Structural and Functional Characterization of Nonstructural Protein 2 forIts Role in Hepatitis C VirusAssembly

摘要

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report (Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81 ,8374 -8383 [] [] []), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectiousvirus production. We also show that serine residue 168 in NS2, implicated inthe phosphorylation and stability of this protein, is dispensable for virionformation. In addition, we determined the NMR structure of the first TMS ofNS2 and show that the N-terminal segment (amino acids 3-11) forms a putativeflexible helical element connected to a stable α-helix (amino acids12-21) that includes an absolutely conserved helix side in genotype 1b. Byusing this structure as well as the amino acid conservation as a guide for afunctional study, we determined the contribution of individual amino acidresidues in TMS1 for HCV assembly. We identified several residues that arecritical for virion formation, most notably a central glycine residue atposition 10 of TMS1. Finally, we demonstrate that mutations in NS2 blockingHCV assembly can be rescued by trans-complementation.