Abstract
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 infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.
Highlights
12–21) that includes an absolutely conserved helix side in genotype 1b
In this study we characterized the role of NS2 for hepatitis C virus (HCV) particle production
The structure of TMS1 was probed by CD, and NMR in membrane mimetics and TMS1 residues that are critical for assembly have been identified
Summary
Sequence Analyses and Structure Predictions—Sequence analyses were performed using the website tools of the European HCV data base (euHCVdb [33]) and Network Protein Sequence Analysis (NPSA, [34]) available at the Institut de Biologie et Chimie des Proteines. To discriminate in TCID50 assays between the virus genome and the helper RNA, a 324-nt-long deletion (nt 7322–7645 of JFH1) was introduced into domain III of NS5A. The fold reduction of core release was found with the mutant selected structures were compared by pairwise root mean carrying the ubiquitin insertion (Jc1-2Ubi3) This result square deviation (r.m.s.d.) over the backbone atom coordinates indicates that the latter construct has a defect in virus pro-. Structural analyses were performed with MOLMOL ver- Kinetics of release of infectious virus was determined by sion 2.6 [55], AQUA version 3.2, and PROCHECK-NMR ver- using TCID50 assays with culture supernatants harvested 24, sion 3.5.4 [56]
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