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Journal of Virology, June 2008, p. 5967-5980, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02737-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast{triangledown}

Judit Pogany and Peter D. Nagy*

Department of Plant Pathology, University of Kentucky, Lexington, Kentucky

Received 23 December 2007/ Accepted 6 April 2008


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ABSTRACT
 
To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ~30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.


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INTRODUCTION
 
Plus-strand RNA [(+)RNA] viruses replicate their genomes via a multistep process in the infected cells. After translation of the invading viral RNA, which leads to production of the viral replication proteins, the viral RNA is selected/recruited for replication. This is followed by the assembly of the viral replicase on subcellular membrane surfaces and complementary minus-strand synthesis. Then, the (–)RNA intermediate is used by the viral replicase to synthesize an excess amount of new (+)RNA progeny, which are released from the site of replication to the cytosol (1, 24). To understand the mechanism of (+)RNA virus replication, several model RNA viruses, including plant viruses, have been exploited in this intensively studied area in recent years (1, 3, 19, 39, 44).

Assembling the viral replicase complex on the cytosolic surfaces of intracellular membranes is a poorly understood process (39). The viral replicase consists of virally encoded RNA-dependent RNA polymerase (RdRp) and viral auxiliary replication proteins as well as co-opted host proteins, whose contribution to the viral replication process is the least understood. To identify the host factors present in the viral replicase complex, a recent proteomics approach revealed that 4 to 10 host proteins, including molecular chaperone Ssa1/2p (yeast homologue of heat shock protein 70 [Hsp70]), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA binding protein), and Pdc1p (pyruvate decarboxylase), were part of the highly purified functional tombusvirus replicase (41, 47). Similarly, the purified Tobacco mosaic virus (ToMV) replicase preparation contained at least four host proteins, including RNA-binding protein GCD10, which is one of the subunits of the 10-component eukaryotic initiation factor 3 (eIF-3) complex (23). Additional host proteins in the ToMV replicase preparation might be translation elongation factor 1A, the seven-path membrane protein Tom1p, and HSP70 (13, 50). The highly purified Brome mosaic virus (BMV) replicase contained ~10 host proteins, including the p41 subunit of the eIF-3 complex (36). Dissection of the functions of the viral and host proteins in the replicase complex would be greatly facilitated by in vitro approaches with either purified components or cellular extracts.

In addition to the complex nature of the viral replicase, another interesting feature is that the RdRps of several (+)RNA viruses, such as p92pol of Tomato bushy stunt virus (TBSV), 2apol of BMV, P2 of Alfalfa mosaic virus, 180K of ToMV, and the hepatitis C virus NS5B, become activated only after the assembly of the viral replicase in membraneous spherules or vesicles (31, 32, 35, 46). Although the assembly of the replicase and mechanism of RdRp activation are currently unknown, the viral RNA plays a key role in these processes.

Because (+)RNA viruses replicate on subcellular membrane surfaces, nonionic detergents with or without RNase treatment (to remove the endogenous viral RNA) have been used to obtain soluble template-dependent viral replicases to dissect cis-acting RNA elements and trans-acting protein factors involved in initiation, elongation, and termination of viral RNA synthesis or in RNA recombination (12). Most of the obtained replicase preparations, however, suffer from limitations, such as their inability to use exogenously added RNA templates or lost template specificity, or they are capable only of cRNA synthesis (not the full replication cycle). To circumvent some of the above problems, development of cell-free replication assays using extracts obtained from noninfected cells followed by coupled translation/replication is useful to study the assembly of viral replicase complexes, including the roles of the viral RNA, viral and host replication proteins, and cellular membranes. Indeed, coupled translation/replication-based cell-free systems have been developed for poliovirus and ToMV replication (2, 10, 14, 45). These cell-free assays have been used to dissect important features of the viral replicases in replication and recombination.

In the current paper, we have developed for the first time a cell-free assay for tombusvirus replication based on Saccharomyces cerevisiae, an excellent model host, which is superior in genetic and biochemical studies. Yeast cells expressing the tombusviral p33 and p92pol replication proteins were used to obtain cell extracts that can successfully utilize the TBSV replicon RNA (repRNA) or the genomic RNA (gRNA) of three different tombusviruses for complete RNA replication and RNA recombination. We show that the yeast extract can be used to recapitulate many steps of the replication process and to characterize biochemical features of the tombusviral replicase.


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MATERIALS AND METHODS
 
Yeast strains and expression plasmids. The expression plasmids pGAD-His92 (containing the p92pol gene of Cucumber necrosis virus [CNV] and the LEU2 marker) and pHisGBK-His33 (expressing CNV p33 from the ADH1 promoter) have been previously described (32). The CNV p33 and p92pol replication proteins are as efficient as the highly homologous TBSV replication proteins in supporting TBSV DI-72 RNA replication in plants or in yeast (18, 25, 48). To construct pGSTGADHis92s, we first made pGADHis92s by isolating the Bsp1407I-BamHI fragment from pGBK-His33 (32) and inserting it into pGAD-His92 digested with SphI and BamHI. To make pGST/GADHis92s, the glutathione S-transferase coding region was amplified by PCR from the pGEX vector using oligonucleotide 2026 (CGCGGGATCCATGTCCCCTATACTAGGTTATTG) and oligonucleotide 2027 (CCGGGGATCCACGCGGAACCAG) (the restriction enzyme site is in italics). The PCR product was digested with BamHI and, after gel isolation, it was ligated into pGADHis92s cleaved with BamHI, treated with shrimp alkaline phosphatase. The correct orientation was confirmed by PCR using oligonucleotide 2026 and oligonucleotide 956 (GCCCACCATGGCTATTTCACACCAAGGGACTCA).

Preparation of cell extract. Yeast strain BY4741 was transformed with the combination of (i) pGSTGADHis92s and pHisGBKHis33, (ii) pGAD and pHisGBKHis33, or (iii) pGSTGADHis92s and pESC. The yeast transformants were grown in 200 ml LH-glucose medium at 29°C for ~24 h with shaking (25, 32). The cells were harvested at an optical density of 1 by centrifugation at 4,000 rpm for 4 min at 4°C. The pellet was suspended in 50 ml ice-cold sterile water, followed by centrifugation at 4,000 rpm at 4°C for 4 min in a swinging-bucket rotor. This step was repeated. Then, the cells were resuspended in 10 ml ice-cold buffer A (0.3 ml of 1 M HEPES-KOH, pH 7.4, 1 ml of 1 M potassium acetate, and 0.02 ml of 1 M magnesium acetate), followed by centrifugation at 4,000 rpm and 4°C for 4 min in a swinging-bucket rotor. This step was repeated. Then, 0.5 g of pellet was suspended in 0.75 ml buffer A containing 1.5 µl 1 M dithiothreitol and 3 g prechilled glass beads. The yeast cells were broken by shaking for 5 min. This was followed by centrifugation at 500 x g and 4°C for 5 min in a swinging-bucket rotor. The supernatant was then moved to a new tube, followed by centrifugation at 500 x g at 4°C for 5 min. The supernatant was then stored at –80°C until use.

Replication assay using the cell extract. The above yeast cell extract (1 µl) was preincubated on ice for 10 min in 10 µl cell-free replication buffer containing 50 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.2 M sorbitol, and 0.4 µl actinomycin D (5 mg/ml). Then, the reaction volume was adjusted to 20 µl with 1x cell-free replication buffer also containing 2 µl of 150 mM creatine phosphate; 2 µl of 10 mM ATP, CTP, and GTP and 0.25 mM UTP; 0.3 µl of [32P]UTP, 0.2 µl of 10-mg/ml creatine kinase, 0.2 µl of RNase inhibitor, 0.2 µl of 1 M dithiothreitol, and 0.5 µg RNA transcript. The reaction mixture was incubated at 25°C for 3 h. The reaction was terminated by adding 110 µl stop buffer (1% sodium dodecyl sulfate [SDS] and 0.05 M EDTA, pH 8.0), followed by phenol-chloroform extraction, isopropanol-ammonium actetate precipitation, and a washing step with 70% ethanol as described earlier (18, 37).

RNA and protein analysis. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the 32P-labeled RNA products of the repRNAs and gRNAs from the cell extract was performed in a 5% acrylamide gel containing 0.5x Tris-borate-EDTA (TBE) buffer with 8 M urea or in a 4.3% acrylamide gel containing 1x TBE buffer with 8 M urea.

To test the ratio of (+)repRNA versus (–)repRNA products in the cell-free assay, we blotted onto a Hybond XL membrane (Amersham) equal amounts of unlabeled in vitro transcripts of plus-stranded [DI-72(+)] and minus-stranded [DI-72(–)] DI-72 repRNAs (prepared by T7 RNA transcription in vitro), which were denatured separately by heating for 5 min at 85°C in Tris-EDTA buffer and formamide (in a 1:1 ratio). This was followed by cross-linking with UV (GS Gene Linker; Bio-Rad) and hybridization with the heat-denatured 32P-labeled cell-free assay products in Ultrahyb solution (Ambion) at 68°C (11, 32).

Western blot analysis of p33 and p92 replication proteins was performed using anti-His6 (Amersham) as the primary antibody and the alkaline phosphatase-conjugated anti-mouse immunoglobulin G antibody (Sigma) as the secondary antibody as described previously (25, 32).

Detection of double-stranded RNA (dsRNA) in the samples from the cell-free assay was done by electrophoresis in a 5% acrylamide-8 M urea gel containing 0.5x TBE buffer at low voltage (150 V). Samples were divided into two; one half was loaded onto the gel without heat treatment in the presence of 25% formamide, and the other half was loaded after denaturation for 5 min at 85°C in the presence of 50% formamide. The dsRNA standard was obtained via hybridization of plus- and minus-stranded 32P-labeled probes made by T7 RNA polymerase reaction according to reference 30.

To test the replicase activity in the membrane fraction, we centrifuged the cell extract for 10 min at 21,000 x g and 4°C to separate the membrane-enriched and soluble fractions. The membrane-enriched fraction was dissolved in buffer A. The centrifugation was done either before the assay or after the completion of the reaction (see details in the figure legends).

Nuclease and proteinase treatment. Micrococcal nuclease digestion of the samples was performed using three different nuclease concentrations together with CaCl2 (1 mM, final concentration) at various time points as described in the legends to the figures. The treatment with micrococcal nuclease was done at 25°C for 15 min, and then EGTA (2.5 mM, final concentration) was added to terminate the activity of the micrococcal nuclease, followed by further incubation of the cell-free replicase assay mixture at 25°C. In the control assay, only CaCl2 and EGTA were added to the reaction mixture in the absence of the nuclease.

Proteinase K digestion was performed using the enzyme in 0.05-, 0.01-, and 0.002-mg/ml final concentrations at various time points as described in the legends to the figures. The cell-free assay was done at 25°C for 3 h.

For the treatment with detergent, we used Triton X-100 in 0.25, 0.125, 0.063, 0.031, 0.016, 0.008, 0.004, and 0.002% final concentrations. Triton X-100 was added to the cell-free assay mixture at the 40-min time point. The products of the control assays, which lacked Triton X-100, were harvested at the 40-min and the 180-min time points.


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RESULTS
 
Replication of the added viral (+)repRNA in cell-free yeast extract containing p33 and p92pol replication proteins. Replication of tombusvirus RNA depends on viral and host proteins and cellular (e.g., peroxisomal) membranes in the yeast model host (19, 47, 49). To test if tombusvirus RNA replication can take place in a yeast extract, we prepared a cell extract from yeast cells as described in Materials and Methods, followed by testing for replicase activity in vitro. Interestingly, when the cell extract was obtained from yeast cells coexpressing p33 and p92pol replication proteins from plasmids, we observed the incorporation of [32P]UTP into full-length RNA products after programming the cell extract with the 621-nucleotide-long (nt) of DI-72(+) repRNA (Fig. 1A, lane 1). On the other hand, no comparably sized radiolabeled RNA was observed when the RNA template was omitted (Fig. 1A, lane 2), excluding the possibility that the repRNA-sized product is derived from an endogenous yeast RNA. The in vitro replicase activity correlated with the added (+)repRNA transcripts (Fig. 1A, lanes 3 to 10), with the exception of the highest concentration, which indicates the saturation level for the repRNA usage by the viral replicase in the cell extract. Also, we did not detect replicase activity in cell extracts derived from yeast expressing only one of the two replication proteins (Fig. 1B, lanes 1 to 4). These data confirmed that the observed activity in the cell extract was an authentic tombusviral replicase activity that is dependent on both p33 and p92pol replication proteins as well as on the added repRNA. Moreover, the lack of 32P-labeled repRNA product from the cell-free assay performed in the absence of either p33 or p92pol replication proteins excludes the possibility that the repRNA was labeled due to the presence of a yeast polymerase or terminal transferase. Western blot analysis of the yeast extract showed that p33 and/or p92pol replication proteins were present in the yeast extracts (Fig. 1C). Altogether, these data have established that the cell extract contains the functional tombusvirus replicase, which can use the (+)repRNA as a template.


Figure 1
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  		FIG. 1. Replication of the TBSV repRNA in the cell-free yeast extract. (A, left) PAGE analysis of the 32P-labeled RNA products obtained when the 621-nt DI-72(+) repRNA was added to the cell extract (lane 1) and in the absence of repRNA (lane 2). The cell extract was obtained from yeast expressing p33 and p92pol replication proteins. Asterisks indicate putative TBSV repRNA-derived recombinants. (Right) Effect of the amount of (+)repRNA on RNA synthesis by the replicase assembled in the yeast cell extract. The repRNA was used in amounts of 1,000, 500, 250, 125, 62, 31, 15, and 8 ng in a standard assay. (B) Dependence of repRNA replication on the copresence of p33 and p92pol replication proteins expressed in yeast. The cell extract was obtained from yeast expressing p33 only or p92pol only or coexpressing p33 and p92pol replication proteins as shown. (C) Western blot analysis of the cell extract used for the in vitro assay shown in panel B. The p33 and p92pol replication proteins carry His tags, which were detected with monoclonal antibody.

To test if mutated repRNA can be used as a template in the cell extract, we programmed the cell-free preparation with two well-characterized repRNA mutants (Fig. 2A). First, the DI-72(+) repRNA carried a C99-to-G mutation [DI-72(+)C99G] within the internally located region II stem-loop structure [RII(+)-SL], which is absolutely required for the recruitment of the viral RNA for replication in plant and yeast cells (17, 34). The DI-72(+)C99G mutant lacked template activity in the cell extract (Fig. 2B, lane 3), suggesting that RNA recruitment is required for successful replication in the cell extract. This is in contrast with the abilities of the yeast-expressed/purified recombinant or the plant-expressed/solubilized tombusvirus replicases, both of which used the DI-72(+)C99G mutant as efficiently as the full-length DI-72(+) repRNA in an in vitro assay (Fig. 2B, lane 6, and data not shown) (31). Thus, unlike the detergent-solubilized and purified in vitro replicases, the tombusvirus replicase in the cell extract depended on the wild-type RII(+)-SL, suggesting that active RNA recruitment to the viral replicase could occur in the cell extract.


Figure 2
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  		FIG. 2. The cell-free yeast extract shows high template specificity compared with the detergent-solubilized tombusvirus replicase prepara- tion. (A) Schematic representation of DI-72(+) repRNA and its derivatives. Single point mutations within the C·C mismatch in RII(+)-SL or the (–)RNA initiation promoter (termed gPR) are shown. Note that the mutation within the gPR interrupted the essential base pairing between the replication silencer element (RSE) (33) and gPR elements. (B) Denaturing PAGE analysis of the labeled repRNA products obtained in a standard cell-free replicase assay (left) or with the detergent-solubilized tombusvirus replicase obtained from CNV-infected N. benthamiana plants (18, 27) (right) using the (+)repRNAs shown. S1 shows the treatment with S1 ssRNA-specific nuclease. Arrows, expected repRNA product. The cell extract was programmed with 0.5 µg repRNA. (C) Denaturing PAGE analysis of the labeled repRNA products obtained in a standard cell-free replicase assay programmed with truncated (+)repRNAs. These repRNAs lack the entire RI and the 5' 69 nt of RII of the DI-72(+) repRNA (7). The further details are the same as described for panel B. (D) The replicase assays were programmed with the DI-72(–) repRNA. The further details are the same as described for panel B. The RNA bands migrating faster than the full-length RNA product (arrow) are due to internal initiation and 3' terminal extension (27, 28). (E) The in vitro replicase assays were programmed with satC(+) RNA associated with TCV. The RNA bands migrating faster than the full-length satC RNA product are due to internal initiation and 3' terminal extension (18, 37). The further details are the same as described for panel B.

The second mutant of (+)repRNA (named DI-{Delta}69RII, missing both RI and 69 nt from the 5' end of RII) had a critical mutation within the gPR minus-strand initiation promoter (G618 to C), which is required for the assembly/activation of the functional tombusvirus replicase in vivo (31). However, the same gPR mutation increases template activity in the solubilized/purified-replicase assay by opening up the RNA structure (Fig. 2C, lanes 5 and 7, and data not shown) (33). Interestingly, the DI-{Delta}69RII(+)G618C mutant had low template activity in the cell extract (Fig. 2C, lanes 3 and 4), suggesting that the wild-type gPR sequence is required for successful replication in the cell extract, likely due to its role in the assembly/activation of the viral replicase. Altogether, unlike the solubilized replicase preparations, which support cRNA synthesis on the added mutated (+)repRNA templates (31-33), the cell-free yeast extract did not support the replication of the above mutants of (+)repRNA, thus showing characteristics similar to those of replication occurring in plant and yeast cells (17, 31, 33).

To further compare the cell-free replicase preparation with the activity of the in vivo replicase, we programmed the yeast cell extract with the DI-72(–) repRNA, which cannot initiate replication in yeast or plant cells (T. Panavas and P. D. Nagy, unpublished). Excitingly, DI-72(–) repRNA was not used as a template by the cell-free replicase preparation (Fig. 2D, lanes 1 and 2), albeit the plasmid-borne replication proteins were present in the cell extract to provide replication in trans. These data suggest that RNA recruitment and/or the plus-stranded TBSV RNA-driven activation of the tombusvirus replicase did not take place in the cell-free replicase preparation in the presence of DI-72(–) repRNA. This is contrary to the observations obtained with the solubilized/purified in vitro replicases, which used the DI-72(–) repRNA efficiently under in vitro conditions (Fig. 2D, lanes 3 and 4) (18, 32).

To analyze the template specificity of the yeast cell extract containing p33 and p92pol replication proteins, we tested if the heterologous Turnip crinkle virus (TCV)-associated satellite RNA, termed satC, can be replicated in vitro. These experiments revealed no detectable level of replicase activity on a plus-stranded satC [satC(+)] template (Fig. 2E, lanes 1 and 2). On the other hand, satC(+) was used as a template by the solubilized/purified in vitro replicase (Fig. 2E, lanes 3 and 4) (18, 32). Altogether, the yeast cell extract showed template selectivity similar to that in the in vivo case, where satC is not replicated by the TBSV helper virus.

Complete cycle of replication of the viral (+)repRNA in the cell extract. The detergent-solubilized TBSV or CNV replicases obtained from infected plants and the recombinant purified CNV replicase from yeast are deficient in their abilities to utilize viral repRNA templates for a complete cycle of replication in vitro, which is the production of (+)RNA progeny from (+)RNA template via a (–)RNA intermediate. Instead, detergent-solubilized TBSV or CNV replicases are mostly capable of synthesizing the cRNA on the added RNA template (18, 32). This is certainly not the case during in vivo replication of TBSV DI-72(+) RNA, which is used efficiently by the tombusvirus replicase to produce excess amounts of DI-72(+) RNA progeny starting from the DI-72(+) RNA template in plant and yeast cells (18, 25, 26, 31, 32).

To test if the cell-free replicase preparation is capable of a complete cycle of replication on the TBSV (+)repRNA, we tested the nature of the 32P-labeled full-length RNA products obtained when the cell-free replicase preparation was programmed with (+)repRNA. First, S1 nuclease (a single-stranded RNA [ssRNA]-specific nuclease) digestion led to the degradation of most of the 32P-labeled full-length RNA products, suggesting the presence of unprotected ss TBSV DI-72 repRNA (Fig. 2C, lane 2). Similarly, the shorter-than-full-length repRNA DI-{Delta}69RII(+) produced partially S1-sensitive RNA products (Fig. 2B, lane 2). This is not the case with the solubilized/purified in vitro replicases, which produced mostly ssRNase-resistant 32P-labeled dsRNA products (Fig. 2B, lane 5, and 2C, lanes 6 and 8), suggesting that the solubilized/purified in vitro replicases are capable of cRNA synthesis only (20, 26-28, 32).

Since a complete cycle of replication leads to production of both plus- and minus-strand RNAs, which can form dsRNA after phenol-chloroform extraction, we checked for dsRNA products from the cell-free assay. When the 32P-labeled repRNA was analyzed under nondenaturing conditions, the presence of small amounts of extra 32P-labeled RNA, comigrating with the dsRNA standard, was detected from the cell-free assays programmed with DI-72(+) or DI-{Delta}69RII(+) repRNAs (Fig. 3A, lanes 2 and 6). These 32P-labeled repRNA products were heat sensitive, as expected from their dsRNA nature (Fig. 3A, lanes 1, 3, 5, and 7 versus 2, 4, 6, and 8). Interestingly, the DI-{Delta}69RII(+) sample gave a slowly migrating dsRNA product representing recombinant RNA (recRNA) (see below). Altogether, these data are consistent with the model that a portion of the newly made 32P-labeled repRNA is present as dsRNA under nondenaturing conditions after phenol-chloroform extraction.


Figure 3
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  		FIG. 3. Asymmetrical RNA replication takes place in the cell extract programmed with DI-72(+) repRNA. (A) Detection of dsRNA products of the repRNAs in the cell-free system. A standard cell-free assay was performed with 0.5 µg repRNA as shown, followed by nondenaturing PAGE analysis of samples without heat treatment (lanes 2, 4, 6, and 8), or a portion of the same samples were heated at 85°C for 5 min prior to loading on the gels (lanes 1, 3, 5, and 7). Note that the gel standards (lanes 3, 4, 7, and 8) were obtained via annealing of plus- and minus-stranded T7-transcripts of 32P-labeled repRNAs. Arrows, ssRNA or dsRNA products, including those for the recRNA (lanes 5 and 6). (B) The 32P-labeled RNA products obtained in the cell-free yeast extract programmed with DI-72(+) repRNA were used as probes to hybridize with equal amounts of unlabeled DI-72(+) and (–)RNAs, resembling the shape of "+" and "||," respectively, blotted on the membranes. The ratio of (+)RNA and (–)RNA products was calculated using ImageQuant software.

The presence of the ssRNA nuclease-sensitive 32P-labeled ssRNA products (Fig. 2B) and the small amount of dsRNA product (Fig. 3A) suggests that the majority of the 32P-labeled RNA products synthesized in our cell extract were ssRNA. To determine the polarity of the ssRNA product made in the cell-free replicase preparation, we used the 32P-labeled RNA from our cell extract programmed with DI-72(+) repRNA as a probe against equal amounts of unlabeled DI-72(+) and (–)RNA transcripts blotted on the membrane (Fig. 3B). This assay revealed that most of the 32P-labeled RNA products from the cell-free replicase preparation programmed with DI-72(+) RNA were of plus-stranded polarity (Fig. 3B). We estimated that the plus-stranded/minus-stranded ratio was at least 30:1 from the cell-free replicase preparation, which is consistent with the ratio of ~22:1 to 40:1 observed with the yeast- and plant-derived membrane-bound tombusvirus replicases (31, 32). This number is also consistent with the presence of only a small amount of dsRNA products, which is less than 5% of the ssRNA product (Fig. 3A, lane 2) under nondenaturing conditions. Based on these data, we suggest that the cell-free replicase preparation from yeast is capable of performing a complete cycle of replication on the added DI-72(+) RNA in a manner comparable with that from previous in vivo studies.

Recombination of the tombusvirus (+)RNA in the cell-free yeast extract. In addition to full replication of the DI-72(+) repRNA in the cell-free replicase preparation, we also observed the emergence of novel, longer-than-full-length RNA species in the cell-free replicase preparation (Fig. 1A, lane 1). These longer-than-full-length RNA species were readily detectable when the gels were exposed to the screens for long periods (Fig. 4, lane 1). Similar-size RNAs have also been detected in vivo in yeast and plant cells during replication of DI-72(+) repRNA (7, 42). Sequencing the in vivo-generated longer-than-full-length RNA species has conclusively demonstrated the recombinant nature of these RNAs, which consisted of either a full-length head-to-tail dimer or the shorter dimeric RNA with 5' truncations (the recRNAs were missing the 5' RI and partly RII sequences) (4, 7, 42). These recRNA-like RNAs were not present when no DI-72(+) repRNA was added to the assay mixture (Fig. 1A, lane 2) or when DI-72(+) repRNA was used to program the cell-free replicase preparation obtained from yeast expressing only p33 or p92pol (not shown), suggesting that the formation of these RNAs depends on active replication of DI-72(+) repRNA in the cell-free replicase preparation.


Figure 4
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  		FIG. 4. Efficient RNA recombination occurs in the cell extract programmed with repRNAs. Denaturing PAGE analysis of the 32P-labeled RNA products obtained in the cell-free yeast extract programmed with DI-72(+) or DI-{Delta}69RII(+) repRNAs is shown. The slow-migrating recRNAs are bracketed, whereas the repRNA products are indicated by arrows.

To obtain evidence on the nature of the putative recRNAs, we programmed the cell-free replicase preparation with DI-{Delta}69RII(+) repRNA, which was cloned from naturally emerging recombination intermediates carrying a major recombination hot-spot sequence [i.e., the RII(+)-SL (43)] at a 5' proximal position. DI-{Delta}69RII(+) repRNA recombines efficiently in yeast and plant cells, forming an abundant amount of dimer and also higher multimers, the latter in smaller amounts (7, 42). As expected, DI-{Delta}69RII(+) repRNA generated the dimer- and multimer-sized RNA products efficiently in the cell-free replicase preparation (Fig. 4, lane 2). The recRNA nature of the multimeric RNAs was confirmed by reverse transcription-PCR, which selectively amplifies only the recRNA (7, 42). Based on these data, we conclude that the cell-free replicase preparation is capable of authentic RNA recombination, generating recRNAs comparable to those seen in vivo in yeast and plant cells (4, 7, 42).

TBSV replication and recombination occur in a membraneous compartment in the cell extract. TBSV replication takes place on the cytosolic face of the peroxisomal membranes in infected plant cells and in yeast cells (16, 22, 24, 38). In addition, N-terminal truncations within the p33 replication protein, which interfered with its localization to the peroxisome and resulted in cytosolic p33, eliminated TBSV replication, suggesting a critical role for subcellular membranes in TBSV replication (16, 24). To test if membranes are also involved in TBSV replication in the cell extract, we separated the soluble and membrane fractions of the cell extract prior to adding the TBSV (+)repRNA. These experiments revealed that the membrane fraction contained ~20-fold higher replicase activity than the soluble fraction in vitro (Fig. 5A, compare lanes 2 and 3). The recRNAs were also formed in the membrane fraction, suggesting that recombination requires replicase activity. As expected, the p33 and p92pol replication proteins were localized to the membrane compartment in the yeast cell extract (Fig. 5A, lanes 5 and 6).


Figure 5
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  		FIG. 5. Membranes are required for TBSV repRNA replication in the cell extract. (A) The cell extract was fractionated to membrane and soluble fractions via centrifugation prior to the in vitro assay. The reactions were performed in 20-µl volumes containing 0.5 µg of DI-72(+) repRNA. The denaturing PAGE is shown on the left, whereas the Western blot based on anti-His antibody is on the right. CF, centrifugation. (B) The standard replicase assay mixture with the cell extract contained final Triton X-100 concentrations of 0.016, 0.03, 0.06, 0.12, and 0.25% and final SDS concentrations of 0.002, 0.01, 0.05, and 0.25%. (C) The cell extract programmed with DI-72(+) repRNA was fractionated to membrane and soluble fractions via centrifugation after the standard in vitro assay. The denaturing PAGE is shown on the left, whereas the Western blot based on anti-His antibody is on the right. (D) Denaturing PAGE analysis of the RNase sensitivity of repRNA products made in the cell extract. After a 180-min incubation, the cell extract programmed with DI-72(+) repRNA was treated with either RNase I or micrococcal nuclease. (E) Denaturing PAGE analysis of the RNase sensitivity of repRNA products in the membrane and soluble fractions in the absence or presence of Triton X-100. After the standard in vitro replicase assay, 1% Triton X-100 was added, followed by RNase I treatment (final concentration, 0.01 U/µl) for 15 min.

To further test the role of the membranes in TBSV replication, we added various amounts of detergents to the cell-free replicase assay mixture prior to the reaction. Interestingly, the detergents, such as Triton X-100 and SDS, inhibited the replicase activity even when present in small amounts (Fig. 5B). This underscores the importance of intact membranes in the cell extract for replicase activity.

Another interesting feature of TBSV replication in vivo is the efficient release of the newly synthesized progeny (+)RNAs from the membraneous compartment to the cytosol (24). To test if the cell extract can also release the progeny RNAs, we fractionated the cell extract after the replicase assay. Interestingly, the soluble fraction contained three times as much labeled progeny RNA as the membrane fraction (Fig. 5C, compare lanes 5 and 6 with 3 and 4). Since the soluble fraction does not have significant replicase activity (Fig. 5A, lane 2), we conclude that the progeny RNAs were actively released during/after RNA synthesis in our cell extract. Indeed, the p33 and p92pol replication proteins were localized mostly in the membrane fraction of the yeast cell extract at the end of the replicase assay (Fig. 5C, lanes 8 and 9). Overall, this phenomenon that the newly synthesized RNA is released to the soluble fraction further strengthens the similarities between in vivo replication (24) and the replication of TBSV RNA in our cell extract.

TBSV RNA is protected from nuclease degradation in the membraneous compartment of the cell extract. Since TBSV replication takes place in the membraneous compartment, it is possible that TBSV RNA might be inaccessible and thus protected from degradation by host-derived RNases. To test the level of protection of the repRNA from degradation, the cell extract was treated with ssRNA-specific RNase after the in vitro replicase assay. We found that 11 to 22% of the total labeled repRNA was insensitive to RNase when the RNase digestion was done at the end of the assay (Fig. 5D). This level of protection of the RNA products could come from the inaccessibility of the repRNA due to its location or from the structure of the repRNA, which might form dsRNA between plus and minus strands. However, the dsRNA form cannot represent more than ~5 to 6% of the total labeled RNA products (Fig. 3A), even if all (–)RNA could participate in dsRNA formation based on our estimation of the (–)RNA present after the in vitro replicase assay (Fig. 3B). To address these points, we treated the membrane and soluble fractions of the cell extract with 1% Triton X-100 followed by RNase I digestion after completion of the in vitro replicase assay. The labeled repRNA in the membrane fraction became RNase I sensitive after treatment with Triton X-100 (Fig. 5E, lane 5), suggesting that the labeled repRNA is protected by the membraneous compartment in the absence of detergents and that the protection from the RNase is not likely to be due to the putative dsRNA structure/form. Moreover, the labeled full-length repRNA in the soluble fraction was completely sensitive to RNase I, confirming that this repRNA represents the ssRNA form (Fig. 5E, lanes 1 and 3).

The role of de novo translation in the cell extract in TBSV replication and recombination. In our cell-free yeast extract, the majority of p33 and p92pol is membrane bound (Fig. 5A, lanes 5 and 6), likely forming a preexisting pool of viral/host protein complex that lacks the repRNA template. To test if new protein synthesis plays a role in TBSV replication in the cell extract, which supports limited translation (Fig. 6D, lane 3), we used cycloheximide to inhibit translation during the in vitro assay. Adding cycloheximide to the in vitro assay mixture resulted in inhibition of TBSV replication by two- to threefold compared to the control containing a comparable amount of solvent (dimethyl sulfoxide [DMSO]) (Fig. 6A, lanes 2 and 3). To further test the role of de novo translation, we also used puromycin to inhibit translation in the cell-free yeast extract (Fig. 6D, lanes 2 and 4). These experiments revealed that puromycin inhibited TBSV repRNA replication by up to 95% (Fig. 6B, lane 5). Thus, de novo protein synthesis is important for TBSV replication in our cell-free yeast extract, probably due to the role of the newly made p33 in recruitment of the added (+)repRNA to the site of preassembled replicase (i.e., the membrane compartment). It is also possible that these inhibitors might bind to translation factors that are recruited or utilized by TBSV for its replication (11, 29).


Figure 6
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  		FIG. 6. Inhibition of protein translation in the cell-free assay reduces repRNA replication. (A) Denaturing PAGE analysis of repRNA replication in the presence of 2.5 µg/µl (1x) or 1.25 µg/µl (0.5x) cycloheximide. Note that DMSO was used as a control since cycloheximide was dissolved in DMSO. The repRNA levels were calculated using ImageQuant software. (B) Denaturing PAGE analysis of repRNA replication in the presence of 0.19, 0.375, 0.75, and 1.5 µg/µl puromycin. (C) Denaturing PAGE analysis of repRNA replication in the membrane fraction in the presence of 2.5 µg/µl cycloheximide. The fractionation was performed prior to the in vitro replication assay. (D) In vitro translation assay with the cell-free yeast extract. SDS-PAGE analysis was used to analyze the protein synthesis products on the endogenous mRNAs in the presence of 0.75 µg/µl puromycin (samples 2 and 4 only) and 35S-labeled methionine. Samples 1 and 2 contained an amino acid (aa) mixture without methionine, whereas 3 and 4 were obtained without added amino acid mixture during the in vitro translation.

To test if the protein translation in the cell extract takes place in the membrane fraction, we removed the soluble fraction from the cell-free yeast extract via centrifugation prior to the assay. This was followed by addition of cycloheximide in combination with the (+)repRNA to the TBSV replicase assay mixture. These experiments revealed that the cycloheximide inhibited TBSV repRNA replication in the membrane fraction of the cell extract by approximately threefold (Fig. 6C, lane 1) compared with the DMSO control (Fig. 6C, lane 2). These data suggest that active protein translation on the membranes facilitates TBSV replication in our cell-free assay.

Effect of temperature on TBSV replication and recombination in the cell-free yeast extract. To test the dependence of the cell-free replicase preparation on temperature, we performed the in vitro assay between 25 and 37°C. The maximum level of RNA accumulation was at 25°C, and only a minor drop in activity was observed at 30°C (to 85% of the activity at 25°C; Fig. 7, lanes 1 and 2). On the other hand, replication was inefficient at 37°C in the cell-free yeast extract (Fig. 7, lane 3). In addition, we observed that the formation of recRNA was temperature sensitive. These results are similar to those obtained in vivo using yeast or plant cells, whereas the solubilized replicase preparation was as active at 37°C as at 25°C (37), suggesting that the already-assembled and activated replicase present in the solubilized replicase preparation is not temperature sensitive.


Figure 7
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  		FIG. 7. Replication of the repRNA in the cell extract is temperature sensitive. The standard cell-free assay programmed with DI-72(+) repRNA was performed at the shown temperature until termination of the assay at the 3-h time point. In selected experiments, the temperature was shifted after 10 min of incubation at 37°C to 25°C (lane 4) or after 20 min (lane 6) and 30 min of incubation (lane 7) at 25°C to 37°C for a total of 3 h. The repRNA levels were analyzed with denaturing PAGE and quantified using ImageQuant.

The replicase activity was also low in the experiment involving a temperature shift from 37°C to 25°C after 20 min of incubation at the higher temperature (Fig. 7, lane 4), suggesting that TBSV replication is temperature sensitive in the early stages in the cell-free assay. The replicase activity was more robust (43 to 50%) in the experiment with temperature shifting from 25°C to 37°C after 20 min of incubation, suggesting that the replicase assay is the most sensitive to high temperature during the early stages, such as the assembly of the replicase complex.

Time requirement of TBSV replication in the cell-free yeast extract. To test the time requirement for full replication of DI-72(+) repRNA in the cell-free replicase preparation, we took samples at various time points as shown in Fig. 8A. The first full-length DI-72 repRNAs were detected after 40 min of incubation (Fig. 8A, lane 4). The accumulation of DI-72 repRNA increased for an additional 100 min before reaching maximum level (Fig. 8A, lane 9). First detection of recRNAs happened after 40 to 60 min of incubation (Fig. 8A and B, lanes 4 and 5), and recRNA accumulation reached the maximum level between 140 and 360 min (Fig. 8A and B, lanes 9 to 12). The ratio between recRNA and repRNA varied only slightly, between 0.13 to 0.18, when estimated from the 80-min until the 360-min time point (not shown). Altogether, these data are consistent with the model that the recRNAs are produced during RNA replication.


Figure 8
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  		FIG. 8. Time course experiments to study the replication of repRNA in the cell extract. (A) Denaturing PAGE analysis of the standard cell extract programmed with DI-72(+) repRNA shows the amount of repRNA and recRNAs produced during the specified lengths of incubation. (B) The repRNA and recRNA levels at various time points were quantified using ImageQuant. The 32P-labeled repRNA product at the 540-min time point was taken as 100%. (C) The cell extract programmed with DI-72(+) repRNA was fractionated to membrane and soluble fractions at the end of incubation as shown. The repRNA levels were analyzed with denaturing PAGE and quantified using ImageQuant. To enhance the visibility of the recRNAs, we show a longer exposure of the top portion of the image at the bottom. (D) The repRNA levels in the total, membrane, and soluble fractions at various time points were quantified using ImageQuant.

Time course experiments were also performed to analyze when the ssRNA is released from replication. As expected, more labeled repRNA was present in the membrane fraction than in the soluble fraction at the 40-min point, whereas the two fractions contained approximately the same amount of labeled repRNA at the 60-min time point (Fig. 8C, lanes 6 and 7 and 11 and 12). The soluble fraction contained approximately twice as much labeled repRNA at the latter time point as the membrane fraction (Fig. 8C, lanes 8 to 10 and 13 to 15). Thus, these data suggest that the newly made ssRNA is released continuously from the membraneous compartment to the soluble fraction during the in vitro assay. Interestingly, we detected only the recRNAs in the membrane-containing fraction at the late time points (Fig. 8C, lanes 8 to 10, bottom), indicating that the release of the recRNA from the membrane might be a limiting step.

Sensitivity of TBSV replication to proteinase and RNase in the cell-free yeast extract. The above time course experiments revealed that the production of new progeny RNAs can be detected first around the 40-min time point (Fig. 8A). This suggests that the assembly/activation of the tombusvirus replicase complex occurs between the 20- and 40-min time points. To test whether replicase complex formation in the yeast cell extract causes some changes in the accessibility of the repRNA and/or viral replication proteins to RNases or proteinase, we performed in vitro experiments at the 0-min and 40-min time points. We found that a 15-min treatment with the ssRNA-specific calcium-dependent micrococcal nuclease, which can be inactivated by adding EGTA to the reaction mixture, abolished TBSV repRNA replication when applied at the 0-min time point (Fig. 9A, lanes 2 to 4). On the other hand, a 15-min treatment with the micrococcal nuclease at the 40-min time point inhibited repRNA replication by 10 to 20% only (Fig. 9A, compare lanes 6 to 8 with the control lane 5). Moreover, the level of repRNA replication was approximately threefold higher at the 180-min time point in the samples treated with the micrococcal nuclease at the 40-min time point than the level when the reaction was terminated at the 40-min time point (Fig. 9A, lane 9). This suggests that replication continued efficiently after the treatment with the micrococcal nuclease at the 40-min time point. These data argue that repRNA was located in a compartment which made the RNA mostly micrococcal nuclease resistant at the 40-min time point, while the repRNA was susceptible at the zero time point. Based on these results, we propose that the repRNA was likely protected within the membrane-bound replicase complex at the 40-min time point, whereas the repRNA was exposed at the start of the assay.


Figure 9
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  		FIG. 9. Sensitivity of repRNA replication in the cell extract to RNase, proteinase, cycloheximide, and Triton X-100 detergent. (A) Micrococcal nuclease (0.250, 0.004, and 0.001 U/µl) was added at the 0-min or 40-min time points to the cell extract programmed with DI-72(+) repRNA for 15 min, followed by inactivation of the micrococcal nuclease with EGTA and continued incubation for a total of 180 min. The proteinase K (final concentrations, 0.05, 0.01, and 0.002 µg/µl) was added at the 0-min or 40-min time point to the cell extract, followed by continued incubation for a total of 180 min. Note that the proteinase K remained active during the whole length of incubation. Cycloheximide (2.5 µg/µl) was added at the 0-min or 40-min time point to the cell extract, followed by continued incubation for a total of 180 min. The control experiment mixtures were incubated either for 40 min (lane 9) or 180 min (lane 10). The repRNA levels were analyzed with denaturing PAGE and quantified using ImageQuant. (B) The proteinase K (final concentrations, 0.01 and 0.002 µg/µl) was added at the 0-min or 20-min time point to the cell extract, followed by continued incubation for a total of 180 min. (C) Triton X-100 (final concentrations, 0.25, 0.125, 0.0625, 0.03125, 0.016, 0.008, 0.004, and 0.002%) was added at the 40-min time point to the cell extract, followed by continued incubation for a total of 180 min.

Treatment of the cell extract at the 0-min time point with proteinase K, followed by incubation for a total of 180 min, revealed that the viral replication proteins or the replicase complex itself was present in a proteinase-sensitive form, resulting in a low level of repRNA replication in the cell extract (Fig. 9A, lanes 11 to 13). On the other hand, treatment with proteinase K, especially at low concentrations, at the 40-min time point inhibited repRNA replication by only 20 to 30% (Fig. 9A, compare lanes 14 to 16 with the control lane 10). Overall, the level of repRNA replication after proteinase K treatment at the 40-min time point (note that the level of repRNA was measured at the 180-min time point) was approximately fourfold higher than that measured at the 40-min time point (Fig. 9A, lane 9). Therefore, we conclude that replication continued during the proteinase K treatment, arguing that the membrane-bound tombusvirus replicase was present in a relatively proteinase-insensitive stage between the 40-min and 180-min time points, while the replication proteins were susceptible at the zero time point. To test if the proteinase insensitivity of replication might arise even earlier, we also performed the assay by adding proteinase K at the 20-min time point, followed by incubation up to 180 min. Interestingly, replication was already insensitive to proteinase K at the 20-min time point (Fig. 9B, lanes 4 and 5). Based on these results, we propose that the membrane-bound replicase complex forms a "protected" structure between the 20- and 40-min time points, which protects both the repRNA and the replication proteins from degradation.

To test if the replicase complex forms a detergent-resistant structure at the 40-min time point, we treated the cell-free replicase preparation with various amounts of Triton X-100 at the 40-min time point, followed by incubation up to 180 min. Interestingly, treatment with Triton X-100 inhibited greatly the activity of the viral replicase (Fig. 9C). The level of repRNA synthesized remained similar to the level of repRNA measured at the 40-min time point (Fig. 9C, lanes 2 to 8), suggesting that treatment with Triton X-100 stopped the replicase activity rapidly. The obtained in vitro data indicate that the viral replicase complex is sensitive to the presence of a detergent in the cell-free assay. This underscores the importance of the membrane in formation of the above proposed "protected" replication complex.

Replication and subgenomic RNA synthesis of three different tombusvirus plus-stranded gRNAs in the cell-free yeast extract. To test if the full-length gRNAs of related tombusviruses can replicate in the cell extract, we added purified virion RNAs (vRNAs) to the in vitro assay mixture. As expected, both the TBSV and the related Carnation Italian ringspot virus (CIRV) vRNAs supported the production of full-length labeled RNAs in the cell extract (Fig. 10A, compare lanes 1 and 2 with 3 and 7). High-resolution denaturing PAGE analysis revealed the presence of gRNA and subgenomic RNA1 (sgRNA1) and to a lesser extent sgRNA2 among the RNA products from the cell extract programmed with CIRV vRNA (Fig. 10B, lane 2). Comparably sized RNA products were absent in the control assay that lacked tombusvirus vRNA (Fig. 10B, lane 1).


Figure 10
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  		FIG. 10. Replication of three tombusvirus gRNAs in the cell extract. (A) The standard cell extract was programmed with the vRNAs of TBSV or CIRV or with T7 transcripts of CNV, TBSV, and CIRV as shown. Note that the gRNA and sgRNA1 comigrate in the standard denaturing PAGE, whereas the position of sgRNA2 is marked. (B) High-resolution denaturing PAGE analysis to separate gRNA and sgRNA1 from samples shown in panel A. Low-intensity nonspecific bands are marked with asterisks. These likely represent residual DNA-to-RNA transcription of rRNAs in the yeast extract.

Programming the cell extract with T7 RNA polymerase-made transcripts of TBSV, CIRV, and CNV (which is the closest relative of TBSV) gRNAs resulted in the efficient production of labeled full-length RNA (Fig. 10A, lanes 4 to 6). High-resolution denaturing PAGE analysis revealed the presence of sgRNA1 (Fig. 10B, lanes 5 and 6), which is produced during replication in tombusviruses (8). On the other hand, only a small amount of sgRNA2 was detected, suggesting that sgRNA2 synthesis occurs with low efficiency from the gRNA transcripts in our cell extract. Nevertheless, the cell extract is capable of using the gRNAs for replication and sgRNA production without the occurrence of major RNA degradation. Thus, the yeast cell-free replication assay will likely be useful in future analysis of the replication of gRNA and production of sgRNA1 of various tombusviruses under in vitro conditions.


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DISCUSSION
 
Complete and asymmetrical TBSV RNA replication in the cell extract. In the current paper, we have developed a novel cell-free, template-dependent tombusvirus replication assay based on yeast, an excellent model host, which is superior for genetic and biochemical studies. The cell extracts were prepared from yeast cells expressing the tombusviral p33 and p92pol replication proteins, which could utilize the added TBSV repRNA or the gRNA of three different tombusviruses for a complete cycle of RNA replication. Evidence for a complete cycle of replication of the tombusvirus RNA in the new in vitro system is conclusive. For example, adding TBSV (+)repRNA to the cell-free yeast extract leads to efficient RNA synthesis of full-sized RNA products, of which ~81 to 90% are sensitive to ssRNA-specific RNase, likely representing the (+)ssRNA progeny produced (Fig. 2 and 3). Also, using the labeled RNA from the cell-free assay as a probe revealed that (+)RNA progeny is generated in ~30-fold excess over the (–)RNA intermediate (Fig. 3), suggesting that the cell extract can support the complete RNA replication cycle, which is asymmetrical, leading to excess amounts of (+)RNA over (–)RNA in vitro. Interestingly, a similar 20- to 40-fold asymmetry in (+)RNA synthesis is also a feature of TBSV replication in plant protoplasts (single cells) or in yeast, a model host (31, 32), indicating that replication in the cell-free system is comparable to in vivo replication. Labeling of the added (+)repRNA is not due to the activity of a terminal transferase or a yeast RNA polymerase since we observed labeled full-length (+)repRNA only when the cell extract was obtained from yeast coexpressing both p33 and p92pol replication proteins (Fig. 1). Moreover, (+)repRNA carrying mutations in critical cis-acting replication elements, the (–)repRNA, or the heterologous TCV-associated satC(+) RNA was unlabeled under comparable conditions (Fig. 2). Also, 10 to 19% of the new RNA products were RNase insensitive, suggesting that this portion of the RNA was part of the replicase complex. However, even this RNA product was degraded in the presence of 1% Triton X-100, which resulted in solubilization of the membrane/replicase complex (Fig. 5E) (18, 32). Overall, the obtained results can be explained by the presence of an authentic tombusvirus-related replicase activity in the cell-free yeast extract.

The full replication with the added RNA template is a unique feature of the new cell extract because the previous tombusvirus replicase preparations obtained by solubilization of the replicases from membranes with nonionic detergents were able to synthesize only cRNA products on the exogenously added RNA templates (Fig. 2) (18, 32). The published detergent-solubilized tombusvirus replicase preparations were obtained from Nicotiana benthamiana plants infected with TBSV or CNV or highly purified from yeast replicating TBSV repRNA (18, 32). The inability to complete the full replication cycle by these solubilized replicase preparations might be due to the absence of host factors lost during solubilization or the disruption of the membraneous environment (41). In contrast to the detergent-solubilized replicase preparations, the previous membrane-enriched CNV replicase preparations supported both plus- and minus-strand synthesis, but they worked only with the endogenous RNA and could not take up the exogenously added RNAs (11, 31, 32). Altogether, the novel template-dependent cell-free replicase preparation developed in this paper combines the advantages of previous systems by being capable of supporting full tombusvirus replication on added RNA templates.

A remarkable feature of the obtained cell extract is the template specificity of the tombusviral replicase in vitro, which was comparable to those observed in vivo. For example, the (–)repRNA, which is an excellent template with the detergent-solubilized replicase from TBSV- or CNV-infected plants or with the highly purified recombinant CNV replicase from yeast (18, 32, 41), was not used as a template in our cell extract (Fig. 2D). Similarly, the heterologous TCV-associated satC(+) RNA cannot be replicated in the cell extract (Fig. 2E), suggesting that template selection/recruitment for replication is stringent in the cell-free system. This was further supported by the inability of the cell-free replicase extract to support the replication of a (+)repRNA mutant that carried mutations in the RII(+)-SL recruitment element (Fig. 2B) (31). The requirement of cis elements in (+)repRNA for template selection/recruitment and replicase assembly in the cell extract will certainly be useful in future studies to dissect the core RNA elements and protein factors for template selection and the assembly of the functional replicase complex.

Another important characteristic of the cell extract is the ability to generate recRNAs which are similar in sizes and abundance to those observed in vivo (7, 42). A modified repRNA, DI-{Delta}69RII(+), which has the RII(+)-SL hot-spot sequence at a 5' proximal position, supported the efficient generation of recRNAs in the cell extract (Fig. 4), suggesting that recombination is as frequent in vitro as seen in vivo (7, 42, 43). Recombination in the yeast cell extract is much higher than in the previous in vitro system based on the detergent-solubilized tombusvirus replicase (5). This makes the cell extract highly suitable for RNA recombination studies.

Membrane requirement of TBSV RNA replication in the cell extract. TBSV replication took place in the membrane fraction of the cell extract (Fig. 5A), suggesting that the added (+)repRNA was recruited to a membraneous compartment in vitro. The recruitment of the (+)repRNA might be facilitated by the ongoing translation, which stimulated repRNA replication in the cell extract (Fig. 6A and B). Interestingly, cycloheximide or puromycin inhibited repRNA replication in the membrane fraction of the cell extract (Fig. 6A and B), suggesting that translation of p33/p92pol could take place on membrane-bound ribosomes. However, further experiments will be necessary to analyze the subcellular compartment for p33/p92pol translation, which is currently unknown. Altogether, the dependence of repRNA replication on de novo translation in the cell extract could be due to the role of the newly translated p33, p92pol, and/or a host factor(s) in facilitating the recruitment of the (+)repRNA to the membranes, the site of replication.

The requirement for the cellular membranes in TBSV replication demonstrates that the membrane is a critical factor in viral replication. Treatment with SDS or Triton X-100 prevented repRNA replication, likely due to destruction of the membranes utilized by the viral replicase in the cell extract. This is further supported by the observation that the detergent was still effective in inhibiting repRNA replication at the 40-min time point, when the role of de novo translation and RNA recruitment was insignificant. The membrane might provide the scaffold for the viral replicase, as shown for BMV and flock house virus (15, 40), or protect the viral RNA from degradation. Indeed, viral replication took place in the presence of RNase or protease after the 40-min time point in the cell extract (Fig. 9). Future studies will address the role of the membrane in the function of the tombusvirus replicase.

Rapid assembly of the functional tombusvirus replicase in the cell extract. A major advantage of the yeast-based cell-free tombusvirus replication assay is that it can be used to characterize important biochemical features of replicase complex formation and its activities, which should be useful during future screening for replicase inhibitors. For example, time course experiments revealed that replicase assembly and initial RNA synthesis take place during the first 40 min (Fig. 9). At the beginning of the assay, the replication proteins were proteinase K sensitive and the viral RNA was RNase sensitive, suggesting that the replicase complex is present in an "accessible" structure that is not yet protected from proteinase or RNase. However, after 20 to 40 min of incubation, the structure of the replicase complex might change to a "protected" form, which is relatively insensitive to proteinase or RNase (Fig. 11). We propose that the membranes present in the cell extract might be involved in switching the replicase from the accessible to the protected form based on the sensitivity of the replicase to treatment with Triton X-100 even after 40 min of incubation.


Figure 11
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  		FIG. 11. Model of the activities taking place in the cell extract programmed with repRNA and sensitivity of the replicase-RNA complex to RNase, proteinase, and Triton X-100. We propose that the tombusvirus replicase-RNA complex switches from an "accessible" structure (proteinase/nuclease sensitive) to a "protected" (proteinase/nuclease-insensitive) membrane-bound structure in the cell extract. Then, the protected replicase structure is capable of minus- and plus-strand synthesis as well as of the release of the (+)repRNA from the replicase. The asterisk indicates that the replicase-bound repRNA in the membraneous compartment is insensitive, whereas the released (+)repRNA is RNase sensitive.

An additional finding is that protein translation in the cell extract increased replicase activity on added (+)repRNA (Fig. 6). This is supported by the observation that puromycin- or cycloheximide-based inhibition of translation had an inhibitory effect on repRNA replication. When translation in the cell extract was inhibited by adding cycloheximide at the beginning of the assay, but not after 40 min of incubation, then repRNA replication decreased by approximately threefold. This suggests that translation is important during the first 40 min in the cell extract, likely by producing p33 molecules that facilitate recruitment of the repRNA to the site of replication (to the membranes that are still present in the cell extract) (Fig. 11).

The cell extract supports the release of repRNA progeny from the replicase. One of the intriguing features of the cell-free replicase assay is the ability of the assembled replicase complex to release the repRNA progeny from the replicase complex to the soluble fraction. Unlike the total amount of labeled repRNA, which increased continuously from the 40-min time point, the amount of repRNA in the membrane-bound replicase reached high levels after 40 to 80 min of incubation (Fig. 8C and D), suggesting that the replicase complex in the cell extract has the ability to efficiently release most of the newly synthesized repRNA products from the membrane fraction to the soluble fraction in vitro. Accordingly, the amount of released repRNA increased more than the level of membrane (replicase)-bound repRNA over time (Fig. 8C and D), suggesting that the release of the repRNA product takes place continuously during RNA synthesis. The release of the (+)repRNA from the peroxisomal membrane to the cytosol has also been observed in yeast cells (24), suggesting similarities between the cell extract and the intact cells during the release of repRNA from replication.

Dependence of RNA recombination on the replicase activity in the cell extract. (+)RNA viruses recombine with various frequencies, likely due to differences in RNA structures (hot spots versus cold spots) and in the abilities of the viral replicases to switch templates (21). This work provides data supporting an important role for the viral replicase in RNA recombination. Indeed, RNA recombinants were detected in the membraneous fraction, where the replicase is also localized (Fig. 8C). Also, the recRNAs were partially protected from RNase I in the membraneous fraction (Fig. 5E, lane 7), suggesting that they are present in the replicase complex. Moreover, we found that the release of the recRNAs from the replicase seems to be less efficient than the release of the repRNA (Fig. 8C), suggesting possibly increased stability of the interaction between the replicase and the recRNAs. This work, however, does not exclude the possibility that recRNAs are generated at the start of incubation via a replicase-independent mechanism, followed by replication of the resulting recRNAs via the tombusvirus replicase. Previous works using detergent-solubilized tombusvirus replicase preparations demonstrated more vigorously that RNA recombination in tombusviruses is driven mostly by the replicase-driven template-switching mechanism (5, 6). Therefore, it is very likely that a similar mechanism operates in the cell-free system as well. Altogether, the yeast extract-based cell-free assay will be useful to study viral and host factors affecting TBSV RNA recombination.

Comparison with other cell-free systems capable of full viral RNA replication. Cell-free coupled translation/replication assays using extracts obtained from noninfected cells have been developed for poliovirus, ToMV, BMV, and TCV (2, 9, 14). The coupled translation/replication-based cell-free systems were capable of supporting full virus replication for poliovirus and ToMV and recombination in the case of poliovirus (10, 45). The major advantage of these systems is their suitability to study the mechanism of replicase assembly. The yeast cell extract developed for TBSV replication in this paper is also capable of authentic, full cycles of replication and recombination. The replicase activity in the cell extract depends on the added repRNA, suggesting that template selection/recruitment and/or the activation/assembly of the viral replicase occurs in the cell-free system. Unlike the above systems, however, our cell extract is based on yeast that will allow the use of yeast genetics and advanced biochemistry tools to dissect the function of host proteins in TBSV replication and recombination.


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ACKNOWLEDGMENTS
 
This work was supported by grants from NSF (MCB0078152), NIH-NIAID, and the University of Kentucky to P.D.N.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, S-305, 201F Plant Science Building, Lexington, KY 40546. Phone: (859) 257-7445. Fax: (859) 323-1961. E-mail: pdnagy2{at}uky.edu Back

{triangledown} Published ahead of print on 16 April 2008. Back


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Journal of Virology, June 2008, p. 5967-5980, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02737-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Pogany, J., Stork, J., Li, Z., Nagy, P. D. (2008). In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70. Proc. Natl. Acad. Sci. USA 105: 19956-19961 [Abstract] [Full Text]  

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