title: Hepatitis C virus polymerase-polymerase contact interface: significance for virus replication and antiviral design.
abstract: The hepatitis C virus (HCV) replicates its genome in replication complexes located in micro-vesicles derived from endoplasmic reticulum. The composition of these replication complexes indicates that proteins, both viral and cellular in origin, are at high concentrations. Under these conditions, protein-protein interactions must occur although their role in the replication pathways is unknown. HCV RNA-dependent RNA-polymerase (NS5B) initiates RNA synthesis in these vesicles by a de novo (DN) mechanism. After initiation, newly synthesized dsRNA could induce conformational changes that direct the transition from an initiating complex into a processive elongation complex. In this report, we analyze the role played by NS5B-NS5B intermolecular interactions controlling these conformational rearrangements. Based on NS5B protein-protein docking and molecular dynamics simulations, we constructed mutants of residues predicted to be involved in protein-protein interactions. Changes at these positions induced severe defects in both the activity of the enzyme and the replication of a subgenomic replicon. Thus, mutations at the interaction surface decreased both DN synthesis initiation and processive elongation activities. Based on this analysis, we define at an atomic level an NS5B homomeric interaction model that connects the T-helix in the thumb subdomain of one monomer, with the F-helix of the fingers subdomain in other monomer. Knowing the molecular determinants involved in viral replication could be helpful to delineate new and powerful antiviral strategies.
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain.
authors.forename: Alberto José
authors.initials: AJ
authors.lastname: López-Jiménez
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Pharmacy, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Clemente-Casares
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Medicine, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Sabariegos
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Llanos-Valero
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain.
authors.forename: Itxaso
authors.initials: I
authors.lastname: Bellón-Echeverría
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche 03202, Spain.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Encinar
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, The State University of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, United States.
authors.forename: Neerja
authors.initials: N
authors.lastname: Kaushik-Basu
authors.affiliation: Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, K.U. Leuven, Belgium.
authors.forename: Mathy
authors.initials: M
authors.lastname: Froeyen
authors.affiliation: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Pharmacy, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain; Unidad de Biomedicina, CSIC-UCLM, Spain. Electronic address: Antonio.Mas@uclm.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Mas
mesh_terms: D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism*; D015195:Drug Design; D016174:Hepacivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D050505:Mutant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Antiviral drugs; HCV; NS5B; Protein–protein interactions