Restoration of the Activated Rig-I Pathway in Hepatitis C Virus (HCV) Replicon Cells by HCV Protease, Polymerase, and NS5A InhibitorsIn Vitroat Clinically Relevant Concentrations

ABSTRACTDevelopment of persistent hepatitis C virus (HCV) infection may be mediated by HCV NS3 · 4A protease-dependent inhibition of host innate immunity. When double-stranded RNA (dsRNA) is detected in virus-infected cells, host innate immunity mounts an antiviral response by upregulating production of type I interferons (α/β interferon [IFN-α/β]); HCV counters by cleaving the IFN-β stimulator 1 (IPS-1) adaptor protein, decreasing synthesis of IFN-α/β. We evaluated HCV protease (telaprevir, boceprevir, and TMC435350), polymerase (HCV-796 and VX-222), and NS5A (BMS-790052) inhibitors for the ability to restore IPS-1-mediated Rig-I signaling by measuring Sendai virus-induced IFN-β promoter activation in HCV replicon cells after various exposure durations. All direct-acting HCV antivirals tested restored mitochondrial localization of IPS-1 and rescued Sendai virus-induced IRF3 signaling after 7 days by inhibiting HCV replication, thereby reducing the abundance of HCV NS3 · 4A protease. With 4-day treatment, HCV protease inhibitors, but not polymerase inhibitors, restored mitochondrial localization of IPS-1 and rescued IFN-β promoter activation in the presence of equivalent levels of NS3 protein in protease or polymerase inhibitor-treated cells. The concentrations of HCV protease and polymerase inhibitors needed to rescue IRF3-mediated signalingin vitrowere in the range of those observedin vivoin the plasma of treated HCV patients. These findings suggest that (i) HCV protease, polymerase, and NS5A inhibitors can restore virus-induced IRF3 signaling by inhibiting viral replication, thereby reducing NS3 protease levels, and (ii) HCV protease inhibitors can restore innate immunity by directly inhibiting NS3 protease-mediated cleavage of IPS-1 at clinically achievable concentrations.

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Synergy of a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01208-07 ◽

2008 ◽

Vol 52 (5) ◽

pp. 1862-1864 ◽

Cited By ~ 31

Author(s):

David L. Wyles ◽

Kelly A. Kaihara ◽

Robert T. Schooley

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Polymerase Inhibitors ◽

Resistance Pathway ◽

Ns3 Protease Inhibitors ◽

Hcv Protease ◽

Ns3 Protease ◽

Emergence Of Resistance ◽

Rapid Emergence ◽

Specific Inhibitors

ABSTRACT Rapid emergence of resistance to monotherapy with virus-specific inhibitors necessitates combination therapy. ACH-806 is a hepatitis C virus NS4A inhibitor with a novel mechanism of action and resistance pathway. This compound was synergistic with NS3 protease inhibitors and NS5B nucleoside and nonnucleoside polymerase inhibitors.

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Debio 025, a Cyclophilin Binding Molecule, Is Highly Efficient in Clearing Hepatitis C Virus (HCV) Replicon-Containing Cells When Used Alone or in Combination with Specifically Targeted Antiviral Therapy for HCV (STAT-C) Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00939-08 ◽

2008 ◽

Vol 53 (3) ◽

pp. 967-976 ◽

Cited By ~ 95

Author(s):

Lotte Coelmont ◽

Suzanne Kaptein ◽

Jan Paeshuyse ◽

Inge Vliegen ◽

Jean-Maurice Dumont ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Therapy ◽

Drug Candidate ◽

Development Of Resistance ◽

Polymerase Inhibitors ◽

Phase Ii Studies ◽

Hcv Protease ◽

Ns3 Protease

ABSTRACT Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log10 units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log10 units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 μM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

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Selection of Replicon Variants Resistant to ACH-806, a Novel Hepatitis C Virus Inhibitor with No Cross-Resistance to NS3 Protease and NS5B Polymerase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01548-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2043-2052 ◽

Cited By ~ 36

Author(s):

Wengang Yang ◽

Yongsen Zhao ◽

Joanne Fabrycki ◽

Xiaohong Hou ◽

Xingtie Nie ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Protease Inhibitors ◽

Cross Resistance ◽

Polymerase Inhibitors ◽

Ns3 Protease Inhibitors ◽

Ns3 Protease ◽

Ns5b Polymerase ◽

Selection Of

ABSTRACT We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.

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Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity

Journal of Virology ◽

10.1128/jvi.01097-15 ◽

2015 ◽

Vol 89 (19) ◽

pp. 9758-9764 ◽

Cited By ~ 1

Author(s):

Miguel Angel Martinez ◽

Maria Nevot ◽

Ana Jordan-Paiz ◽

Sandra Franco

Keyword(s):

Genetic Diversity ◽

Human Immunodeficiency Virus ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Enzymatic Activity ◽

Immunodeficiency Virus ◽

Hcv Protease ◽

Ns3 Protease ◽

Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299 clones). Single-nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 × 10−3± 0.4 × 10−3; HCV, 2.1 × 10−3± 0.5 × 10−3) (P= 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 ± 0.6667 and 2.183 ± 0.9048, respectively) (P= 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 ± 0.02 and 0.83 ± 0.12, respectively) (P= 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection is not responsible for the higher worldwide genetic diversity observed in HCV.IMPORTANCEHCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection is responsible for the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in infected individuals. Our results indicate that HIV-1 and HCV protease quasispecies have very similar genetic diversity and comparable rugged enzymatic activity landscapes. Therapy for HCV has expanded, with new therapeutic agents such as the direct-acting antivirals (DAAs). DAAs, which target HCV NS3 protease and other virus proteins, have improved cure rates. However, major questions remain to be elucidated regarding the virologic correlates of HCV eradication. The findings shown here may help our understanding of the different therapeutic responses observed during chronic HCV infection.

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Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity

Journal of Hepatology ◽

10.1016/j.jhep.2013.03.019 ◽

2013 ◽

Vol 59 (1) ◽

pp. 52-58 ◽

Cited By ~ 92

Author(s):

Qiang Ding ◽

Xuezhi Cao ◽

Jie Lu ◽

Bing Huang ◽

Yong-Jun Liu ◽

...

Keyword(s):

Innate Immunity ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Host Innate Immunity

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Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A

Journal of Virology ◽

10.1128/jvi.00420-20 ◽

2020 ◽

Vol 94 (19) ◽

Author(s):

Cho-Han Chiang ◽

Yen-Ling Lai ◽

Yu-Ning Huang ◽

Chun-Chiao Yu ◽

Christine C. Lu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Necessary Condition ◽

Cleavage Sites ◽

Dual Functional ◽

Genotype 2 ◽

Hcv Protease ◽

Ns3 Protease

ABSTRACT Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein. IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

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Virus-Specific Cofactor Requirement and Chimeric Hepatitis C Virus/GB Virus B Nonstructural Protein 3

Journal of Virology ◽

10.1128/jvi.74.9.4291-4301.2000 ◽

2000 ◽

Vol 74 (9) ◽

pp. 4291-4301 ◽

Cited By ~ 34

Author(s):

Nancy Butkiewicz ◽

Nanhua Yao ◽

Weidong Zhong ◽

Jacquelyn Wright-Minogue ◽

Paul Ingravallo ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Central Region ◽

Protease Activity ◽

Nonstructural Protein ◽

Protease Domain ◽

Hcv Protease ◽

Nonstructural Protein 3 ◽

Ns3 Protease

ABSTRACT GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.

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