Guan Zhanwen, Zhong Ling, Li Chunyan, Wu Wenbi, Yuan Meijin, Yang Kai
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou, China.
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou, China
J Virol. 2016 Mar 28;90(8):4115-4126. doi: 10.1128/JVI.02885-15. Print 2016 Apr.
Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly.
Baculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.
杆状病毒DNA在感染细胞核内的一个位点（称为病毒发生基质）合成并插入预先形成的衣壳中，形成核衣壳。苜蓿银纹夜蛾多核多角体病毒（AcMNPV）的核衣壳组装需要主要衣壳蛋白VP39和九种次要衣壳蛋白，包括VP1054。然而，VP1054如何参与核衣壳组装仍不清楚。在本研究中，缺失了编码VP1054的基因（ac54）以产生ac54基因敲除的AcMNPV（vAc54KO）。在vAc54KO转染的细胞中，核衣壳组装被破坏，导致形成异常拉长的衣壳结构。有趣的是，与用缺乏其他次要衣壳蛋白的AcMNPV突变体转染的细胞不同，在那些细胞中衣壳结构分布在病毒发生基质内，ac54缺失导致衣壳结构和VP39在细胞核周边的独特定位。在用缺乏BV/ODV-C42的AcMNPV转染的细胞或用细胞松弛素处理的AcMNPV感染的细胞中也观察到了衣壳结构分布模式的改变。BV/ODV-C42与PP78/83一起已被证明可促进核丝状肌动蛋白（F-肌动蛋白）形成，这是核衣壳组装的另一个必要条件。用鬼笔环肽进行的免疫荧光显示，核F-肌动蛋白的形成和分布不受ac54缺失的影响。然而，免疫电子显微镜显示，在没有VP1054的情况下，BV/ODV-C42、PP78/83和38K未能整合到衣壳结构中，免疫沉淀进一步证明，在瞬时表达试验中，VP1054与BV/ODV-C42和VP80相互作用，但不与VP39相互作用。我们的研究结果表明，VP1054在核衣壳组装过程之前将衣壳蛋白运输到核衣壳组装位点中发挥重要作用。
杆状病毒是大型DNA病毒，其复制发生在宿主细胞核内。衣壳定位到衣壳组装位点需要病毒诱导的核F-肌动蛋白；核F-肌动蛋白形成的抑制导致衣壳结构保留在细胞核周边。在本文中，我们注意到次要衣壳蛋白VP1054对于衣壳结构、主要衣壳蛋白VP39和次要衣壳蛋白38K定位到衣壳组装位点至关重要。此外，VP1054对于核F-肌动蛋白因子BV/ODV-C42和PP78/83正确靶向衣壳成熟至关重要。然而，核F-肌动蛋白的形成和分布不受VP1054缺失的影响。我们进一步揭示VP1054与BV/ODV-C42和一种衣壳运输相关蛋白VP80相互作用。综上所述，我们的研究结果表明VP1054在衣壳蛋白运输途径中发挥独特作用。
