Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly

During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein-protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.