Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.
Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.
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DOI：
10.1016/j.virol.2015.07.011
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发表时间：
2015-11
期刊：
影响因子：
3.7
通讯作者：
Robert J
中科院分区：
文献类型：
--
作者：
Andino Fde J;Grayfer L;Chen G;Chinchar VG;Edholm ES;Robert J
为了鉴定蛙病毒的毒力基因，我们设计了青蛙病毒3型（FV 3）敲除（KO）突变体，这些突变体缺陷于一种假定的病毒半胱天冬酶激活和募集结构域（CARD）蛋白（Δ 64 R-FV 3）和一种β-羟基类固醇脱氢酶同系物（Δ 52 L-FV 3）。与野生型（WT）FV 3相比，用Δ 64 R-或Δ 52 L-FV 3感染爪蟾蝌蚪导致显著较低的死亡率和病毒复制水平。我们进一步鉴定了这些突变体和两个早期的KO突变体，它们缺乏即刻早期18 kDa蛋白（FV 3-Δ 18 K）或截短的eIF-2α病毒同源物（FV 3-ΔvIF-2α）。所有KO突变体在非两栖类细胞系中的复制与WT-FV 3一样好，而在非洲爪蟾A6肾细胞中，ΔvCARD-、ΔvβHSD-和ΔvIF-2α-FV 3的复制显著降低。此外，Δ 64 R-和ΔvIF-2α-FV 3对干扰素比WT和Δ18-FV 3更敏感。值得注意的是，Δ 64 R-、Δ 18 K-和ΔvIF-2α-而不是Δ 52 L-FV 3比WT FV 3触发更多的凋亡。这些数据表明，vCARD（64 R）和vβ-HSD（52 L）基因有助于病毒的发病机制。
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.