Effects of deletion of MIT domains of host Vta1 on replication of Autographa californica multiple nucleopolyhedrovirus
Effects of deletion of MIT domains of host Vta1 on replication of Autographa californica multiple nucleopolyhedrovirus
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DOI：
10.13343/j.cnki.wsxb.20180112
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Li, Zhaofei
中科院分区：
文献类型：
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作者：
Sun, Yu;Li, Yuying;Li, Zhaofei
[目的]分离草地贪夜蛾Vta1，并检测苜蓿银纹夜蛾多重核多角体病毒（AcMNPV）复制对Vta1的需求。 [方法]采用反转录PCR从Sf9细胞中分离Vta1。 Vta1 的两个突变瞬时表达，它们去除了第一或第二微管相互作用和转运结构域 (MIT)。通过双分子荧光互补 (BiFC) 检测 Vta1 及其突变体与 Vps4、Vps46 和 Vps60 的相互作用。使用病毒互补测定，确定了 Vtal 突变体对 AcMNPV 复制的影响。 [结果]获得了草地贪夜蛾的Vta1。昆虫和酵母的Vta1之间或昆虫和人类的Vtal之间的氨基酸同一性约为20％或50％。 Western blotting 分析显示 GFP 标记的 Vta1 及其突变体在 Sf9 细胞中表达。 BiFC分析表明，MIT1或MIT2的缺失显着降低了Vtal突变体与Vps4、Vps46或Vps60的相互作用。 Vta1突变体的过表达显着降低了感染性AcMNPV出芽病毒粒子的产生，但对报告基因LacZ和GUS的表达没有影响，这两个基因分别由AcMNPV ie1和p6.9早期或晚期启动子控制。 [结论]Vta1可能参与AcMNPV子代病毒粒子的组装和/或出芽。
[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 Vtal 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 Vtal 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 Vtal 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.