Specific Interaction between the Hepatitis C Virus NS5B RNA Polymerase and the 3′ End of the Viral RNA

ABSTRACT Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3′ viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5′ noncoding region and very poorly by the 3′ conserved 98-nucleotide tail. A 3′ coding region with conserved stem-loop structures rather than the 3′ noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3′ coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.

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Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/3196140 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11

Author(s):

Marina K. Kukhanova ◽

Vera L. Tunitskaya ◽

Olga A. Smirnova ◽

Olga A. Khomich ◽

Natalia F. Zakirova ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Recombinant Protein ◽

Rna Polymerase ◽

Reducing Agents ◽

Cysteine Residues ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Redox Switches ◽

Ns5b Protein

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

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Functional Analysis of RNA Binding by the Hepatitis C Virus RNA-dependent RNA Polymerase

Journal of Biological Chemistry ◽

10.1074/jbc.m508145200 ◽

2005 ◽

Vol 280 (45) ◽

pp. 38011-38019 ◽

Cited By ~ 30

Author(s):

Young-Chan Kim ◽

William K. Russell ◽

C. T. Ranjith-Kumar ◽

Michael Thomson ◽

David H. Russell ◽

...

Keyword(s):

Functional Analysis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Binding ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention

Nucleic Acids Research ◽

10.1093/nar/gku632 ◽

2014 ◽

Vol 42 (14) ◽

pp. 9399-9409 ◽

Cited By ~ 23

Author(s):

Abdelhakim Ahmed-Belkacem ◽

Jean-François Guichou ◽

Rozenn Brillet ◽

Nazim Ahnou ◽

Eva Hernandez ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Binding ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics

10.1101/2021.07.21.453274 ◽

2021 ◽

Author(s):

Xuanting Wang ◽

Carolina Q. Sacramento ◽

Steffen Jockusch ◽

Otávio Augusto Chaves ◽

Chuanjuan Tao ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Viral Rna ◽

Combination Strategy ◽

Rna Dependent Rna Polymerase ◽

Culture Studies ◽

Unique Structure ◽

Combination Drugs

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

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De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

Journal of Virology ◽

10.1128/jvi.78.22.12207-12217.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12207-12217 ◽

Cited By ~ 21

Author(s):

C. T. Ranjith-Kumar ◽

R. T. Sarisky ◽

L. Gutshall ◽

M. Thomson ◽

C. C. Kao

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

De Novo ◽

Specific Interaction ◽

Binding Pocket ◽

Hcv Rna ◽

Elongation Complex ◽

Rna Dependent Rna Polymerase ◽

Template Switch

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 μM, all of the mutations except one had Km s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

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Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis

Journal of Virology ◽

10.1128/jvi.77.16.9020-9028.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 9020-9028 ◽

Cited By ~ 40

Author(s):

Vincent J.-P. Lévêque ◽

Robert B. Johnson ◽

Stephen Parsons ◽

Jianxin Ren ◽

Congping Xie ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Rna Binding ◽

Dimensional Structure ◽

Synthesis Rate ◽

Rna Dependent Rna Polymerase ◽

Hcv Ns5b ◽

Ns5b Polymerase

ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

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Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4813-4821.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4813-4821 ◽

Cited By ~ 41

Author(s):

Anita Y. M. Howe ◽

Johnathan Bloom ◽

Carl J. Baldick ◽

Christopher A. Benetatos ◽

Huiming Cheng ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

High Throughput Screening ◽

Viral Rna ◽

Alpha Interferon ◽

Hcv Rna ◽

Binding Studies ◽

Rna Dependent Rna Polymerase ◽

Multiple Treatments

ABSTRACT A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

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Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity

Journal of Virology ◽

10.1128/jvi.01185-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11523-11533 ◽

Cited By ~ 20

Author(s):

Mounavya Aligeti ◽

Allison Roder ◽

Stacy M. Horner

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Molecular Mechanisms ◽

Genetic Interaction ◽

Infectious Clone ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Adaptive Mutations ◽

Viral Titers

ABSTRACTThe molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly.IMPORTANCEHepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.

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