Chronic infection with hepatitis C virus (HCV) is associated with progressive liver
damage, including the development of cirrhosis and hepatocellular carcinoma, and HCV
is a leading cause of liver dysfunction worldwide. The current therapy for chronic
HCV infection, interferon-alpha (IFN), is effective in a minority of HCV-infected
patients. Several studies have demonstrated a correlation between therapeutic outcome
and the amino acid sequence of a small region of the HCV non-structural 5A (NS5A)
gene product. It has been suggested that this region, termed the interferon sensitivity-determining
region (ISDR), may mediate IFN resistance by directly interacting with one or more
cellular proteins associated with the IFN-mediated antiviral response.
In an attempt to define the molecular mechanism by which the NS5A protein and the
ISDR might contribute to HCV resistance to IFN, we examined whether NS5A could regulate
the IFN-induced protein kinase, PKR, a primary mediator of the IFN-induced antiviral
Multiple approaches, including in vitro assays using recombinant proteins, the transfection
of recombinant clones into cultured cells, and in vivo studies in yeast, were used
to examine the interaction of NS5A with PKR, as well as the functional significance
of the interaction. An ISDR deletion mutant was prepared to evaluate the importance
of the ISDR in mediating the NS5A-PKR interaction and the requirement of this region
for PKR inhibition.
NS5A repressed PKR activity through a direct interaction with the protein kinase catalytic
domain. Both PKR repression and interaction required the presence of the ISDR.
Inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects
of IFN. Thus,therapeutic strategies designed to block the NS5A-PKR interaction may
increase the efficacy of IFN therapy in HCV-infected individuals.