Complex formation between hepatitis C virus NS2 and NS3 proteins

Hepatitis C virus (HCV) NS2 and NS3 proteins as well as the NS3 protease cofactor NS4A are essential for the replication of the virus. The presence of in vivo heterodimeric complex between HCV NS2 and NS3 has been suggested by biochemical studies. Detailed characterization of the interactions between these viral proteins is of great importance for better understanding their role in viral replication cycle and represents attractive target for antiviral agents. In this study, we demonstrated in vivo interactions between HCV NS2 and NS3 proteins using an epitope tagging technique. For this purpose NS2, NS3 and NS4A were expressed in fusion with two different tags in Cos7 cells. Immunofluorescence analysis and co-immunoprecipitation with tag-specific antibodies revealed the existence of biologically important NS3/NS4A and NS3/NS2 complexes. Similar complexes were detected also in Huh7 cells infected with Semliki Forest virus vectors expressing NS2 and NS3 or NS23 precusor polyprotein. The formation of complex between NS2 and NS3 was found not to depend on whether the proteins were expressed individually or in form of common precursor. This observation suggests the existence of direct interaction between these two proteins that may have importance for the formation of the whole HCV replication complex.