title: The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo.
abstract: Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, California 90033, USA.
authors.forename: Ki Jeong
authors.initials: KJ
authors.lastname: Lee
authors.forename: Jinah
authors.initials: J
authors.lastname: Choi
authors.forename: Jing-Hsiung
authors.initials: JH
authors.lastname: Ou
authors.forename: Michael M C
authors.initials: MM
authors.lastname: Lai
mesh_terms: D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D051943:Antennapedia Homeodomain Protein; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase