Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors

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 genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the hepatitis C virus 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 with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <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 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis

Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation.

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts. Deletion of single stem-loop structures gave progeny retaining near-wild-type accumulation levels. Constructions representing deletion of two or three stem-loops substantially lowered the accumulation of progeny RNA-3 relative to wild-type levels. RNA-3 mutants bearing deletions of longer sequences or of the entire region (delta PsKs RNA-3) replicated poorly, yielding no detectable RNA-3 or RNA-4 progeny. Levels of RNA-1 and RNA-2, in the presence of a mutant RNA-3, were found to increase relative to the accumulation observed in a complete wild-type transfection. The stability of delta PsKs RNA-3 in protoplasts was somewhat lower than that of wild-type RNA during the first 3 h postinoculation. Little difference in translatability in vitro of wild-type and RNA-3 constructs bearing deletions within the stem-loop region was observed, and Western immunoblot analysis of viral coat protein produced in transfected protoplasts showed that protein accumulation paralleled the amount of RNA-4 message produced from the various sequences evaluated. These results indicate that the RNA-3 pseudoknot region plays a minor role in translational control but contributes substantially to the overall replication of the brome mosaic virus genome