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Flexible and rapid construction of viral chimeras applied to hepatitis C virus

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...

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Autores principales: McClure, C. Patrick, Urbanowicz, Richard A., King, Barnabas J., Cano-Crespo, Sara, Tarr, Alexander W., Ball, Jonathan K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Microbiology Society 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042125/
https://www.ncbi.nlm.nih.gov/pubmed/27329374
http://dx.doi.org/10.1099/jgv.0.000530
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author McClure, C. Patrick
Urbanowicz, Richard A.
King, Barnabas J.
Cano-Crespo, Sara
Tarr, Alexander W.
Ball, Jonathan K.
author_facet McClure, C. Patrick
Urbanowicz, Richard A.
King, Barnabas J.
Cano-Crespo, Sara
Tarr, Alexander W.
Ball, Jonathan K.
author_sort McClure, C. Patrick
collection PubMed
description 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.
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spelling pubmed-50421252017-09-01 Flexible and rapid construction of viral chimeras applied to hepatitis C virus McClure, C. Patrick Urbanowicz, Richard A. King, Barnabas J. Cano-Crespo, Sara Tarr, Alexander W. Ball, Jonathan K. J Gen Virol Short Communication 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. Microbiology Society 2016-09 2016-09 /pmc/articles/PMC5042125/ /pubmed/27329374 http://dx.doi.org/10.1099/jgv.0.000530 Text en © 2016 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
spellingShingle Short Communication
McClure, C. Patrick
Urbanowicz, Richard A.
King, Barnabas J.
Cano-Crespo, Sara
Tarr, Alexander W.
Ball, Jonathan K.
Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title_full Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title_fullStr Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title_full_unstemmed Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title_short Flexible and rapid construction of viral chimeras applied to hepatitis C virus
title_sort flexible and rapid construction of viral chimeras applied to hepatitis c virus
topic Short Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042125/
https://www.ncbi.nlm.nih.gov/pubmed/27329374
http://dx.doi.org/10.1099/jgv.0.000530
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