THE NS2 PROTEIN OF HEPATITIS-C VIRUS IS A TRANSMEMBRANE POLYPEPTIDE

The NS2 protein of hepatitis C virus (HCV) is released from its polypr otein precursor by two proteolytic cleavages. The N terminus of this p rotein is separated from the E2/p7 polypeptide by a cleavage thought t o be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize th e biogenesis of NS2 encoded by the BK strain of HCV, we have defined t he minimal region of the polyprotein required for efficient cleavage a t the NS2-3 site and analyzed the interaction of the mature polypeptid e with the membrane of the endoplasmic reticulum (ER). We have observe d that although cleavage can occur in vitro in the absence of microsom al membranes, synthesis of the polyprotein precursor in the presence o f membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing v aries among different HCV strains and that host proteins located on th e ER membrane, and in particular the signal recognition particle recep tor, are required to sustain efficient proteolysis. By means of sedime ntation analysis, protease protection assay, and site-directed mutagen esis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.