Hepatitis C virus NS5B and host cyclophilin A share a common binding site on NS5A.

ABSTRACT: Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2 and NS5A-D3), CypA, and NS5B(?21). We show that three regions of NS5A-D2 (residues 250-262 (region A), 274-287 (region B), and 306-333 (region C)) interact with NS5B(?21), whereas NS5A-D3 does not. We show that both NS5B(?21) and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has been detected by NMR spectroscopy between HCV NS5B(?21) and host CypA. We show that cyclosporine A added to a sample containing NS5B(?21), NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B(?21). A high quality heteronuclear NMR spectrum of HCV NS5B(?21) has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight the potential of using NMR of NS5B(?21) as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins, inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and CypA, three essentials proteins for HCV replication.

SUBMITTER: Rosnoblet C 

PROVIDER: S-EPMC3531740 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature