Environment determines fidelity for an RNA virus replicase

The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.     

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.     

The 2b protein of cucumoviruses has a role in promoting the cell-to-cell movement of pseudorecombinant viruses

Pseudorecombinant viruses (i.e., those containing a reassorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.     

Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.     

In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5′ end and lack polyadenylation at the 3′ terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3′ end of their genomic RNAs, a prominent feature of bromo- and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3′ end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. ​(Fig.1)1) turned out to be the replicative form of the 3′ termini (19, 20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16-18). Although other models have been forwarded (9), we have proposed that switching between these two conformations, mediated by CP binding, plays a fundamental role in the life cycle of AMV and ilarviruses by regulating the competing processes of translation and replication of the viral RNAs.Open in a separate windowFIG. 1.The CPB and the TL conformations of the AMV RNA3 3′ terminus. The two conformers of AMV RNA3 3′ 145 nt are shown. (A) CPB conformer. The two major CP binding sites are indicated by brackets. Base pairing between loop D and stem A promotes TL conformation. (B) Secondary structure of the TL conformer.In the present study, the distribution between CPB and TL conformers was further investigated. We addressed how changes in this distribution would affect recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding ezyme) and the viral polymerase in vitro. We also monitored how the binding affinity to CP is affected by this conformational switch in vivo using the yeast three-hybrid (Y3H) system (2, 11, 24). Together, the in vitro and in vivo data clearly demonstrate the existence and function of the conformational switch in the 3′ UTR of AMV RNAs.     

The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that a cis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.     

Satellite RNA reduces expression of the 2b suppressor protein resulting in the attenuation of symptoms caused by Cucumber mosaic virus infection

Satellite RNAs (satRNAs) depend on cognate helper viruses for replication, encapsidation, movement and transmission. Many satRNAs with different symptom modulation effects have been reported. The pathogenicity of satRNAs is thought to be the result of a direct interaction among the satRNA, helper viruses and host factors by unknown mechanisms. To understand the effect of satRNA of Cucumber mosaic virus (a severe field ShanDong strain, SD-CMV) on pathogenicity, and the possible involvement of host RNA silencing pathways in pathogenicity, we constructed biologically active CMV cDNA clones and a CMV-Δ2b mutant lacking the open reading frame of 2b, a silencing suppressor protein, in order to infect Nicotiana benthamiana and Arabidopsis with or without SD-satRNA. We found that SD-satRNA reduced the accumulation of the 2b protein and its coding RNA4A and attenuated the yellowing caused by SD-CMV infection. Small RNA analysis indicated that the 2b protein interfered with RNA silencing, specifically in the synthesis of CMV RNA3-derived small interfering RNAs (R3-siRNAs). The accumulation of R3-siRNAs in CMV-Δ2b infection was reduced in the presence of satRNA, for which greater accumulation of satRNA-derived siRNAs (satsiRNAs) was detected. Our results suggest that abundant SD-satRNA serving as target for RNA silencing may play a role in protecting helper CMV RNA, especially, subgenomic RNA4, from being targeted by RNA silencing. This compensates for the increase in RNA silencing resulting from the reduction in expression of the 2b suppressor in the presence of satRNA. Our data provide evidence that a plant silencing mechanism is involved in the pathogenicity of satRNA.     

In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.     

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

Homologous Terminal Sequences in the Double-Stranded RNA Genome Segments of Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori

The 3′-terminal regions (20 to 32 residues) of the genome double-stranded RNA (dsRNA) segments of cytoplasmic polyhedrosis virus were sequenced. The dsRNAs, which were labeled at their 3′ termini by incubation with [5′-³²P]pCp and T4 RNA ligase, were denatured and resolved into the plus and minus strands by agarose-urea gel electrophoresis. Ten single-stranded RNAs thus obtained from the five dsRNA segments IV, V, VIII, IX, and X were sequenced by postlabeling methods. Common 3′-terminal sequences, -GUUAGCC and -UUACU, were found in the plus and minus strands, respectively, of all five dsRNA segments. However, adjacent sequences diverged and were considerably variable. The homologous sequences found in the 3′ end may be important recognition signals for viral RNA polymerases and for assembly of the genome segments.     

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3′ terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3′ terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3′ cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3′ termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3′ terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3′ vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.     

Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.     

Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.