Specificity and Mechanism Analysis of Hepatitis C Virus RNA-Dependent RNA Polymerase

Johnson, Robert B.; Sun, Xin-Lai; Hockman, Michelle A.; Villarreal, Elcira C.; Wakulchik, Mark; Wang, Q.May

doi:10.1006/abbi.2000.1749

Cited by 28 publications

(56 citation statements)

References 19 publications

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“…8A) (30,38,39), the displacement of Mg 2ϩ (or Mn 2ϩ ) by iron can explain the inhibition of the RdRp activity of NS5B. A direct iron-mediated inhibition of NS5B activity is indeed illustrated by the data in Fig.…”

Section: Iron-mediated Inhibition Of Rdrp Activity In a Cell-free Sysmentioning

confidence: 76%

“…This experiment showed that Fe 2ϩ competes with Mg 2ϩ for binding to the protein. Because Mg 2ϩ (or Mn 2ϩ , or a combination of both) are absolutely required for NS5B to function as an RdRp (30,38,39), we reasoned that the binding of iron may impair its enzymatic activity. This was directly assessed by a primer-independent RNA synthesis assay (Fig.…”

Section: Iron Binds Specifically To Purified Hcv Rna Polymerase and Imentioning

confidence: 99%

“…Interestingly, other divalent metal cations, such as Cu 2ϩ , Zn 2ϩ , Co 2ϩ , and Ni 2ϩ , have also been reported to inhibit NS5B in vitro (30,38,39). Thus, one would expect that the treatment of replicon cells with Cu 2ϩ , Zn 2ϩ , or Co 2ϩ salts might also impair the expression of viral proteins, such as NS5A.…”

Section: Iron-mediated Inhibition Of Rdrp Activity In a Cell-free Sysmentioning

confidence: 99%

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Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

Fillebeen

Rivas-Estilla

Bisaillon

et al. 2005

Journal of Biological Chemistry

100

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Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a wellestablished model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent K d : 6 and 60 M for Fe 2؉ and Fe 3؉ , respectively) to the protein's Mg 2؉ -binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.

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Section: Iron-mediated Inhibition Of Rdrp Activity In a Cell-free Sysmentioning

confidence: 76%

Section: Iron Binds Specifically To Purified Hcv Rna Polymerase and Imentioning

confidence: 99%

Section: Iron-mediated Inhibition Of Rdrp Activity In a Cell-free Sysmentioning

confidence: 99%

See 1 more Smart Citation

Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

Fillebeen

Rivas-Estilla

Bisaillon

et al. 2005

Journal of Biological Chemistry

100

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“…Expression, Purification, and Intrinsic Fluorescence Properties of HCV NS5B Protein-The HCV NS5B protein contains motifs shared by RNA polymerases, and its activity has been shown to be dependent on the presence of either magnesium or manganese ions (8,14,(22)(23)(24)(25)(26). To further characterize the metal binding activity of the enzyme, the NS5B protein was expressed in E. coli as described under "Experimental Procedures."…”

Section: Resultsmentioning

confidence: 99%

“…By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8). This lack of specificity for HCV RNA supports the notion that additional viral or cellular factors are required for specific recognition of the viral replication signal.Previous reports showed that both magnesium and manganese ions can support the polymerase activity (8,14,(22)(23)(24)(25)(26), although manganese ions appear to be more effective in promoting optimal catalytic action (14). No information is currently available on the precise affinity of the enzyme for metal ions nor on their possible roles in catalysis, but manganese has recently been found in the crystal structure of NS5B (28).…”

mentioning

confidence: 99%

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

Bougie

Charpentier

Bisaillon

2003

Journal of Biological Chemistry

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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...

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Solid‐Phase Synthesis and Screening of Macrocyclic Nucleotide‐Hybrid Compounds Targeted to Hepatitis C NS5B

Smietana

Johnson

Wang

et al. 2003

Chemistry A European J

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A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modified ribonucleosides (such as 2'-OMe-ribonucleotides), as well as threoninol derivatives. This methodology allows a fully automated assembly by means of standard phosphoramidite chemistry and is based on a recently published procedure for the preparation of cyclic oligodinucleotides in the DNA series (M. Smietana, E. T. Kool, Angew. Chem. 2002, 114, 3856-3859; Angew. Chem. Int. Ed. Engl. 2002, 41, 3704-3707). A library of potential cyclic hybrid inhibitor compounds targeting hepatitis C virus NS5B enzyme (the replicating polymerase of HCV) was generated by means of the parallel-pool strategy. Screening of the library revealed that cyclic hybrid c(C(OME)EthenodA) was a significant inhibitor of NS5B, with an IC(50) of 40 microM. Preliminary structure-activity studies of this lead compound are described.

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Specificity and Mechanism Analysis of Hepatitis C Virus RNA-Dependent RNA Polymerase

Cited by 28 publications

References 19 publications

Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus

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

Solid‐Phase Synthesis and Screening of Macrocyclic Nucleotide‐Hybrid Compounds Targeted to Hepatitis C NS5B

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