title: The NS2 protein of hepatitis C virus is a transmembrane polypeptide.
abstract: The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, 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.
mesh_terms: D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: C097010:NS2 protein, Hepatitis C virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins