We previously identified the NS5A/HSP70 binding site to be a hairpin moiety at C-terminus of NS5A domain I and showed a corresponding cyclized polyarginine-tagged synthetic peptide (HCV4) significantly blocks virus production. Here, sequence comparison confirmed five residues to be conserved. Based on NS5A domain I crystal structure, Phe171, Val173, and Tyr178 were predicted to form the binding interface. Substitution of Phe171 and Val173 with more hydrophobic unusual amino acids improved peptide antiviral activity and HSP70 binding, while similar substitutions at Tyr178 had a negative effect. Substitution of non-conserved residues with arginines maintained antiviral activity and HSP70 binding and dispensed with polyarginine tag for cellular entry. Peptide cyclization improved antiviral activity and HSP70 binding. The cyclic retro-inverso analog displayed the best antiviral properties. FTIR spectroscopy confirmed a secondary structure consisting of an N-terminal beta-sheet followed by a turn and a C-terminal beta-sheet. These peptides constitute a new class of anti-HCV compounds.

Patients with chronic hepatitis C virus (HCV) infection risk complications of cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Previously, our proteomic examination of hepatocytes carrying a HCV-replicon revealed that deregulation of cytoskeletal dynamics may be a potential mechanism of viral-induced HCC growth. Here, we demonstrate the effect of HCV replication on the microtubule regulator stathmin (STMN1) in HCC cells. We further explore how the altered activity or synthesis of stathmin affects cellular proliferation and sensitivity to apoptosis in control HCC cells (Huh7.5) and experimental HCV-replicon harboring HCC cells (R-Huh7.5). The HCV-replicon harboring HCC cells (R-Huh 7.5) lack viral structural genes/proteins for acute infectivity and thus is the standard model for in vitro chronic infection study. Knockdown of endogenous stathmin reduced sensitivity to apoptosis in replicon cells. Meanwhile, constitutively active stathmin increased sensitivity to apoptosis in replicon cells. In addition, overexpression of constitutively active stathmin reduced cell proliferation in both control and replicon cells. These findings implicate, for the first time, a novel role for stathmin in viral replication-related apoptosis. Stathmin's potential role in HCV replication and HCC make it a candidate for the future study of viral-induced malignancies.