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

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.

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

2000 ◽  
Vol 74 (9) ◽  
pp. 4291-4301 ◽  
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.

scholarly journals Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

2014 ◽  
Vol 58 (6) ◽  
pp. 3485-3495 ◽  
Author(s):  
Julie A. Lemm ◽  
Mengping Liu ◽  
Robert G. Gentles ◽  
Min Ding ◽  
Stacey Voss ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleoside Analogs ◽  
Synergistic Activity ◽  
Genotype 1 ◽  
Allosteric Inhibitor ◽  
Genotype 2 ◽  
Hcv Genotype 1 ◽  
Ns3 Protease

ABSTRACTBMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposuresin vivoin several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

scholarly journals Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
Author(s):  
Marco Binder ◽  
Doris Quinkert ◽  
Olga Bochkarova ◽  
Rahel Klein ◽  
Nikolina Kezmic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Negative Strand ◽  
Virus Rna ◽  
Nontranslated Region ◽  
Genotype 2 ◽  
Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

scholarly journals 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

2013 ◽  
Vol 57 (9) ◽  
pp. 4417-4426 ◽  
Author(s):  
Gururaj Kalkeri ◽  
Chao Lin ◽  
Jenna Gopilan ◽  
Kevin Sloan ◽  
Rene Rijnbrand ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Sendai Virus ◽  
Mitochondrial Localization ◽  
Promoter Activation ◽  
Polymerase Inhibitors ◽  
Hcv Protease ◽  
Host Innate Immunity ◽  
Ns3 Protease

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.

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

2015 ◽  
Vol 89 (19) ◽  
pp. 9758-9764 ◽  
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.

scholarly journals Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants:In VitroSelection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

2016 ◽  
Vol 60 (6) ◽  
pp. 3563-3578 ◽  
Author(s):  
Stéphanie B. N. Serre ◽  
Sanne B. Jensen ◽  
Lubna Ghanem ◽  
Daryl G. Humes ◽  
Santseharay Ramirez ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hot Spots ◽  
Viral Life Cycle ◽  
Viral Fitness ◽  
Cross Resistance ◽  
Genotype 1 ◽  
Genotype 2 ◽  
Ns3 Protease

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.

Mechanistic Role of an NS4A Peptide Cofactor with the Truncated NS3 Protease of Hepatitis C Virus:  Elucidation of the NS4A Stimulatory EffectviaKinetic Analysis and Inhibitor Mapping

Biochemistry ◽  
1997 ◽  
Vol 36 (31) ◽  
pp. 9340-9348 ◽  
Author(s):  
James A. Landro ◽  
Scott A. Raybuck ◽  
Yu Ping C. Luong ◽  
Ethan T. O'Malley ◽  
Scott L. Harbeson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ns3 Protease

scholarly journals Role of interleukin-28B polymorphisms in the treatment of hepatitis C virus genotype 2 infection in Asian patients

Hepatology ◽  
2011 ◽  
Vol 53 (1) ◽  
pp. 7-13 ◽  
Author(s):  
Ming-Lung Yu ◽  
Chung-Feng Huang ◽  
Jee-Fu Huang ◽  
Ning-Chia Chang ◽  
Jeng-Fu Yang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Genotype ◽  
Interleukin 28B ◽  
Asian Patients ◽  
Genotype 2

scholarly journals Development of a High Throughput Scintillation Proximity Assay for Hepatitis C Virus NS3 Protease That Reduces the Proportion of Competitive Inhibitors Identified

2000 ◽  
Vol 5 (3) ◽  
pp. 153-158 ◽  
Author(s):  
Anne Fowler ◽  
Molly Price-Jones ◽  
Kelvin Hughes ◽  
John Anson ◽  
Russell Lingham ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Substrate Concentration ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Screening Assay ◽  
Scintillation Proximity Assay ◽  
Competitive Inhibitors ◽  
Ns3 Protease

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC50 values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.

scholarly journals Mutational Analysis of Hepatitis C Virus Nonstructural Protein 5A: Potential Role of Differential Phosphorylation in RNA Replication and Identification of a Genetically Flexible Domain

2005 ◽  
Vol 79 (5) ◽  
pp. 3187-3194 ◽  
Author(s):  
Nicole Appel ◽  
Thomas Pietschmann ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Adaptive Mutations ◽  
Phosphorylation Pattern ◽  
Molecular Masses ◽  
Nonstructural Protein 5A

ABSTRACT Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.

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