The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities

RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3′ untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3′ UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3′ terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3′ UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.     

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication.     

A conserved secondary structure in the hypervariable region at the 5' end of Bamboo mosaic virus satellite RNA is functionally interchangeable

Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV.     

Downregulation of Bamboo mosaic virus replication requires the 5' apical hairpin stem loop structure and sequence of satellite RNA

Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.     

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (−)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (−)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (−)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.     

Use of double-stranded RNA templates by the tombusvirus replicase in vitro: Implications for the mechanism of plus-strand initiation

Plus-stranded RNA viruses replicate efficiently in infected hosts producing numerous copies of the viral RNA. One of the long-standing mysteries in RNA virus replication is the occurrence and possible role of the double-stranded (ds)RNA formed between minus- and plus-strands. Using the partially purified Cucumber necrosis virus (CNV) replicase from plants and the recombinant RNA-dependent RNA polymerase (RdRp) of Turnip crinkle virus (TCV), in this paper, we demonstrate that both CNV replicase and the related TCV RdRp can utilize dsRNA templates to produce viral plus-stranded RNA in vitro. Sequence and structure of the dsRNA around the plus-strand initiation site had a significant effect on initiation, suggesting that initiation on dsRNA templates is a rate-limiting step. In contrast, the CNV replicase could efficiently synthesize plus-strand RNA on partial dsRNAs that had the plus-strand initiation promoter "exposed", suggesting that the polymerase activity of CNV replicase is strong enough to unwind extended dsRNA regions in the template during RNA synthesis. Based on the in vitro data, we propose that dsRNA forms might have functional roles during tombus- and carmovirus replication and the AU-rich nature of the terminus could be important for opening the dsRNA structure around the plus-strand initiation promoter for tombus- and carmoviruses and possibly many other positive-strand RNA viruses.     

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.     

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.     

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.     

Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.     

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.     

Mechanism of DI RNA formation in tombusviruses: dissecting the requirement for primer extension by the tombusvirus RNA dependent RNA polymerase in vitro

Tombusviruses, which are positive-strand RNA viruses of plants, frequently generate defective interfering (DI) RNAs that consist of three to four noncontiguous segments of the parental RNA. Replicase jumping was postulated to cause multiple deletions leading to the de novo formation of DI RNAs in planta. This model was tested using a partially purified RNA-dependent RNA polymerase (RdRp) preparation from tombusvirus-infected plants in vitro. The tombusvirus RdRp was capable of primer extension without the need for sequence complementarity between the primer and the acceptor template in vitro, although the most efficient primer extension was obtained with primers forming a 5-bp duplex with the acceptor region. Primers forming 14- to 20-bp duplexes with the acceptor region were used less efficiently by the tombusvirus RdRp in vitro. In addition, primers with 3' noncomplementary nucleotides were also extended by the tombusvirus RdRp, albeit with a reduced efficiency. The preference of the tombusvirus RdRp for short base-paired primers in vitro is consistent with the lack of extended sequence similarities at the junction sites in the de novo generated tombusvirus-associated DI RNAs. The in vitro experiments also revealed that the acceptor region plays a significant role in primer extension. Comparison of tombusvirus-derived, heterologous and artificial acceptor regions revealed that the conserved regions present in DI RNAs are the best acceptor regions when they are available in the minus-strand orientation. These data suggest that recombination/deletion events may be more frequent at some regions, rather than occurring randomly throughout the parental genome. In addition, these findings support a model that predicts a higher frequency of replicase jumping, i.e., recombination/deletion events, during plus-strand synthesis than during minus-strand synthesis.     

Primer-independent initiation of RNA synthesis by SeMV recombinant RNA-dependent RNA polymerase

Sesbania mosaic virus (SeMV), a single-strand positive-sense RNA plant virus, belongs to the genus Sobemoviruses. Mechanism of replication in Sobemoviruses is poorly understood. In the present study, SeMV RNA-dependent RNA polymerase (RdRp) was overexpressed and purified as a thioredoxin-tagged protein. The recombinant SeMV RdRp could synthesize RNA from genomic or subgenomic RNA templates, even in the absence of the protein primer, VPg. Analysis of the product indicated that it was double-stranded and that the mode of initiation was de novo. Mutational analysis of the 3′ UTR of subgenomic RNA revealed that a stem-loop structure at the 3′ end was important. Further, analysis of this stem-loop showed that the SeMV RdRp was capable of recognizing stem-loop structures of various lengths and forms. These results demonstrate that the SeMV RdRp is capable of primer-independent RNA synthesis in vitro.