Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Abstract

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.

Divergent roles of β‐ and γ‐actin isoforms during spread of vaccinia virus

“…Wedlich-Soldner, 2008), and when bound to GFP enables visualization of the F-actin cytoskeleton in live cells. Lifeact-GFP was cloned and inserted between VACV genes C10L and C11R by homologous recombination as previously described (Marzook et al, 2014).…”

“…Lifeact is a 17‐amino acid peptide that is used to visualize actin (Riedl, Crevenna, Kessenbrock, Yu, Neukirchen, Bista, … Wedlich‐Soldner, ), and when bound to GFP enables visualization of the F‐actin cytoskeleton in live cells. Lifeact‐GFP was cloned and inserted between VACV genes C10L and C11R by homologous recombination as previously described (Marzook et al, ).…”

Phototracking Vaccinia Virus Transport Reveals Dynamics of Cytoplasmic Dispersal and a Requirement for A36R and F12L for Exit from the Site of Wrapping

“…A slightly different modular approach was used for construction of pF13L-Dendra2. Using the F13L targeting vector [ 28 ], the GFP open reading frame was replaced with Dendra2 following the amplification of Dendra2-N vector with primer pair den_3/den_4 (see Table 1 ).…”

“…Plaques able to grow under GPT selection that displayed red fluorescence imaged with the TxRed filter of an Olympus BX51 Microscope were picked and purified three times by plaque assay with GPT selection throughout. These intermediate viruses were then allowed to intra-genomically recombine in a GPT selection negative infection, producing viruses which do not encode selection genes [ 28 ]. 24 hpi virus was collected and a plaque assay was performed to isolate the desired fluorescent recombinant which displayed green fluorescence imaged with a FITC filter.…”

Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

“…The presence of the protein encoded by gpt allows the recombinant viruses to grow in the presence of mycophenolic acid, xanthine and hypoxanthine [ 31 ]. Subsequently, after this first metabolic selection, a second recombination event must occur to eliminate the selection marker, maintaining the mutation introduced into the VACV genome ( Figure 3 ) [ 32 ]. In contrast to the method described above, in the TDS technique the marker should not be flanked by homologous regions of the VACV genome [ 30 ].…”