Researchers have uncovered the mechanisms of drugs that inhibit the hepatitis C virus (HCV) non-structural protein 5A (NS5A), described in the August issue of Gastroenterology. In a quantitative analysis of the kinetics of NS5A inhibitors, David R. McGivern et al. show that these agents rapidly inhibit intracellular assembly of virions and inhibit formation of functional replicase complexes, but have no activity against preformed replicase.
NS5A inhibitors are likely to become an important component of hepatitis C treatment regimens, yet their exact mechanism of action are unclear. NS5A has no known enzymatic activity and no cellular orthologs or viral homologs, other than NS5A proteins of other hepaciviruses. However, NS5A functions in multiple aspects of the HCV life cycle.
McGivern et al. performed detailed kinetic analyses of specific steps in the HCV life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors.
The authors found that the NS5A inhibitors blocked HCV RNA synthesis more slowly than 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, occurring within 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.
McGivern et al. provided direct evidence for an immediate (<3 hours) effect of NS5A inhibitors on the intracellular assembly of HCV. In the absence of inhibitors, NS5A is distributed between membrane-bound viral replicase complexes derived from the endoplasmic reticulum, where NS5A functions in RNA replication, and lipid droplets, to which it is recruited by core protein and functions in viral assembly. NS5A inhibitors induce a redistribution of NS5A within the cell, into large, cytoplasmic aggregates. The authors propose that the inhibitors bind NS5A in proximity to the lipid droplet, given their effect on virus assembly.
In addition to their immediate effect on virus assembly, NS5A inhibitors potently induced an early (<12 hours), but only partial block in viral RNA synthesis.
In an editorial that accompanies the article, Menashe Elazar and Jeffrey S. Glenn explain that although NS5A has independent roles in RNA replication and particle production, these functions might be tightly linked. Inhibition of RNA replication by other drugs could lead to a NS5A-mediated immediate shut down of particle assembly (see figure). This type of linkage could provide a mechanism for HCV to prevent further packaging of viral genomes and instead divert them to serve as templates, allowing them overcome the inhibition of RNA replication.
Although NS5A inhibitors have not been approved for treatment of hepatitis C, there are several in clinical phases of development. These include daclatasvir, ledipasvir, and MK-8742. McGivern et al. state that these inhibitors show substantial therapeutic promise, with 50% effective concentrations in replicon assays in the low picomolar range, making them the most potent class of anti-HCV agent.
The authors conclude that their findings complement recent efforts to develop multiscale mathematical models of the clinical response to antiviral agents. They state that further research is needed to elucidate the full viral life cycle, which would increase our understanding of antiviral activity and resistance.
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