Only a Small Fraction of Purified Hepatitis C RNA-Dependent RNA Polymerase Is Catalytically Competent:  Implications for Viral Replication and in Vitro Assays

Carroll, Steven S.; Sardana, Vinod; Yang, Zhucheng; Jacobs, Amanda R.; Mizenko, Christine; Hall, Dawn L.; Hill, Lorraine A; Zugay-Murphy, Joan; Kuo, Lawrence C.

doi:10.1021/bi991992s

Cited by 62 publications

(63 citation statements)

References 22 publications

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“…These results might help explain the biochemical data reported previously. For example, Carroll and colleagues reported that a large fraction of the purified HCV NS5B protein was catalytically incompetent (6). If inactive NS5B proteins can form a complex with the active enzyme, as seen in Fig.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Wang

Hockman

Staschke

et al. 2002

J Virol

136

127

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The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K d , of Ϸ22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.Hepatitis C virus (HCV) belongs to the Flaviviridae family and is responsible for a significant proportion of acute and chronic hepatitis in humans worldwide (7,8). Similar to other flaviviruses, HCV is a small positive-stranded RNA virus with a genome size of Ϸ9.6 kb encoding a single polyprotein (30). This viral polyprotein is processed by both host and virally encoded proteases to generate mature structural and nonstructural proteins essential for virus replication (see references 9, 34, and 39 for review). One of the nonstructural proteins, designated NS5B, is the virally encoded RNA-dependent RNA polymerase (RdRp), which contains the GDD signature motif of RNA polymerases (3).It has been shown that NS5B is a membrane-associated protein, which contains a C-terminal domain comprising Ϸ21 hydrophobic amino acids that is responsible for membrane anchorage (41). NS5B may form a complex with cellular proteins (37) or other HCV nonstructural proteins, including NS3, the viral protease and helicase; NS4A, a cofactor of NS3 protease activity; and NS5A, a phosphoprotein containing a putative interferon sensitivity region (15). Although the HCV replication mechanism is not clearly understood, the essential role of NS5B polymerase in the HCV replication and infection process has been demonstrated in chimpanzees (22). Accordingly, it has been viewed as an attractive target for antiviral intervention.Recombinant HCV NS5B polymerase has been produced and purified from both bacterial and insect cells by several groups (3,11,16,19,25,27,31,41). The availability of highly purified protein has facilitated the biochemical characterization of HCV NS5B polymerase. Similar to other viral RdRps, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (3,11,16,19,25,27,31,41). In this regard, two RNA synthesis reaction modes have been described for this enzyme: RNA elongation using a preannealed primer and RNA in...

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Section: Discussionmentioning

confidence: 99%

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Wang

Hockman

Staschke

et al. 2002

J Virol

136

127

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“…The RNA polymerase activity of the HCV NS5B protein has been characterized extensively (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The protein is believed to be responsible for the genome replication of HCV and is thus a critical protein of the virus.…”

Section: Discussionmentioning

confidence: 99%

“…The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26).…”

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confidence: 99%

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Benzaghou

Bougie

Bisaillon

2004

Journal of Biological Chemistry

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The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp 220 , Asp 318 , and Asp 319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzymebound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.Recent estimates indicate that more than 170 million people worldwide are infected with the hepatitis C virus (HCV) 1 (1). It is estimated that 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). There is thus an urgent need for the development of antiviral drugs aimed at inhibiting this pathogen.The HCV nonstructural 5B protein (NS5B) has been shown to be an RNA-dependent RNA polymerase (6 -11). The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (3). 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. The HCV RNA polymerase activity has been shown to be supported by both magnesium and manganese ions (7, 13, 21-25). However, the re...

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“…The last 21 residues at the C terminus of NS5B are required for endoplasmic reticulum membrane anchoring (11,12) and in vivo HCV replication (13), but they are not absolutely required for its enzymatic activity in vitro (14,15). NS5B exhibits a similar "right-hand" shape as other RNA-dependent RNA polymerases, with thumb, palm, and finger subdomains (16 -18).…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

Rosnoblet¹,

Fritzinger²,

Legrand

et al. 2012

Journal of Biological Chemistry

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Background: HCV replication requires the interaction of the viral polymerase NS5B with both viral and host proteins. Results: We performed the molecular characterization of the interactions between HCV NS5B, NS5A, and host CypA. Conclusion: HCV NS5B and host CypA share a binding site on HCV NS5A. Significance: The NS5A-D2 site, which interacts with both the HCV polymerase NS5B and the host CypA, might regulate the HCV replication.

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Only a Small Fraction of Purified Hepatitis C RNA-Dependent RNA Polymerase Is Catalytically Competent: Implications for Viral Replication and in Vitro Assays

Cited by 62 publications

References 22 publications

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

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