Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

Ferrari, Eric; He, Zhiqing; Palermo, Robert E.; Huang, Hsiao‐Ching

doi:10.1074/jbc.m803094200

Cited by 34 publications

(44 citation statements)

References 52 publications

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“…We therefore rule out that the 18 base pair DNA: RNA duplex segment contributes to NS5B binding. These observations are consistent with the reported narrow binding tunnel associated with the closed conformation of NS5B, which is unable to accommodate a double stranded segment (7,38,39).…”

Section: Resultssupporting

confidence: 82%

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Karam

Powdrill

Liu

et al. 2014

Journal of Biological Chemistry

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Background:The dynamics associated with RNA binding by the hepatitis C virus (HCV) polymerase remain elusive. Results: Single molecule experiments reveal changing populations of binary RNA-enzyme complexes. Conclusion: Rapid enzyme conformational changes facilitate sliding/wrapping of the polymerase along its RNA substrate. Significance: This study provides novel insight into mechanisms associated with viral replication and its inhibition.

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Section: Resultssupporting

confidence: 82%

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Karam

Powdrill

Liu

et al. 2014

Journal of Biological Chemistry

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“…The first two stages are rate limiting and are collectively called initiation events. The K m s for NTPs for initiation nucleotides are significantly lower for elongation than for initiation (15,47), consistent with the idea of a conformational change in the RdRp between initiation and elongation in RNA synthesis.…”

Section: Discussionsupporting

confidence: 62%

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

Chinnaswamy

Murali

et al. 2010

J Virol

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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the ⌬1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the lowresolution conformations of a dimer of a de novo initiation-competent HCV RdRp.Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the templatedependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.Several model systems for biochemical study of viral RNAdependent RNA synthesis exist (4,19,20,25,37,42). Wellcharacterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed c...

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“…The data are given in picomoles of incorporated NMP per microgram of enzyme at the given incubation time. 15), and most of the reaction products had gained the template length of 9.7 kb after 60 min, corresponding to a polymerization velocity of ca. 150 to 200 bases per minute.…”

Section: Resultsmentioning

confidence: 99%

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Simister

Schmitt

Geitmann

et al. 2009

J Virol

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The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5-to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame flanked by nontranslated regions (NTRs), which is primarily translated into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membraneassociated replication complex in which NS5B is the RNAdependent RNA polymerase (RdRp), the key enzyme of viral-RNA replication.Studies of HCV have long been hampered by the lack of efficient cell culture systems. The advent of subgenomic replicons derived from genotype 1 isolates allowed for the first time a concise analysis of authentic viral-RNA replication (29).However, these isolates required adaptive mutations for efficient propagation in cells (9,21,27). Based on these adapted viral sequences, it was not possible to generate infectious virus, since most of these mutations seemed to interfere with virus assembly (38), and only one cell culture-adapted genotype 1 isolate has yet given rise to moderately efficient production of infectious virus (49). This restriction was abolished by a particu...

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Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

Cited by 34 publications

References 52 publications

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

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