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

Date Published: June 8, 2017

Publisher: Public Library of Science

Author(s): Tiffany Benzine, Ryan Brandt, William C. Lovell, Daisuke Yamane, Petra Neddermann, Raffaele De Francesco, Stanley M. Lemon, Alan S. Perelson, Ruian Ke, David R. McGivern, Glenn Randall.


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.

Partial Text

Direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) include specific inhibitors of the NS3/4A protease/helicase, the NS5B RNA-dependent RNA polymerase and the NS5A protein. Combination therapies with two or more DAAs can result in a sustained virological response (SVR) in most infected persons and have revolutionized treatment of chronic hepatitis C in the USA and other developed countries.

Our previous studies show that when cells infected with the gt1a HCV clone H77S.3 are cultured with NS5A inhibitors, the decline in intracellular viral RNA abundance and new viral RNA synthesis is relatively slow compared to other classes of DAA such as protease and polymerase inhibitors [2]. These unique kinetics of RNA synthesis inhibition suggested a model where NS5A inhibitors block assembly of new RCs but are unable to inhibit RNA synthesis from pre-existing RCs in infected cells. In contrast to our findings, other studies using reporter viruses based upon the gt2a HCV clone JFH-1 found that NS5A inhibitors resulted in more rapid decline in RNA abundance compared to other classes of DAA [7].

Assuming that an NS5A inhibitor binds to NS5A and blocks formation of new membrane-protected RCs soon after its addition to infected cell cultures, the rate of decline of RNA synthesis should reflect the rate of turnover of existing functional RCs. In this study, we have demonstrated that RNA synthesis by LPOR viruses such as H77D show a faster decline in RNA synthesis following treatment with NS5A inhibitors, cyclophilin inhibitors or PI4KIIIα inhibitors compared to LPOS viruses such as H77S.3.




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