The role of the NS2 protein in the Hepatitis C virus life-cycle

Hepatitis C virus (HCV) is a small, enveloped RNA virus, classified in the Flaviviridae family, Hepacivirus genus. HCV encompasses a positive sense single stranded RNA (+ssRNA) of ~9.6 kb that encodes a polyprotein of about 3000 amino acids (a.a.). Cellular and viral proteases co- and post-translationally cleave the polyprotein into at least 10 structural (core, E1 and E2) and non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. NS2 is a hydrophobic protein containing 3 transmembrane domains (TMDs) at the N-terminus and a cysteine protease at the C-terminus. So far, only limited data have been published on the function of this difficult to study protein and the exact roles of NS2 in the HCV life-cycle are not fully understood. In this study, to investigate the role of the N-terminus of NS2 in the HCV life-cycle, based on computational modeling, two NS2 deletion mutants were generated and examined. Data showed that JFH1-∆NT-NS2, lacking TMDs 1 and 2 of NS2, but not JFH1-∆FL-NS2, with deletion of the entire NS2 sequence, was replication competent and the replication level of this mutant was comparable to the JFH1-wild type (wt). However, this mutant failed to secrete infectious virions into the supernatant. In addition, when the cells were infected with cleared cell lysate from JFH1-∆NT-NS2 RNA transfected cells no intra-cellular infectivity was detected, suggesting that the first two TMDs of NS2 play a critical role in HCV morphogenesis. Trans-complementation of the JFH1-∆NT-NS2 mutant with either a helper virus (i.e. JFH1-∆E1-E2, lacking the E1 and E2 genes), or over-expression of recombinant Myc-tagged rescued the infectivity of JFH1-∆NT-NS2, showing that NS2 can be successfully supplied in trans. Imaging studies demonstrated that mature Myc-NS2 was expressed and localized to the endoplasmic reticulum (ER). The Huh-7.5.1 cell line (Huh-7 derived) was shown to be less permissive for infectious virus production although it was more permissive for HCV RNA replication. Huh-7.5.1 had all elements required for HCV entry but apparently lacks key element(s) or pathway(s) important for HCV morphogenesis. Using computational modeling, 22 residues at the NS2Pro dimer interface were identified. Based on the free binding energy, the top five ranked in silico mutations (V162A, M170A, I175A, D186A and I201A) were selected for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0, 3.2, 3.0, 2.8 and 1.5-fold increase, respectively in the monomer:dimer ratio compared to wt, confirming a role in homodimer stability. JFH1 mutants expressing the M170A, I175A, D186A or I201A mutations adversely affected replication of the viral genome. The greatest decrease was noted for the I201A and M170A (conferring a 100-fold decrease in replication compared to wt) whereas the replication of the I175A and D186A mutants were 10-fold lower than that of wt. In contrast the V162A mutant demonstrated no replication defect. An infectivity experiment revealed that apart from V162A, the mutants showed 100-1000-fold decrease in infectivity compared to wt. These results identified residues at the NS2Pro dimer interface that modulate NS2Pro homodimerization and demonstrated that abrogation of NS2Pro homodimerization results in defects in HCV replication and infectious virus release.