Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viral RNA template during RNA replication

The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5' ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.     

Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.     

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.     

Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3' end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.     

Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.     

Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase

The kinetic, thermodynamic, and structural basis for fidelity of nucleic acid polymerases remains controversial. An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness. Here we show that a conformational change step preceding phosphoryl transfer is a key fidelity checkpoint for the poliovirus RdRp (3Dpol). We provide evidence that this conformational change step is orientation of the triphosphate into a conformation suitable for catalysis, suggesting a kinetic and structural model for RdRp fidelity that can be extrapolated to other classes of nucleic acid polymerases. Finally, we show that a site remote from the catalytic center can control this checkpoint, which occurs at the active site. Importantly, similar connections between a remote site and the active site exist in a wide variety of viral RdRps. The capacity for sites remote from the catalytic center to alter fidelity suggests new possibilities for targeting the viral RdRp for antiviral drug development.     

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.     

ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication

The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.     

Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA.

We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3Dpol was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3Dpol binds with high affinity.     

An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.     

A novel, highly selective inhibitor of pestivirus replication that targets the viral RNA-dependent RNA polymerase

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.     

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.