Improved Knockout Methodology Reveals That Frog Virus 3 Mutants Lacking either the 18K Immediate-Early Gene or the Truncated vIF-2 α Gene Are Defective for Replication and Growth In Vivo

Abstract

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.

Prominent Amphibian (Xenopus laevis) Tadpole Type III Interferon Response to the Frog Virus 3 Ranavirus

“…The production and characterization of recombinant FV3 bearing site-specific deletions of the 18K (ORF 82R) and vIF-2 genes has been previously described (27), while the characterization of ⌬vCARD FV3 (where vCARD is viral caspase activation and recruitment domain; open reading frame 64R, nucleotides 75529 to 75816) is presently in review as a separate manuscript. The two recombinant FV3s were generated by homologous recombination; target genes (FV3 genomic location for ORF 52L, nucleotides 57481 to 58548; ORF 64R, 75529 to 75816) were PCR amplified from the FV3 genome and cloned into right (restriction sites XhoI and ClaI) and left (restriction sites SacI and SpeI) sides of cassettes bearing a puromycin (Puro) resistance gene fused with the coding sequence of enhanced green fluorescent protein (EGFP) under the control of FV3 immediate early (IE) 18K gene promoter (18Kprom-Puro-EGFP cassette).…”

Virus genomes and virus-host interactions in aquaculture animals

“…Besides the controllable recombinant virus technique mentioned above [77], gene knockout methodology has been also used to investigate gene function in iridoviruses [78]. Expression inhibition of a structural protein gene and RNA polymerase gene by morpholino knockdown or gene-specific silencing has been observed to cause a significant reduction in the yield of virus progeny [79,80].…”

Rana grylio virus TK and DUT gene locus could be simultaneously used for foreign gene expression

“…Briefly, selection markers were inserted into the locus of a target gene through homologous recombination. With the help of selection markers, like resistance to drugs or fluorescence, knockout mutants were generated through successively purification (Chen et al, 2011;Cheng et al, 2014). Finally, gene functions were inferred by changes in viral replication and virulence.…”

“…Among them, rBIV and ATV 57R used neomycin resistance gene for selection (Pallister et al, 2007;Jancovich and Jacobs, 2011). FV3-vIF-2␣, FV3-18K, 64R-FV3, 52L-FV3 and ESV DHFR were generated basing on fluorescence marker coupled with resistance gene (Chen et al, 2011;Andino et al, 2015;Martín et al, 2015). The other six, including 67R-RGV, TK-RGV, i53R-RGV-lacIO, i2L-RGV-lacIO, EGFP-STIV and rCIV-157L-gfp, were purified by successive rounds of plaque isolation using fluorescence selection (He et al, 2012;Huang et al, 2014;Ozgen et al, 2014;He et al, 2013;Huang et al, 2011;He et al, 2014).…”