缺失宿主Vta1蛋白的MIT结构域对杆状病毒AcMNPV复制的影响

Effects of deletion of MIT domains of host Vta1 on replication of Autographa californica multiple nucleopolyhedrovirus

摘要:

[目的]克隆草地贪夜蛾（Spodoptera frugiperda）Vta1基因，检测Vta1在苜蓿银纹夜蛾核多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）复制中的作用。[方法]利用反转录-PCR与PCR方法筛选草地贪夜蛾Vta1基因及缺失Vta1 N-端MIT结构域的突变体并构建其瞬时表达质粒，通过转染Sf9细胞检测表达；构建Vta1及其突变体的双分子荧光互补表达质粒，并通过瞬时转染检测其与Vps4及ESCRT-III亚基Vps46与Vps60的相互作用；共转染gp64与Vta1及其突变体瞬时表达质粒，检测瞬时表达Vta1突变体对AcMNPV出芽型病毒产量及病毒基因启动子指导报告基因表达的影响。[结果]获得了草地贪夜蛾Vta1基因。氨基酸序列相似性分析表明，昆虫、酵母与人类Vta1同源蛋白的相似性分别约为20%与50%。Western blotting分析表明GFP标签的Vta1及其突变体均能在瞬时转染的Sf9细胞中表达。双分子荧光互补分析发现，缺失第1个或第2个MIT结构域显著降低Vta1突变体与Vps4、Vps46或Vps60的相互作用。此外，瞬时表达Vta1突变体显著降低了AcMNPV感染性出芽型病毒的产量，但并未影响AcMNPV ie1基因早期启动子和p6.9基因晚期启动子指导的LacZ和GUS报告基因的表达。[结论]Vta1可能参与杆状病毒AcMNPV子代病毒粒子的组装和/或出芽释放过程。

Abstract:

[Objective] To isolate Vta1 of Spodoptera frugiperda and to detect the requirement of Vta1 in replication of Autographa californica multiple nucleopolyhedrovirus(AcMNPV).[Methods] Vta1 was isolated from Sf9 cells using reverse-transcription PCR.Two mutations of Vta1,which removed the first or second microtubule-interacting and transport domain (MIT) were transiently expressed.The interaction of Vta1 and its mutants with Vps4,Vps46 and Vps60 was detected with bimolecular fluorescence complementation (BiFC).Using a viral complementation assay,the effect of Vta1 mutants on replication of AcMNPV was determined.[Results] We obtained Vta1 of S.frugiperda.The amino acid identities between Vta1 of insect and yeast or between Vta1 of insect and human are about 20% or 50%.Western blotting analysis showed GFP-tagged Vta1 and its mutants were expressed in Sf9 cells.BiFC analysis revealed that deletion of MIT1 or MIT2 significantly reduced the interaction of Vta1 mutants with Vps4,Vps46 or Vps60.Overexpression of Vta1 mutants significantly decreased the infectious AcMNPV budded virions production but had no effect on the expression of the reporter genes LacZ and GUS,which separately controlled by AcMNPV ie1 and p6.9 early or late promoter.[Conclusion] Vta1 might be involved in assembly and/or budding of progeny virions of AcMNPV.