharose 4B gel filtration chroma­tography allows for substantial purification of the replicase. We are now attempting further biochemical separation techniques starting with the partially purified replicase preparation, including velocity sedimentation centrifugation and affinity chromatography. Purified TMV replicase will enable the identification of its protein components, as well as the examination of its template specificity, since studies on the response of the replicase to added RNA templates are impossible until the replicase is completely free of contaminating host-encoded RdRp.

Subcellular localization of TMV replicase has also been remarkably difficult to accomplish. Focusing on the nucleus, we have shown that nuclear DNA and the minus strand of TMV, which is diagnostic for TMV replication, are not localized in the same subcellular fraction, strongly suggesting that TMV replication does not occur in the nucleus. In collaboration with G. Hills & R. H. Plaskitt of The John Innes Institute we are currently following up these experiments by immunocyto- chemical examination of TMV replication using antibodies directed against the 126K protein of TMV and nucleic acid probes specific for the minus strand of TMV. Preliminary results with immunogold labelling show that the 126K protein is found

284 N. Young, J . Forney and M. Zaitlinexclusively in ‘viroplasms’ in the cytoplasm which resemble elements of the well- known ‘X body’ inclusions of TMY-infected cells (Matthews, 1981).

We thank D r Peter Palukaitis for his interest and advice in some of these studies. The work was supported in part by Grant 8500279 from the Competitive Grants Program of the United States Department of Agriculture and Grant 84-09851 from the National Science Foundation.

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