The Putative Pocket Protein Binding Site of Autographa californica Nucleopolyhedrovirus BV/ODV-C42 Is Required for Virus-Induced Nuclear Actin Polymerization
The Putative Pocket Protein Binding Site of Autographa californica Nucleopolyhedrovirus BV/ODV-C42 Is Required for Virus-Induced Nuclear Actin Polymerization
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苜蓿银纹夜蛾核多角体病毒 BV/ODV-C42 的假定口袋蛋白结合位点是病毒诱导的核肌动蛋白聚合所必需的
DOI：
10.1128/jvi.00174-10
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发表时间：
2010-08-01
影响因子：
5.4
通讯作者：
Chen, Xinwen
中科院分区：
文献类型：
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作者：
Li, Kun;Wang, Yun;Chen, Xinwen
核丝状肌动蛋白（F-actin）在鳞翅目核多角体病毒的核衣壳形态发生中起重要作用。在此之前，我们已经证明加州自噬多核多角体病毒（AcMNPV） BV/ODV-C42 （C42）通过招募P78/83参与核肌动蛋白聚合，P78/83是一种AcMNPV orf9编码的N-WASP同源蛋白，能够激活肌动蛋白相关蛋白2/3 （Arp2/3）复合物以启动肌动蛋白聚合。为了进一步研究C42在病毒诱导的肌动蛋白聚合中的作用，我们构建了C42敲除、p78/83标记有核定位信号编码序列、egfp作为报告基因、Pp10启动子调控的重组bacmid vacp78 / 83nns -gfp，并将其转染到Sf9细胞。在转染vacp78 / 83nns -gfp的细胞细胞核中，不存在聚合的f -肌动蛋白丝，而存在其他肌动蛋白聚合元件（即P78/83， g -肌动蛋白和Arp2/3复合物）。这一体内证据表明，C42除了参与招募P78/83进入细胞核外，还作为一个关键元件积极参与核肌动蛋白聚合过程。为了收集C42参与肌动蛋白聚合的体外证据，我们使用抗C42抗体来中和能够在体外启动肌动蛋白聚合的病毒核衣壳。pyrenee -actin聚合动力学和phalloidin对F-actin特异性染色均表明，与对照抗体相比，抗c42能显著降低F-actin的形成效率。此外，我们已经确定了C42上假定的口袋蛋白结合序列（PPBS），这是C42在核肌动蛋白聚合中发挥其功能所必需的。
ABSTRACT Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAcp78/83nls-gfp, with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAcp78/83nls-gfp-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.