Peripheral association of a polyprotein precursor form of the RNA-dependent RNA polymerase of Tomato ringspot virus with the membrane-bound viral replication complex

Replication of Tomato ringspot virus (ToRSV) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleotide triphosphate-binding protein (NTB) of ToRSV is an ER-targeted protein and that an intermediate polyprotein containing the domains for NTB and for the genome-linked viral protein (VPg) is associated with the replication complex. We now report the detection of a 95-kDa polyprotein that contains the domains for the RNA-dependent RNA polymerase (Pol), the proteinase (Pro) and the VPg. This polyprotein appears to be a truncated version of the full-length 111-kDa VPg-Pro-Pol polyprotein and was termed VPg-Pro-Pol'. A subpopulation of VPg-Pro-Pol' was peripherally associated with ER-derived membranes active in viral replication. However, the VPg, Pro and Pol domains did not target to membranes in the absence of viral infection. We propose a model in which VPg-Pro-Pol' is brought to the site of replication through interaction with a viral membrane protein.     

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.     

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.     

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.     

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.     

A long-distance RNA-RNA interaction plays an important role in programmed -1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus

Programmed − 1 ribosomal frameshifting (− 1 PRF) is one viral translation strategy to express overlapping genes in positive-strand RNA viruses. Red clover necrotic mosaic virus (RCNMV) uses this strategy to express its replicase component protein p88. In this study, we used a cell-free translation system to map cis-acting RNA elements required for − 1 PRF. Our results show that a small stem-loop structure adjacent to the cap-independent translation element in the 3′ untranslated region (UTR) of RCNMV RNA1 is required for − 1 PRF. Site-directed mutagenesis experiments suggested that this stem-loop regulates − 1 PRF via base-pairing with complementary sequences in a bulged stem-loop adjacent to the shifty site. The existence of RNA elements responsible for − 1 PRF and the cap-independent translation of replicase proteins in the 3′ UTR of RNA1 might be important for switching translation to replication and for regulating the ratio of p88 to p27.     

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC(50) values lower than 1 microM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC(50) = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA.     

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.     

Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3' stem-loop structure

Dengue virus (DEN) is a major public health problem worldwide and causes a spectrum of diseases, for which no antiviral treatments exist. Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) complementary to the DEN 5' stem-loop (5'SL) and to the DEN 3' cyclization sequence (3'CS) inhibit DEN replication, presumably by blocking critical RNA-RNA or RNA-protein interactions involved in viral translation and/or RNA synthesis. Here, a third P-PMO, complementary to the top of the 3' stem-loop (3'SLT), inhibited DEN replication in BHK cells. Using a novel DEN2 reporter replicon and a DEN2 reporter mRNA, we determined that the 5'SL P-PMO inhibited viral translation, the 3'CS P-PMO blocked viral RNA synthesis but not viral translation, and the 3'SLT P-PMO inhibited both viral translation and RNA synthesis. These results show that the 3'CS and the 3'SL domains regulate DEN translation and RNA synthesis and further demonstrate that P-PMOs are potentially useful as antiviral agents.     

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.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

Dengue virus (DENV) and West Nile virus (WNV) are members of the Flavivirus genus of positive-strand RNA viruses. RNA sequences and structures, primarily in the untranslated regions, have been shown to modulate flaviviral gene expression and genome replication. Previously, we demonstrated that a structure in the DENV coding region (cHP) enhances translation start codon selection and is required for viral replication. Here we further characterize the role of the cHP in the DENV life cycle. We demonstrate that the cHP is required for efficient viral RNA synthesis in a sequence-independent manner. Viruses with a disrupted cHP are rescued by a spontaneous compensatory mutation that restabilizes the structure. Furthermore, the cHP, which is predicted to be conserved among arthropod-borne flaviviruses, is required for WNV replication. We propose that the cHP is a multifunctional determinant of flavivirus replication, functioning in both translation and RNA synthesis.