Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

Abstract

The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

“…NS2 has been implicated in virus particle assembly, although its precise role in this process is not understood (6,9,12,13,32,37,42,45). The W879 residue in NS2 is conserved in 100% of the genotype 2 and genotype 3 NS2 sequences listed in the Los Alamos HCV database.…”

Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells

“…The mouse antibodies C7-50 (anticore [28]) and 9E10 (anti-NS5A [7]), the human anti-E2 antibody CBH23 (29), and sheep anti-ADRP antiserum (30) were generous gifts from D. Moradpour (University of Lausanne), C. M. Rice (Rockefeller University), S. Foung (Stanford University), and J. McLauchlan (Glasgow University), respectively. The mouse anti-NS2 6H6 antibody and rabbit anti-NS3 4949 antiserum were previously reported (31,32) and were kindly provided by C. M. Rice and R. Bartenschlager, respectively.…”

Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

“…Samples were heated at 95°C for 10 min, centrifuged at 10,000 ϫ g for 5 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% nonfat milk and incubated with primary monoclonal antibodies to core (C7-50; Affinity Bioreagents, Golden, CO), E1 (A4 [11]; a kind gift from Jean Dubuisson), E2 (3/11 [14]; a kind gift from Jane McKeating), NS2 (6/H6 [10]; a kind gift from Charles M. Rice), NS3 (9G2; Virogen, Watertown, MA), and NS5A (9E10 [38]). Membranes were washed extensively with TBS with 1% Tween 20, incubated with secondary IgG-horseradish peroxidase (HRP) antibodies, and developed using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).…”

“…Several cell culture-adaptive mutations that increase virus yields have been mapped to NS2 (16,21,25,60,76), and mutagenesis of NS2 identified several residues that are important for infectious virus production (10,23,55,71,77). We previously identified second-site mutations in E1 (A78T), E2 (I360T), NS3 (Q221L), and NS4A (E42G) that suppressed defects in virus particle assembly caused by specific NS2 mutations (55).…”