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

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.Author summary: Inhibitors targeting the HCV NS5A protein are a key component of highly effective interferon-free combination therapies for chronic hepatitis C. Despite their high potency against HCV, the precise details of their mode of action are poorly understood. They are known to block assembly and release of virus particles from infected hepatocytes, resulting in a rapid drop in viral RNA in the blood. Additionally they block formation of intracellular membrane structures that are the site of viral RNA synthesis in infected hepatocytes. By preventing membrane remodeling, NS5A inhibitors effectively block formation of new RCs within the cell. Following addition of NS5A inhibitors to infected cell cultures, the kinetics of antiviral suppression were found to vary between different HCV strains, independent of specific differences in NS5A sequence. Using an integrated experimental and mathematical modeling approach, we provide evidence that the rate of decline of viral RNA abundance in infected cells treated with NS5A inhibitors is determined by the stability or half-life of the functional HCV RC.