Canonical 3'-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase.

Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication.

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).