Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes

- Generation of new FV3 knockout mutants identifies two virulence genes vCARD (64R) and vβ-HSD (52L).
- Δ64R- and Δ52L-FV3 induce less mortality and replicate more poorly in Xenopus tadpoles than WT-FV3.
- Δ64R- and ΔvIF-2α-FV3 are more sensitive to interferon than WT and Δ18-FV3.
- Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 trigger more apoptosis than WT FV3.
- vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

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 Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.