Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase

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267

201

Ribavirin is administered in combination with interferon-␣ for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection. Hepatitis C virus (HCV)1 establishes a persistent infection of the liver, which leads to the development of cirrhosis and cancer (1). Currently, at least four million Americans are carriers of HCV. The available therapies are not very effective in clearing virus. The current therapeutic regimen includes a combination of interferon-␣ (IFN-␣) and ribavirin (2). IFN-␣ increases the nonadaptive antiviral response in cells (3). Ribavirin is a broad-spectrum antiviral nucleoside that is converted to the mono-, di-, and triphosphorylated forms inside of cells (4). Until this year, it was thought that the mechanism of action of ribavirin involved a decrease in cellular GTP pools resulting from inhibition of inosine monophosphate dehydrogenase by ribavirin monophosphate (5). The mechanism of action for ribavirin has been expanded to include lethal mutagenesis of the viral genome as a result of ribavirin triphosphate utilization by the viral RNA-dependent RNA polymerase and incorporation into viral RNA (6, 7). As shown in Fig. 1, the pseudo base of ribavirin can pair equivalently with both cytosine and uracil, thus providing a molecular basis for the mutagenic potential of ribavirin. Given the need for more effective strategies to control HCV infection, a precise understanding of the mechanism of action of existing antivirals should provide direction for the development of new anti-HCV therapeutics.Full-length HCV polymerase (NS5B) contains a very hydrophobic carboxyl terminus. To increase the solubility of this enzyme, the carboxyl-terminal 21 amino acids have been removed (8). This enzyme has been referred ...

Section: Resultsmentioning

confidence: 99%

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

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Hepatitis C Virus RNA-dependent RNA Polymerase (NS5B) as a Mediator of the Antiviral Activity of Ribavirin

Maag¹,

Castro²,

Hong³

et al. 2001

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267

201

“…The activity has been extensively studied because it is one of the major targets for the development of antiviral drugs. The NS5B protein can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (27). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8).…”

Section: Currently Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Characterization of the Metal Ion Binding Properties of the Hepatitis C Virus RNA Polymerase

Bougie

Charpentier

Bisaillon

2003

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg 2؉ or Mn 2؉ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K d values for the binding of Mg 2؉ and Mn 2؉ ions to the free enzyme were 3.1 and 0.3 mM, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ioninduced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state. Currently, hepatitis C virus (HCV)1 is the leading etiological agent of non-A non-B hepatitis, with more than 170 million people worldwide being infected with HCV (1). About 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2-5). HCV is a positive, single-stranded RNA virus of the Flaviviridae family. The genome is ϳ10,000 nucleotides long and encodes a single polyprotein of about 3,010 amino acids (6). The polyprotein is processed by both host cell and viral proteases into three major structural proteins and several nonstructural proteins necessary for viral replication (6).One key enzyme encoded by HCV is the nonstructural 5B protein (NS5B), which has been shown to be an RNA-dependent RNA polymerase (7-12). The HCV NS5B protein contains characteristic motifs, such as the GDD motif, shared by RNAdependent RNA polymerases (13). The NS5B protein is thus believed to be responsible for the genome replication of HCV. Indeed, polymerase activity has been demonstrated with recombinant NS5B expressed in both insect cells and Escherichia coli (8, 14 -26). The activity has been extensively studied because it is one of the major targets for the development of antiviral drugs. The NS5B protein can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (27). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8). This lack of spec...

“…Like poliovirus (15), the HCV RdRp is capable of initiating viral RNA synthesis in vitro by a primer-dependent mechanism (9,10,16). However, Flaviviridae RdRps have also been shown to initiate RNA synthesis by a de novo mechanism (12,13,(17)(18)(19). De novo initiation of virus replication is the likely preferred mechanism for HCV in infected cells (20,21).…”

mentioning

confidence: 99%

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

Johnston

Gutshall

et al. 2003

In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of ⌬21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNAbound form of ⌬21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.