NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

PLoS Pathogens

William C. Lovell¹  Ruian Ke²  Ryan Brandt²  Raffaele De Francesco³  Stanley M. Lemon³  Daisuke Yamane⁴  Alan S. Perelson⁴  Petra Neddermann⁴  Tiffany Benzine⁴  David R. McGivern⁵ 

[1] Department of Mathematics, North Carolina State University, Raleigh, North Carolina, United States of America;Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America;INGM -Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy;Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America;Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America

关键词: RNA synthesis;    Viral replication;    Cell cultures;    Hepatitis C virus;    RNA viruses;    Microbial mutation;    Mathematical models;    Lipid peroxidation;   

DOI  :  10.1371/journal.ppat.1006343

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.

【 授权许可】

CC BY