title: Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.
abstract: A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Fournier
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.
authors.forename: François
authors.initials: F
authors.lastname: Helle
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Descamps
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Morel
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: François
authors.affiliation: Laboratory of Cancer Epigenetics, Université Libre de Bruxelles, Faculty of Medicine, Brussels, Belgium.|EA4294, Jules Verne University of Picardy, Amiens, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Dedeurwaerder
authors.affiliation: INSERM U1019, CNRS UMR 8204, Center for Infection and Immunity of Lille, Institut de Biologie de Lille, Lille, France.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.affiliation: EA4294, Jules Verne University of Picardy, Amiens, France.|Virology Department, Amiens University Hospital, South Hospital, Amiens, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Castelain
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D005816:Genetic Complementation Test; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon; D059386:Reverse Genetics; D012641:Selection, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D019065:Virus Assembly; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase