Chronic hepatitis C virus (HCV) infection has become a worldwide health problem, affecting an estimated 170 million people worldwide, including about 4 million Americans (1
). Chronically infected patients often develop progressive liver disease, cirrhosis, hepatic failure, and hepatocellular carcinoma (36
). There is no vaccine available against HCV, and current therapies, including the combination of alpha interferon (IFN-α) and ribavirin, are effective at viral eradication in only a small percentage of patients (26
). However, the development of more effective anti-HCV therapeutic agents has been hampered by the lack of an efficient cell culture system and an adequate animal model for HCV infection and replication.
HCV is a hepacivirus belonging to the Flaviviridae
family. The positive-sense single-stranded enveloped RNA genome is translated in an internal ribosomal entry site-dependent manner to generate a single polyprotein, which is proteolytically processed into at least nine proteins (40
). Among these proteins, the NS5A nonstructural protein is receiving increasing attention as a potential target for anti-HCV therapy. The initial interest in NS5A stemmed from the observation that mutations within a discrete region of NS5A from certain HCV genotypes, termed the IFN sensitivity-determining region, correlated with increased sensitivity to IFN treatment (14
). Although the existence of the IFN sensitivity-determining region has been rather controversial (25
), subsequent studies have shown that NS5A interacted with and inhibited protein kinase R (PKR), which is a key mediator of the host IFN antiviral response (18
). PKR exerts its effects by phosphorylating the GTP-binding eukaryotic initiation factor 2 (eIF2) (11
). The eIF2 facilitates binding of the initiator Met-tRNA
to the 40S ribosomal subunit during translation initiation. Phosphorylation of the α subunit of eIF2 (eIF2α) on Ser51 by PKR converts eIF2 into a competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, resulting in the inhibition of general cellular protein synthesis and hence virus replication. Thus, NS5A-mediated inhibition of PKR may counteract the IFN-induced translational arrest, alluding to a possible mechanism by which HCV induces or sustains resistance to IFN treatment.
More recently, we have found that NS5A binds to a Src-homology 3 (SH3) domain of Grb2 (51
). Grb2 is an adapter protein that mediates intracellular signaling by nucleating the formation of signal transduction complexes. The SH3 domains of Grb2 bind the nucleotide exchange factor Sos to trigger a series of signaling cascades in response to growth factor stimulation (10
). Stimulation with epidermal growth factor (EGF) induces the SH2 domain of Grb2 to bind to the EGF receptor, thereby recruiting Grb2 and Sos into a complex with the receptor and stimulating Sos to activate Ras. Activated Ras triggers the mitogen-activated protein kinase (MAPK) cascades, including the extracellular signal-regulated kinase (ERK) pathway (23
). ERKs phosphorylate a number of substrates, including the transcription factors E1k-1 and c-Jun, as well as the protein kinases Mnk1 and 3pK. Intriguingly, the phosphorylation of eIF4E by Mnk1 suggests a potential mechanism of translational control mediated through growth factor signaling (48
). Furthermore, the ERK pathway may play a role in IFN signaling and/or viral replication efficiency (28
), alluding to another possible mechanism of HCV resistance to IFN. In addition to NS5A, the E2 envelope protein of HCV has also been implicated as an inhibitor of PKR (54
). The exact mechanism by which E2 inhibits PKR is not known, but inhibition may be mediated through E2 sequence homology with the autophosphorylation sites of PKR. Thus, HCV likely employs multiple strategies, including a two-pronged attack on PKR that incorporates the inhibitory functions of both NS5A and E2 proteins, to evade the IFN-induced antiviral response.
The use of cell lines inducibly expressing NS5A to determine the relevance of NS5A-mediated inhibition of PKR in HCV resistance to IFN have led to conflicting results (16
). In this report, we investigated the role of NS5A in PKR inhibition in the setting of a virus infection by using an established recombinant vaccinia virus (VV)-based system. We found that expression of NS5A in this system perturbed PKR-mediated signaling cascades, including the p38 pathway, possibly resulting in opposing effects on mRNA translation. Our results provide for the first time evidence supporting the PKR inhibitory role of NS5A in virus-infected cells and highlight a potential mechanism by which HCV subverts cellular signaling pathways to favor cap-independent translation of its mRNA.