The N-terminal helix α0 of hepatitis C virus NS3 protein dictates the subcellular localization and stability of NS3/NS4A complex

The N-terminal amphipathic helix α0 of hepatitis C virus (HCV) NS3 protein is an essential structural determinant for the protein membrane association. Here, we performed functional analysis to probe the role of this helix α0 in the HCV life cycle. A point mutation M21P in this region that destroyed the helix formation disrupted the membrane association of NS3 protein and completely abolished HCV replication. Mechanistically the mutation did not affect either protease or helicase/NTPase activities of NS3, but significantly reduced the stability of NS3 protein. Furthermore, the membrane association and stability of NS3 protein can be restored by replacing the helix α0 with an amphipathic helix of the HCV NS5A protein. In summary, our data demonstrated that the amphipathic helix α0 of NS3 protein determines the proper membrane association of NS3, and this subcellular localization dictates the functional role of NS3 in the HCV life cycle.

► M21P mutation destroyed HCV NS3 helix α0 structure with intact enzymatic activities. ► M21P mutation abolished HCV replication by reducing the stability of NS3 protein. ► Membrane disassociation of NS3 resulted in the rapid protein degradation. ► Helix α0 was the primary determinant for NS3-4A membrane association and stability.