title: Flexible and rapid construction of viral chimeras applied to hepatitis C virus.
abstract: A novel and broadly applicable strategy combining site-directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using hepatitis C virus (HCV) as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single, site-directed mutagenesis reaction and digested to receive PCR-amplified virus envelope genes by In-Fusion cloning. Using this method, we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses.
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: C Patrick
authors.initials: CP
authors.lastname: McClure
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Urbanowicz
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Barnabas J
authors.initials: BJ
authors.lastname: King
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Cano-Crespo
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tarr
authors.affiliation: School of Life Sciences and NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Ball
mesh_terms: D005818:Genetic Engineering / Q000379:methods*; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D014759:Viral Envelope Proteins