Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: implication for modular evolution of RNA viruses

The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and post-recombinational amplification of new recombinant viruses.     

Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. A partially purified RNA-dependent RNA polymerase (RdRp) preparation of Cucumber necrosis virus (CNV), which is capable of de novo initiation of complementary RNA synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (DI) RNAs. In vitro RdRp assay revealed that the core plus-strand initiation promoter included only the 3'-terminal 11 nucleotides. A hypothetical promoter-like sequence, which has been termed consensus sequence by Wu and White (1998, J. Virol. 72, 9897-9905), is recognized less efficiently by the CNV RdRp than the core plus-strand initiation promoter. The CNV RdRp can efficiently recognize the core plus-strand initiation promoter for a satellite RNA associated with the distantly related Turnip crinkle virus, while artificial AU- or GC-rich 3'-terminal sequences make poor templates in the in vitro assays. Comparison of the "strength" of minimal plus-strand and minus-strand initiation promoters reveals that the latter is almost twice as efficient in promoting complementary RNA synthesis. Template competition experiments, however, suggest that the minimal plus-strand initiation promoter makes an RNA template more competitive than the minimal minus-strand initiation promoter. Taken together, these results demonstrate that promoter recognition by the tombusvirus RdRp requires only short sequences present at the 3' end of templates.     

Internal initiation by the cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences

Tombusviruses, small positive sense RNA viruses of plants, are replicated by the viral-coded RNA-dependent RNA polymerase (RdRp) in infected cells. An unusual feature of the tombusvirus RdRp that is partially purified from cucumber necrosis virus (CNV)-infected plants is the ability to initiate complementary RNA synthesis from several internal positions on minus-strand templates derived from DI RNAs ( Nagy and Pogany, 2000 ). In this study, we used template deletion, mutagenesis, and oligo-based inhibition of RNA synthesis to map the internal initiation sites observed with the in vitro CNV RdRp system. Comparing sequences around the internal initiation sites reveals that they have either (i) similar sequences to the core minus-strand initiation promoter; or (ii) similar structures to the core plus-strand initiation promoter. In addition, we find similarities among the internal initiation sites and the subgenomic RNA initiation sites. These similarities suggest that the mechanism of internal initiation is similar to initiation from the terminal core promoters or the putative subgenomic promoter sequences. We propose that internal initiation on full-length RNA templates may be important in defective interfering (DI) RNA formation/evolution by producing intermediate templates for RNA recombination in tombusviruses. This may explain why tombusviruses are frequently associated with DI RNAs.     

RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp in vitro

Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity.     

Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes

Plus-strand RNA virus replication takes place on distinct membranous surfaces in infected cells via the assembly of viral replicase complexes involving multiple viral and host proteins. One group of tombusviruses, such as Tomato bushy stunt virus (TBSV), replicate on the surfaces of peroxisomal membranes in plant and yeast cells. Surprisingly, previous genome-wide screen performed in yeast demonstrated that a TBSV replicon RNA replicated as efficiently in yeast defective in peroxisome biogenesis as in the wt yeast (Panavas et al., Proc Natl Acad Sci U S A, 2005). To further test how the lack of peroxisomes could affect tombusvirus replication, we used yeast cells missing either PEX3 or PEX19 genes, which are absolutely essential for peroxisome biogenesis. Confocal microscopy-based approach revealed that the wild-type tombusvirus p33 replication protein accumulated in the endoplasmic reticulum (ER) in pex3Delta or pex19Delta yeast, suggesting that tombusvirus replication could take place on the surface of ER membrane. The activities of the isolated tombusvirus replicase preparations from wt, pex3Delta or pex19Delta yeasts were comparable, demonstrating that the assembly of the replicase was as efficient in the ER as in the authentic subcellular environments. The generation/accumulation of tombusvirus recombinants was similar in wt, pex3Delta and pex19Delta yeasts, suggesting that the rate of mistakes occurring during tombusvirus replication is comparable in the presence or absence of peroxisomes. Overall, this work demonstrates that a tombusvirus, relying on the wt replication proteins, can efficiently replicate on an alternative intracellular membrane. This suggests that RNA viruses might have remarkable flexibility for using various host membranes for their replication.     

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.     

Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus

Tombusviruses, which are small plus-strand RNA viruses of plants, require the viral-coded p33 replication co-factor for template selection and recruitment into replication in infected cells. As presented in the accompanying paper [Shapka, N., Stork, J., Nagy, P.D., 2005. Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication. J. Virol. 343, 65-78.], p33 can be phosphorylated in vitro at serine and threonine residues adjacent to its arginine-proline-rich RNA binding site. To test the effect of phosphorylation on p33 function, in this paper, we used phosphorylation-mimicking aspartic acid mutants of Cucumber necrosis virus (CNV) p33 and in-vitro-phosphorylated p33 in gel mobility shift experiments. We found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. In contrast, the nonphosphorylation-mimicking alanine mutants of p33 bound to viral RNA as efficiently as the nonphosphorylated wild type p33 did. In vitro assays with purified CNV replicase preparations revealed that phosphorylation-mimicking mutants of p33 did not support the assembly of functional CNV replicase complexes in yeast, a model host. Based on these results, we propose that the primary function of reversible phosphorylation of p33 is to regulate the RNA binding capacity of p33, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement in infected hosts.     

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.     

In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A

Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with 3'-untranslated region (3'-UTR) of TMV RNA but also directly with RdRp without mediation by the 3'-UTR. The methyltransferase domain of TMV RdRp was indicated to be responsible for the interaction with eEF1A in vitro and in yeast. These results suggest that eEF1A is a component of the virus replication complex of TMV.     

Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus

Defective interfering (DI) RNA associated with Tomato bushy stunt virus (TBSV), which is a plus-strand RNA virus, requires p33 and p92 proteins of TBSV or the related Cucumber necrosis virus (CNV), for replication in plants. To test if DI RNA can replicate in a model host, we coexpressed TBSV DI RNA and p33/p92 of CNV in yeast. We show evidence for replication of DI RNA in yeast, including (i) dependence on p33 and p92 for DI replication; (ii) presence of active CNV RNA-dependent RNA polymerase in isolated membrane-containing preparations; (iii) increasing amount of DI RNA(+) over time; (iv) accumulation of (-)stranded DI RNA; (v) presence of correct 5' and 3' ends in DI RNA; (vi) inhibition of replication by mutations in the replication enhancer; and (vii) evolution of DI RNA over time, as shown by sequence heterogeneity. We also produced evidence supporting the occurrence of DI RNA recombinants in yeast. In summary, development of yeast as a host for replication of TBSV DI RNA will facilitate studies on the roles of viral and host proteins in replication/recombination.     

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3′ end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3′ CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.     

Temperature requirements for initiation of RNA-dependent RNA polymerization

To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage phi12. Interestingly, initiation by both wt and the ts phi12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme. Experiments with the Pol subunit of related cystovirus phi6 revealed a similar differential sensitivity of the initiation and elongation steps. This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable. Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex.     

A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique beta-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this beta-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this beta-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this beta-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a beta-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.     

Classical swine fever virus NS5B-GFP fusion protein possesses an RNA-dependent RNA polymerase activity

Summary.  RNA-dependent RNA polymerase (RdRp) is the replicase of positive-strand RNA viruses. Expression and characterization of the replicase are the first steps in the elucidation of the virus replication mechanism. We expressed nonstructural protein 5B (NS5B) of classical swine fever virus (CSFV) as a fusion protein with green fluorescent protein (GFP) in porcine kidney cells (PK-15 cells), natural host cells of CSFV. The expressed CSFV NS5B-GFP fusion protein possessed RdRp activity. By fluorescence microscope it was observed that the density of the fusion protein near cytoplasmic membranes was higher than that in other parts of cells. This was in contrast to the distribution of the GFP alone which was uniformly distributed throughout the cytoplasm. The GFP is a signal for the location of NS5B in a host cell that allows in vitro and in vivo investigation of the distribution of plus-strand RNA virus RdRp. Received September 24, 2001; accepted March 23, 2002 Published online July 19, 2002     

Enhancement of RNA synthesis by promoter duplication in tombusviruses

Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants, Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses.     

Emerging picture of host chaperone and cyclophilin roles in RNA virus replication

Many plus-strand (+)RNA viruses co-opt protein chaperones from the host cell to assist the synthesis, localization and folding of abundant viral proteins, to regulate viral replication via activation of replication proteins and to interfere with host antiviral responses. The most frequently subverted host chaperones are heat shock protein 70 (Hsp70), Hsp90 and the J-domain co-chaperones. The various roles of these host chaperones in RNA virus replication are presented to illustrate the astonishing repertoire of host chaperone functions that are subverted by RNA viruses. This review also discusses the emerging roles of cyclophilins, which are peptidyl-prolyl isomerases with chaperone functions, in replication of selected (+)RNA viruses.