Functions of hepatitis C virus non-structural protein 2

An estimated 170 million people are infected with hepatitis C virus (HCV). 15-30% of HCV-induced chronic hepatitis progresses to cirrhosis within years to decades after infection, and 3-4% of them will develop hepatocellular carcinoma. There is no vaccine available, and current HCV therapy of pegylated interferon-! in combination with ribavirin leads to a sustained response only in about 50% of infected patients. The HCV non-structural protein NS2 (MW 23 kDa) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. The N terminal region of NS2 interacts with membranes whereas the Cterminal region, together with the N-terminal third of NS3, forms the NS2-3 protease. NS2 is not required for RNA synthesis, although cleavage at the NS2/3 junction is necessary for replication. Further, NS2 has been shown to interact with a number of viral and host proteins; it has been reported to activate transcription factors, inhibit apoptosis, and is a substrate for host kinase phosphorylation and proteosomic degradation. NS2 determinants and their respective function in the HCV life cycle were investigated. Based on the crystal structure of the post-cleavage 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 by preventing NS2/3 cleavage. Supplementary assays indicated a dimerization defect for these mutant constructs, which prevented cleavage and RNA replication. In contrast, alterations in the dimer interface and at the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity, but decreased infectious virus production. Analysis of the NS2/3 cleavage site revealed an additional function for several residues besides cleavage, notably in infectious virus production. A more comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal residue in infectious particle production. Structural data suggests that the C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the NS2 folding and disrupt potential protein-protein interactions important for infectious particle production. Further, we established an NS2- 3 interaction model based on the solved NS2pro and NS3 crystal structures and assessed the importance of proximal residues for viral propagation. We were able to show genetic interactions between the viral proteins NS2 and E1 as well as NS2 and NS3. Additionally a pull down assay of strep-tagged NS2 followed by a mass spectrometry analysis divulged the physical interactions between NS2-E2, and NS2- NS3. Numerous host cell proteins could be identified to interact with NS2, involved in various pathways such as membrane trafficking, actin/myosin interactions or actin polymerization. In this study we dissected the residues of NS2 involved in its multiple essential roles and interactions in the HCV life cycle and established NS2 as a new viable target for HCV-specific inhibitors for future anti-viral therapeutics