title: Modulation of replication efficacy of the hepatitis C virus replicon Con1 by site-directed mutagenesis of an NS4B aminoterminal basic leucine zipper.
abstract: The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is assumed to function as a membrane anchor and protein hub for the viral replication complex. The aim of the current work was to modulate HCV replication efficacy in the subgenomic Con1 replicon by mutations of specific sites within the aminoterminal-located basic leucine zipper (bZIP), a candidate motif for protein-protein interactions involving NS4B. Mutational sites and amino acid substitutes were determined by in-silico sequence analyses of the NS4B-bZIP motif in 357 isolates of HCV genotype 1b from the euHCVdB and LosAlamos database and consecutive analysis of conserved physico-chemical properties at bZIP specific positions. Mutants with predicted minor, medium or major reduction of replication efficacy were tested in the pFKI389neo/NS3-3'/ET plasmid replicon model. Four sites (L25, T29, V39 and W43) of crucial importance for bZIP-mediated protein interaction with predicted apolarity of respective amino acid positions were selected for mutational studies. Substitutes with physico-chemical properties matching the predicted requirements either well (T29A), moderately (L25W, V39W), or insufficiently (T29E, W43E) were associated with slightly improved, moderate and marked decreased replication efficacy, respectively. Spontaneous (T29G) and adaptive (A28G, E40G) mutations occurred in the T29E mutation isolate only and were associated with marked reduction of replication efficacy. The bZIP motif region of NS4B is crucial for RNA replication in the subgenomic Con1 replicon system. RNA replication efficacy can be modulated by site-directed mutagenesis at specific bZIP functional sites. New adaptive amino acid mutations were identified within the HCV NS4B protein.
authors.affiliation: Klinikum der Johann Wolfgang Goethe-Universität, Medizinische Klinik, Theodor-Stern-Kai, Frankfurt am Main, Germany.
authors.forename: M-W
authors.initials: MW
authors.lastname: Welker
authors.forename: S
authors.initials: S
authors.lastname: Susser
authors.forename: C
authors.initials: C
authors.lastname: Welsch
authors.forename: D
authors.initials: D
authors.lastname: Perner
authors.forename: C
authors.initials: C
authors.lastname: Füller
authors.forename: B
authors.initials: B
authors.lastname: Kronenberger
authors.forename: E
authors.initials: E
authors.lastname: Herrmann
authors.forename: S
authors.initials: S
authors.lastname: Zeuzem
authors.forename: C
authors.initials: C
authors.lastname: Sarrazin
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D057809:HEK293 Cells; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016350:Leucine Zippers; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
chemical_list: C080107:NS4B protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins