Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus
Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus
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DOI：
10.1128/jvi.02912-15
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发表时间：
2016-04-01
影响因子：
5.4
通讯作者：
Theilmann, David A.
中科院分区：
文献类型：
--
作者：
Biswas, Siddhartha;Blissard, Gary W.;Theilmann, David A.
苜蓿银纹夜蛾多核多角体病毒（AcMNPV）的核衣壳从细胞核进入质膜，导致出芽病毒（BV）形成的机制尚不清楚。AC 141是BV排出所需的核衣壳相关蛋白，此前已被证明与β-微管蛋白相关。此外，AC 141和VP 39先前通过荧光共振能量转移荧光寿命成像显示直接与果蝇驱动蛋白-1轻链（KLC）的tetratricopeptide repeat（TPR）结构域相互作用。这些结果表明，微管运输系统可能参与杆状病毒核衣壳的出口和BV的形成。在这项研究中，我们研究了鳞翅目微管运输的作用，采用免疫共沉淀，共定位，酵母双杂交，和小干扰RNA（siRNA）分析。我们发现，核衣壳AC 141协会与鳞翅目粉纹夜蛾尼KLC和驱动蛋白-1重链（KHC）的免疫共沉淀和共定位。驱动蛋白-1，AC 141和微管主要共定位于质膜。此外，核壳蛋白VP 39、FP 25和BV/ODV-C42也与T. ni KLC。直接分析了T.通过siRNA下调KLC而抑制N1驱动蛋白-1，导致BV产生显著减少。用与mCherry荧光蛋白的三个拷贝融合的VP 39标记的核衣壳也与微管共定位。酵母双杂交分析表明，驱动蛋白-1和AC 141或VP 39之间没有直接相互作用的证据，这表明可能需要其他核衣壳蛋白或衔接蛋白。这些结果进一步支持AcMNPV BV形成需要微管转运的结论。
The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with beta-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.