The Hepatitis C Virus RNA-Dependent RNA Polymerase Membrane Insertion Sequence Is a Transmembrane Segment

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) belongs to a class of membrane proteins termed tail-anchored proteins. Here, we show that the HCV RdRp C-terminal membrane insertion sequence traverses the phospholipid bilayer as a transmembrane segment. Moreover, the HCV RdRp was found to be retained in the endoplasmic reticulum (ER) or an ER-derived modified compartment both following transient transfection and in the context of a subgenomic replicon. An absolutely conserved GVG motif was not essential for membrane insertion but possibly provides a docking site for transmembrane protein-protein interactions. These findings have important implications for the functional architecture of the HCV replication complex.

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Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.78.23.13278-13284.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13278-13284 ◽

Cited By ~ 97

Author(s):

Darius Moradpour ◽

Volker Brass ◽

Elke Bieck ◽

Peter Friebe ◽

Rainer Gosert ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Rna Polymerase ◽

Insertion Sequence ◽

Rna Replication ◽

Hcv Rna ◽

Membrane Association ◽

Rna Dependent Rna Polymerase ◽

Terminal Domain

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.

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THE QSAR RESEARCH ON AURONE INHIBITORS OF HEPATITIS C VIRUS RNA-DEPENDENT RNA POLYMERASE BY APPLYING MULTI-DIMENSIONAL SCALING METHOD

International Journal of Biomathematics ◽

10.1142/s1793524512500623 ◽

2013 ◽

Vol 06 (01) ◽

pp. 1250062

Author(s):

YONG-HONG HU ◽

BAO-HUA ZHANG

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Hcv Rna ◽

Training Set ◽

Rna Dependent Rna Polymerase ◽

Dimensional Scaling ◽

Naturally Occurring ◽

Virus Rna ◽

Multi Dimensional Scaling

In this paper, we take naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) as an example to study the Multi-dimensional scaling (MDS) method for structure-activity relationship. By analyzing training set molecules, our MDS method combined with a PROXSCAL algorithm can predict inhibitory activity of most compounds correctly. Thus, a new sample's activity can be estimated and judged conveniently, and whether it should be synthesized can be known. The MDS method is applicable to optimize the structure for a compound and to provide suggestions for drug design.

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Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.01106-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2052-2063 ◽

Cited By ~ 5

Author(s):

Amy L. Cherry ◽

Caitriona A. Dennis ◽

Andrew Baron ◽

Leslie E. Eisele ◽

Pia A. Thommes ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

De Novo ◽

Structural Features ◽

Primer Extension ◽

Hcv Rna ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

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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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Preclinical Characterization of JTK-853, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00312-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4250-4256 ◽

Cited By ~ 12

Author(s):

Izuru Ando ◽

Tsuyoshi Adachi ◽

Naoki Ogura ◽

Yukiyo Toyonaga ◽

Kazuyuki Sugimoto ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Rna Polymerase ◽

Antiviral Agents ◽

Hcv Rna ◽

Resistance Mutations ◽

Rna Dependent Rna Polymerase ◽

Virus Rna ◽

Polymerase Inhibitor

ABSTRACTJTK-853 is a novel piperazine derivative nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase. JTK-853 showed potent inhibitory activity against genotype 1 HCV polymerase, with a 50% inhibitory concentration in the nanomolar range, and showed potent antiviral activity against the genotype 1b replicon, with a 50% effective concentration of 0.035 μM. The presence of human serum at up to 40% had little effect on the antiviral activity of JTK-853. Structure analysis of HCV polymerase with JTK-853 revealed that JTK-853 associates with the palm site and β-hairpin region of HCV polymerase, and JTK-853 showed decreased antiviral activity against HCV replicons bearing the resistance mutations C316Y, M414T, Y452H, and L466V in the palm site region of HCV polymerase. JTK-853 showed an additive combination effect with other DAAs (direct antiviral agents), such as nucleoside polymerase inhibitor, thumb pocket-binding nonnucleoside polymerase inhibitor, NS5A inhibitor, and protease inhibitor. Collectively, these data demonstrate that JTK-853 is a potent and novel nonnucleoside palm site-binding HCV polymerase inhibitor, suggesting JTK-853 as a potentially useful agent in combination with other DAAs for treatment of HCV infections.

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Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.10.4305-4314.2005 ◽

2005 ◽

Vol 49 (10) ◽

pp. 4305-4314 ◽

Cited By ~ 93

Author(s):

Hongmei Mo ◽

Liangjun Lu ◽

Tami Pilot-Matias ◽

Ron Pithawalla ◽

Rubina Mondal ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Serine Protease ◽

Hcv Rna ◽

Protease Gene ◽

Polymerase Gene ◽

Rna Dependent Rna Polymerase ◽

Resistant Mutants ◽

Ns5b Polymerase

ABSTRACT Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

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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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The Mechanism of Action of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, a Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 458-464 ◽

Cited By ~ 85

Author(s):

Eisuke Murakami ◽

Congrong Niu ◽

Haiying Bao ◽

Holly M. Micolochick Steuer ◽

Tony Whitaker ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Potent Inhibitor ◽

Nucleoside Diphosphate Kinase ◽

Competitive Inhibitor ◽

Hcv Rna ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5′-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5′-triphosphate of the uridine congener, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5′-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

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Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.77.7.4149-4159.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4149-4159 ◽

Cited By ~ 69

Author(s):

Lu Gao ◽

Hong Tu ◽

Stephanie T. Shi ◽

Ki-Jeong Lee ◽

Miyuki Asanaka ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Hcv Rna ◽

Rna Dependent Rna Polymerase ◽

Subgenomic Replicon ◽

C Terminus ◽

Huh7 Cells

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

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