Methodology for the efficient generation of fluorescently tagged vaccinia virus proteins.
Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.
School of Molecular Bioscience, University of Sydney.
N Bishara
NB
Marzook
Dean J
DJ
Procter
Helena
H
Lynn
Yui
Y
Yamamoto
Jacquelyn
J
Horsington
Timothy P
TP
Newsome
D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins