Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represente d by nonstructural protein 5B (NS5B), is believed to form a membrane-associ ated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membran e association of this essential viral enzyme have not been defined. By doub le label immunofluorescence analyses, NS5B was found in the endoplasmic ret iculum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 ami no acid residues were necessary and sufficient to target NS5B or a heterolo gous protein to the cytosolic side of the ER membrane. This hydrophobic dom ain is highly conserved among 269 HCV isolates analyzed and predicted to fo rm a transmembrane a-helix. Association of NS5B with the ER membrane occurr ed by a posttranslational mechanism that was ATP-independent. These feature s define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttransl ationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.