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(Δ21). 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(Δ21), whereas NS5A-D3 does not. We show that both NS5B(Δ21) 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(Δ21) and host CypA. We show that cyclosporine A added to a sample containing NS5B(Δ21), NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B(Δ21). A high quality heteronuclear NMR spectrum of HCV NS5B(Δ21) 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(Δ21) 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.
Highlights
hepatitis C virus (HCV) replication requires the interaction of the viral polymerase Nonstructural protein 5B (NS5B) with both viral and host proteins
We report NMR spectroscopy analyses at a per residue level of the molecular interactions between NS5A domains 2 and 3, NS5B, and cyclophilin A (CypA). We show that both HCV NS5B and host CypA share a common binding site on NS5A and use NMR of the NS5B enzyme to get a first localization of the NS5A-binding site on NS5B
Interaction of NS5B with NS5A—HCV NS5B is the central enzyme for HCV replication, as it carries the RNA-dependent RNA polymerase activity (8 –10)
Summary
HCV replication requires the interaction of the viral polymerase NS5B with both viral and host proteins. HCV RNA replication occurs in a replication complex that contains endoplasmic reticulum-derived membrane(s), NS3 to NS5B viral proteins [25], and viral RNA as well as several host factors such as hVAP-33 [26], VAP-B [27], and cyclophilin A (CypA) [28]. The latter is a major host factor required for HCV replication [29]. We show that both HCV NS5B and host CypA share a common binding site on NS5A and use NMR of the NS5B enzyme to get a first localization of the NS5A-binding site on NS5B
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