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.
Highlights
We were able to construct gene-shuttle cassettes for generation of cell cultureinfectious JFH-1-based chimeras containing genotype 1–3 E1E2 genes
Using this method we show that E1E2 clones that were not able to support cell entry in the hepatitis C virus (HCV) pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system
Whilst the chosen parental HCV genome presented a fortuitous mutagenesis option, the restriction endonuclease (RE) digest mapping described above could be applied to select a novel non-cutting site, even the same FseI as used in the E1E2 region as this would be lost in the first DNA assembly cloning reaction
Summary
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. Reverse genetics systems developed to study host– pathogen interaction are typically constructed by PCR amplification and gene or genome cloning into plasmid vectors (Das et al, 2013; deCamp et al, 2014; Dutta et al, 2013; Imhof & Simmonds, 2010, 2011; Lindenbach et al, 2005; Pietschmann et al, 2006; Urbanowicz et al, 2015).
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