Dataset Information

A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.

ABSTRACT: The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.

SUBMITTER: Kulkarni AS

PROVIDER: S-EPMC4808174 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.

Kulkarni Anupriya S AS Damha Masad J MJ Schinazi Raymond F RF Mo Hongmei H Doehle Brian B Sagan Selena M SM Götte Matthias M

Antimicrobial agents and chemotherapy 20160325 4

The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibil ...[more]

PMID: 26824949

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Project description:Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Project description:ImportanceTreatment of hepatitis C virus (HCV) infection in patients also infected with human immunodeficiency virus (HIV) has been limited due to drug interactions with antiretroviral therapies (ARTs) and the need to use interferon.ObjectiveTo determine the rates of HCV eradication (sustained virologic response [SVR]) and adverse events in patients with HCV-HIV coinfection receiving sofosbuvir and ribavirin treatment.Design, setting, and participantsOpen-label, nonrandomized, uncontrolled phase 3 trial conducted at 34 treatment centers in the United States and Puerto Rico (August 2012-November 2013) evaluating treatment with sofosbuvir and ribavirin among patients with HCV genotypes 1, 2, or 3 and concurrent HIV. Patients were required to be receiving ART with HIV RNA values of 50 copies/mL or less and a CD4 T-cell count of more than 200 cells/μL or to have untreated HIV infection with a CD4 T-cell count of more than 500 cells/μL. Of the treatment-naive patients, 114 had HCV genotype 1 and 68 had HCV genotype 2 or 3, and 41 treatment experienced participants who had been treated with peginterferon-ribavirin had HCV genotype 2 or 3, for a total of 223 participants.InterventionsTreatment-naive patients with HCV genotype 2 or 3 received 400 mg of sofosbuvir and weight-based ribavirin for 12 weeks and treatment-naive patients with HCV genotype 1 and treatment-experienced patients with HCV genotype 2 or 3 received the same treatment for 24 weeks.Main outcomes and measuresThe primary study outcome was the proportion of patients with SVR (serum HCV <25 copies/mL) 12 weeks (SVR12) after cessation of HCV therapy.ResultsAmong treatment-naive participants, 87 patients (76%) of 114 (95% CI, 67%-84%) with genotype 1, 23 patients (88%) of 26 with genotype 2 (95% CI, 70%-985), and 28 patients (67%) of 42 with genotype 3 (95% CI, 51%-80%) achieved SVR12. Among treatment-experienced participants, 22 patients (92%) of 24 with genotype 2 (95% CI, 73%-99%) and 16 patients (94%) of 17 (95% CI, 71%-100%) achieved SVR12. The most common adverse events were fatigue, insomnia, headache, and nausea. Seven patients (3%) discontinued HCV treatment due to adverse events. No adverse effect on HIV disease or its treatment was observed.Conclusions and relevanceIn this open-label, nonrandomized, uncontrolled study, patients with HIV who were coinfected with HCV genotype 1, 2, or 3 who received the oral, interferon-free combination of sofosbuvir and ribavirin for 12 or 24 weeks had high rates of SVR12. Further studies of this oral regimen in diverse populations of coinfected patients are warranted.Trial registrationclinicaltrials.gov Identifier: NCT01667731.

Project description:Sofosbuvir and ribavirin exert their anti-hepatitis C virus (anti-HCV) activity following metabolic activation in the liver. However, intrahepatic concentrations of the pharmacologically active nucleotide metabolites in humans are poorly characterized due to the inaccessibility of tissue and technical challenges with measuring nucleotide levels. A clinical study assessing the efficacy of sofosbuvir and ribavirin administered prior to liver transplantation to prevent HCV recurrence provided a unique opportunity to quantify nucleotide concentrations in human liver. We analyzed nucleotides using high-performance liquid chromatography coupled to tandem mass spectrometry in liver tissue from 30 HCV-infected patients with hepatocellular carcinoma who were administered sofosbuvir (400 mg/day) and ribavirin (1,000 to 1,200 mg/day) for 3 to 52 weeks prior to liver transplantation. Median total hepatic metabolite concentrations (the sum of nucleoside and mono-, di-, and triphosphates) were 77.1 ?M for sofosbuvir and 361 ?M for ribavirin in patients on therapy at the time of transplantation. Ribavirin and sofosbuvir efficiently loaded the liver, with total hepatic metabolite concentrations exceeding maximal levels in plasma by approximately 30-fold. Ribavirin metabolite levels suggest that its monophosphate is in great excess of its inhibition constant for IMP dehydrogenase and that its triphosphate is approaching the binding constant for incorporation by the HCV NS5B RNA-dependent RNA polymerase. In accordance with the potent antiviral activity of sofosbuvir, these results demonstrate that the liver triphosphate levels achieved following sofosbuvir administration greatly exceed the inhibition constant for HCV NS5B. In conclusion, this study expands the quantitative understanding of the pharmacology of sofosbuvir and ribavirin by establishing efficient hepatic delivery in the clinic. (This study has been registered at ClinicalTrials.gov under identifier NCT01559844.).

Dataset Information

A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.

Publications

A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.

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