Kinetic Analyses Reveal Potent and Early Blockade of Hepatitis C Virus Assembly by NS5A Inhibitors

Abstract

Background & Aims All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus (HCV) genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the HCV life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Results Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared to protease or polymerase inhibitors. By 24 hrs after addition of an NS5A inhibitor, polyprotein synthesis was reduced less than 50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hrs. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing HCV particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of gentoype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against pre-formed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…This is consistent with the recent, elegant kinetic studies from McGivern et al, 17 who provided evidence that NS5A inhibitors target newly forming RCs and early events in virus assembly. Next, the authors use NS3-5B protein expression systems, that mirror virus-encoded NS protein expression uncoupled from HCV RNA replication, to demonstrate that neither NS5A stability nor NS5A dimerization are altered by DCV derivatives.…”

Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle

“…Electroporated cells were cultured for 7 days to allow HCV replication to reach a steady state before seeding to different plate formats for specific assays of the virus life cycle. GLuc assays of HCV genome replication and measurement of infectious HCV production by a focus-forming-unit (FFU) assay have been described previously (26). To measure the effects of protease inhibitors on cell proliferation and viability, a WST-1 assay was used according to the manufacturer's protocol (Roche).…”

“…After drug treatment, cells were harvested by trypsinization, washed twice in PBS, and resuspended in 400 l PBS. Lysates were prepared by multiple freeze-thaw cycles as described previously (26) and subjected to rate-zonal centrifugation. Clarified lysate (350 l) was loaded onto a 10 to 50% sucrose gradient prepared in TNE (10 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA) and centrifuged for 1 h at 40,000 rpm in an SW55 Ti rotor at 4°C.…”

“…Our previous studies suggested that production of HCV particles is a dynamic process that can be rapidly inhibited by DAAs that target viral proteins involved in assembly, such as NS5A (26). Whether inhibitors that target NS3 impact viral assembly or maturation is not known, but genetic and biochemical studies have suggested a role for the helicase domain of NS3 in virus assembly (13).…”

Modeling how reversal of immune exhaustion elicits cure of chronic hepatitis C after the end of treatment with direct‐acting antiviral agents

“…The residual, predominantly non‐infectious virions would be cleared eventually, leading to SVR. Evidence for this hypothesis comes from studies that show a reduction in viral infectivity following exposure to DAAs, including a preferential targeting of infectious over non‐infectious virion production by some DAAs 15, 16, 17. Furthermore, mathematical models of viral kinetics that account for this mode of DAA action have been constructed and shown to describe data of viral load changes during therapy in patients who experience EOT + /SVR, as well as SVR rates elicited by certain DAA combinations 18, 19.…”