Interaction between Nonstructural Proteins NS4B and NS5A Is Essential for Proper NS5A Localization and Hepatitis C Virus RNA Replication

The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions. IMPORTANCE Information about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes.