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

We synthesized the Hepatitis C virus (HCV) RNA 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 preformed 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 (LPO R) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent 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 LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differencesmore » 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. The 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 LPO S and LPO R 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.« less
Authors:
 [1] ;  [2] ; ORCiD logo [3] ;  [3] ; ORCiD logo [4] ;  [4] ;  [1] ; ORCiD logo [5] ;  [2] ; ORCiD logo [1] ;  [6]
  1. Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center and Dept. of Medicine
  2. North Carolina State Univ., Raleigh, NC (United States). Dept. of Mathematics
  3. Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center
  4. National Inst. of Molecular Genetics (INGM), Milan (Italy)
  5. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Biology and Biophysics
  6. Univ. of Chicago, IL (United States)
Publication Date:
Report Number(s):
LA-UR-17-20265
Journal ID: ISSN 1553-7374
Grant/Contract Number:
AC52-06NA25396; R21-AI115207 (DRM); R01-AI095690 (SML); R01-AI078881 (ASP); R01- AI028433 (ASP); OD-011095 (ASP)
Type:
Accepted Manuscript
Journal Name:
PLoS Pathogens
Additional Journal Information:
Journal Volume: 13; Journal Issue: 6; Journal ID: ISSN 1553-7374
Publisher:
Public Library of Science
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
USDOE; National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Biological Science
OSTI Identifier:
1375872

Benzine, Tiffany, Brandt, Ryan, Lovell, William C., Yamane, Daisuke, Neddermann, Petra, De Francesco, Raffaele, Lemon, Stanley M., Perelson, Alan S., Ke, Ruian, McGivern, David R., and Randall, Glenn. NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains. United States: N. p., Web. doi:10.1371/journal.ppat.1006343.
Benzine, Tiffany, Brandt, Ryan, Lovell, William C., Yamane, Daisuke, Neddermann, Petra, De Francesco, Raffaele, Lemon, Stanley M., Perelson, Alan S., Ke, Ruian, McGivern, David R., & Randall, Glenn. NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains. United States. doi:10.1371/journal.ppat.1006343.
Benzine, Tiffany, Brandt, Ryan, Lovell, William C., Yamane, Daisuke, Neddermann, Petra, De Francesco, Raffaele, Lemon, Stanley M., Perelson, Alan S., Ke, Ruian, McGivern, David R., and Randall, Glenn. 2017. "NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains". United States. doi:10.1371/journal.ppat.1006343. https://www.osti.gov/servlets/purl/1375872.
@article{osti_1375872,
title = {NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains},
author = {Benzine, Tiffany and Brandt, Ryan and Lovell, William C. and Yamane, Daisuke and Neddermann, Petra and De Francesco, Raffaele and Lemon, Stanley M. and Perelson, Alan S. and Ke, Ruian and McGivern, David R. and Randall, Glenn},
abstractNote = {We synthesized the Hepatitis C virus (HCV) RNA 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 preformed 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). Furthermore, 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 RNAdependent 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. The 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.},
doi = {10.1371/journal.ppat.1006343},
journal = {PLoS Pathogens},
number = 6,
volume = 13,
place = {United States},
year = {2017},
month = {6}
}