Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes

Abstract

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18 kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α–FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not the Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

Xenopus-FV3 host-pathogen interactions and immune evasion

“…Reminiscent to what was observed in vitro using the A6 kidney cell line (Andino Fde et al, 2015), Δ18K-FV3 had the tendency to exacerbate tadpole IFN responses. In PLs, although high individual variation prevented to reach statistical significance, there was a trend toward higher expression of type I and type II IFN genes at 2 dpi in Δ18K-compared to WT-FV3 infected tadpoles (Suppl.…”

“…However, the absence of sequence similarity with any eukaryotic or prokaryotic genes compared to the high degree of conservation of this gene within the ranavirus genus suggests a biologically important species-specific role of 18K molecules. Based on our previous studies with a Δ18K-FV3 KO mutant, we postulate that 18K play a role in immune evasion by interfering with the IFN response (Andino Fde et al, 2015). We further examined the ability of Δ18K-FV3 to infect tadpole PLs, replicate in kidney and disseminate into the brain.…”

“…Using the technique described above we generated FV3 knock-out (KO) recombinants for 64R and 52L (Andino Fde et al, 2015). Because of its robust immediate early expression pattern and dispensable role for productive infection in cell culture (Cheng et al, 2014; Sample et al, 2007), the FV3 ICP18 or 18K (82R) gene was also selected as a virulence gene candidate and knocked out (Chen et al, 2011).…”

“…While deficiency of each of these viral genes results in different degrees of attenuated viral replication in X. laevis hosts, closer examination in vitro indicates distinct, albeit not fully defined, functions. Infection studies of A6 kidney cells suggest that the CARD-like molecule encoded by 64R interferes with IFN-induced apoptosis, whereas 18K may indirectly alter IFN responses by regulating other viral genes (Andino Fde et al, 2015). The role of the βHSD-like molecule encoded by 52L has remained elusive to date.…”

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

“…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).…”