Structural characterization of the HSP70 interaction domain of the hepatitis C viral protein NS5A.

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.