title: A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication.
abstract: The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Department of Virology, F-75015 Paris, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Benureau
authors.forename: Lucile
authors.initials: L
authors.lastname: Warter
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Malcolm
authors.forename: Annette
authors.initials: A
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D004261:DNA Replication; D018178:Flaviviridae Infections / Q000821:virology; D029406:GB virus B / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006526:Hepatitis C / Q000821:virology; D006525:Hepatitis, Viral, Human / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C084422:NS3 protein, hepatitis C virus; C080107:NS4B protein, flavivirus