转运所需的内体分选复合物(ESCRT)是介导膜重塑和断裂的保守蛋白质机器。在病毒感染的背景下,ESCRT-III复合物的不同成分,作为催化膜裂变的核心机械,涉及多种病毒进入,复制,和/或萌芽。然而,ESCRT-III与病毒生命周期中的病毒因子之间的相互作用,特别是对于大型包膜DNA病毒,基本上是未知的。最近,ESCRT-III组件Vps2B,Vps20,Vps24,Snf7,Vps46和Vps60被确定为进入和/或离开杆状病毒加利福尼亚多核多角体病毒(AcMNPV)。这里,我们确定了节食夜蛾的最后三个ESCRT-III成分Chm7,Ist1和Vps2A。这些蛋白质的显性阴性形式的过表达或其转录物的RNAi下调显着降低了AcMNPV的感染性出芽病毒(BV)的产生。定量PCR以及共聚焦和透射电子显微镜分析显示,在核衣壳的进出过程中,BV的内化和运输需要这些蛋白质。在受感染的Sf9细胞中,9种ESCRT-III成分分布在核膜和质膜上,除Chm7外,其他成分也位于核内环区。Y2H和BiFC分析显示,64种BV相关蛋白中有42种与单个或多个ESCRT-III成分相互作用,其中包括35种BV结构蛋白和7种非BV结构蛋白。通过进一步绘制64个BV相关蛋白的相互作用组,我们建立了ESCRT-III和参与BV进出的病毒蛋白复合物的相互作用网络。重要性从古细菌到真核生物,转运所需的内体分选复合物(ESCRT)-III复合物被许多包膜和无包膜DNA或RNA病毒劫持,以实现有效复制。然而,ESCRT-III招募机制,特别是对于大型包膜DNA病毒,仍然难以捉摸。最近,我们发现了ESCRT-III组件Vps2B,Vps20,Vps24,Snf7,Vps46和Vps60对于进入和/或离开加利福尼亚自拟多核多角体病毒的出芽病毒(BV)是必需的。这里,我们证明了其他三种ESCRT-III成分Chm7,Ist1和Vps2A在BV感染中的作用相似。通过确定感染细胞中ESCRT-III组分的亚细胞定位,并绘制9种ESCRT-III组分与64种BV相关蛋白的相互作用图,我们建立了ESCRT-III和参与BV进出的病毒蛋白复合物的相互作用网络。这些研究为理解ESCRT介导的杆状病毒复制的膜重塑机制提供了基础。
The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses\' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.