Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane
Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane
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DOI：
10.1128/jvi.00215-22
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发表时间：
2022-05-24
影响因子：
5.4
通讯作者：
Blissard，Gary W.
中科院分区：
文献类型：
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作者：
Hodgson，Jeffrey J.;Buchon，Nicolas;Blissard，Gary W.
杆状病毒囊膜蛋白GP64是芽生病毒的重要组成部分，是有效组装病毒粒子所必需的。关于GP64在细胞内向质膜的运输，人们对此知之甚少，在质膜上，它在出口过程中被合并到萌芽中的病毒粒子中。为了确定参与将GP64运送到质膜的宿主蛋白和潜在的细胞转运途径，我们开发并鉴定了一个稳定的、可诱导表达AcMNPV GP64蛋白的果蝇细胞系，并将该细胞系与囊泡蛋白转运途径基因的靶向RNA干扰(RNAi)筛选相结合。在屏幕上的37个初始点击中，我们验证并检查了6个宿主基因，这些基因对于将GP64运输到细胞表面非常重要。验证的命中包括Rab GTP酶Rab1和Rab4、笼状蛋白重链、笼状蛋白适配器蛋白基因AP-1-2β和AP-2μ以及Snap29。两个基因敲除(Rab5和Exo84)导致质膜上GP64的大幅增加(高达2.5倍)。我们发现，少量的GP64在外体中从细胞中释放出来，细胞表面的GP64的一部分被内吞，这表明循环有助于将GP64保持在细胞表面。重要信息虽然关于病毒包膜蛋白在哺乳动物细胞中的运输已知很多，但对昆虫细胞中的这一过程知之甚少。为了开始了解昆虫病毒包膜蛋白的运输需要哪些因子和途径，我们设计了一个黑腹果蝇细胞系，并实施了RNAi筛选，以确定帮助将模型杆状病毒包膜蛋白(GP64)运输到细胞表面的细胞蛋白。为此，我们开发了一个实验系统，利用果蝇可用的大量工具，并进行有针对性的RNAi筛选，以确定参与GP64运输到细胞表面的细胞蛋白。由于病毒包膜蛋白通常是产生传染性后代病毒粒子的关键，这些研究为了解致病昆虫病毒(杆状病毒)或昆虫媒介病毒(例如黄病毒、甲病毒)如何从中肠等组织的细胞中渗出以实现全身病毒感染奠定了基础。
The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stableDrosophilacell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPasesRab1andRab4,Clathrin heavy chain, clathrin adaptor protein genesAP-1-2βandAP-2μ, andSnap29. Two gene knockdowns (Rab5andExo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface.IMPORTANCEWhile much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available forDrosophilaand performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection.