Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

Abstract

We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFPpositive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinasedeficient (Tk À ) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk À rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.

Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression

“…S/B ratios were 76.6 for Cherry and 291.9 for Venus, consistent with the transient transfection results in Figure 1A. Reporter expression allowed for easy identification of recombinant virus during plaque purification and in pooled format, as has been noted earlier (Popov et al, 2009). This is illustrated in Figure 1D, where distinct Venus- and Cherry-expressing foci are clearly visible following low MOI co-infection and spread of LR and LV reporter viruses on BHK-21 cells.…”

“…We were interested in developing a series of fluorescent protein-based Vaccinia viruses for rapid, homogenous assays in higher throughput applications. Fluorescent protein-based reporters have been applied with success in other virus systems, and have previously been incorporated into Vaccinia for various purposes (Hansen et al, 2002; Johnson et al, 2008; Popov et al, 2009; Villa et al,; Ward and Moss, 2001). However a systematic incorporation of fluorescent reporters to query each step of the virus life-cycle has not been undertaken.…”

Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping

“…We attempted to enhance rMVA immunogenicity by increasing transgene expression, through insertion of an IRES between the ATG start codon of the transgene and the pB8 promoter, aiming to overcome the negative effects of decapping activity on the transgene. IRES elements have been used in bicistronic constructs integrated into VACV genome, in previous studies [ 22 ]. Here, the EMCV IRES was tested first in a bicistronic expression system and proved functional in expressing influenza virus proteins in a cap-independent expression.…”

A Novel System for Constructing a Recombinant Highly-Attenuated Vaccinia Virus Strain (LC16m8) Expressing Foreign Genes and Its Application for the Generation of LC16m8-Based Vaccines against Herpes Simplex Virus 2

“…There are many methodological reports using homologous DNA recombination naturally during replication for constructing recombinant VAC with a desired property, (1)(2)(3)(4)(5). Although the recombination naturally occurs, the selection process utilizing a selection marker gene with the desired phenotypic property is usually indispensable, because of the low frequency (1,2).…”