Sehrawat Sapna, Khasa Renu, Deb Arundhati, Prajapat Surendra Kumar, Mallick Suvadip, Basu Anirban, Surjit Milan, Kalia Manjula, Vrati Sudhanshu
Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad-121001, India.
Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad-121001, India.
J Virol. 2021 May 10;95(11). doi: 10.1128/JVI.02336-20. Epub 2021 Mar 17.
Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.
宿主因素为病毒生命周期的各个方面提供关键支持。我们最近鉴定出含缬酪肽蛋白（VCP）/p97，一种具有多种细胞功能的丰富细胞ATP酶，是对日本脑炎病毒（JEV）复制很重要的宿主因素。在培养细胞中，我们使用小干扰RNA（siRNA）介导的蛋白质消耗和药理学抑制剂表明，VCP对三种黄病毒的复制至关重要：JEV、登革热病毒和西尼罗河病毒。一种美国食品药品监督管理局（FDA）批准的VCP抑制剂CB - 5083，在JEV感染的动物模型中延长了小鼠的存活时间。虽然VCP的消耗并未抑制JEV在细胞上的附着，但它延迟了衣壳降解，这可能是通过将内吞的病毒截留在网格蛋白包被小泡（CCV）中实现的。在感染早期，VCP消耗的细胞显示JEV衣壳与网格蛋白的共定位增加，并且在纯化的CCV中病毒RNA水平也更高。我们表明VCP与JEV非结构蛋白NS5相互作用，并且是病毒复制复合体的重要组成部分。主要VCP辅因子UFD - 1的消耗也显著抑制了JEV复制。因此，从机制上讲，VCP影响了JEV生命周期的两个关键步骤——核衣壳释放和RNA复制。我们的研究确定VCP是一种对黄病毒具有广泛抗病毒潜力的常见宿主因素。JEV是东南亚病毒性脑炎流行的主要原因，主要影响儿童，发病率和死亡率很高。因此，鉴定宿主因素对于合理设计作为治疗急需的抗病毒药物至关重要。在这里，我们已经确定VCP蛋白就是这样一种宿主因素。这种蛋白在细胞中高度丰富，并通过其与不同辅因子相互作用的能力参与多种功能和细胞途径。使用siRNA介导的蛋白质敲低，我们表明这种蛋白对于病毒RNA释放到细胞中以便启动复制至关重要。该蛋白在JEV复制复合体的形成中起第二个关键作用。靶向VCP的FDA批准药物在JE疾病模型中提高了小鼠存活率，这表明这可能是黄病毒感染的一个可药物化靶点。
