Defining the functional interface between the ER and flaviviruses

黄病毒是一类相关的正链包膜RNA病毒，显著 影响人类健康，包括登革热(DENV)、寨卡病毒(ZIKV)。西尼罗河(WNV)，日语 脑炎(JEV)和黄热病(YFV)病毒。病毒关键宿主因子的发现 感染揭示了细胞生物学的新方面，错综复杂的病毒-宿主关系，以及潜在的 抗病毒治疗的靶点。在进入细胞并在酸化的内体中融合后， 黄病毒RNA基因组进入细胞质，然后被翻译成多蛋白，并 在内质网(ER)加工。除了利用ER的许多功能来 蛋白质生产，黄病毒广泛重塑ER膜，为RNA创造一个利基- 依赖的RNA复制。ER也是黄病毒组装的场所，这使得 产生和分泌新的传染性病毒。因此，ER作为一个中心点 协调黄病毒感染生命周期中的许多基本步骤。尽管如此，几乎没有什么 了解内质网中的宿主因素和分子机制，这些因素和分子机制是实现最佳内质网 翻译和加工病毒蛋白或组装复制生态位。我们 最近进行了几次基因筛查，以确定这一过程中的重要成分。 我们在昆虫细胞中对西尼罗河病毒进行的全基因组RNAi筛查验证了18个与ER相关的基因 促进感染的生物学。我们的CRISPR/Cas9基因编辑屏幕在感染西尼罗河病毒的人细胞中的应用 还鉴定了12个ER相关基因。这些筛选集中在内质网驻留蛋白上， 是西尼罗河病毒感染的关键，并包括与ER易位和信号相关的基因 肽加工、内质网相关降解(ERAD)、蛋白质糖基化、蛋白质折叠和 脂类代谢。事实上，WNV、ZIKV、JEV、DENV和YFV的感染都需要特异性 用于处理病毒的宿主信号肽复合体(SPC)的亚单位组件 多蛋白，病毒糖蛋白的产生，从而产生新生病毒粒子。这个 这项建议的目的是定义黄病毒利用特定的分子机制 内质网相关宿主蛋白促进病毒翻译、多蛋白加工、RNA复制、 和/或组件。目标1将定义内质网转位蛋白促进 多蛋白的翻译和加工，而Aim 2将剖析内质网相关衰退的作用 (Erad)促进黄病毒复制。我们的长期目标是通过以下方式确定机制 哪些黄病毒利用ER进行复制，因为这将揭示这两个基本方面 病毒学以及抗病毒治疗的新途径。

Flaviviruses are a genus of related positive-stranded enveloped RNA viruses that significantly impact human health, including dengue (DENV), Zika (ZIKV). West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. The discovery of host factors critical for viral infection reveals new aspects of cell biology, intricate virus-host relationships, and potential targets for antiviral therapeutics. After entering cells and fusing in the acidified endosome, the flavivirus RNA genome penetrates into the cytoplasm and is then translated into a polyprotein and processed at the endoplasmic reticulum (ER). In addition to utilizing many functions of the ER for protein production, flaviviruses extensively remodel ER membranes to create a niche for RNA- dependent RNA replication. The ER also is the site for flavivirus assembly, which enables the production and secretion of new infectious viruses. Thus, the ER serves as a central point for orchestrating many of the essential steps in the flavivirus infection life cycle. Despite this, little is known about the host factors and molecular mechanisms at the ER that are required for optimal translation and processing of the viral proteins or for the assembly of the replication niche. We recently have performed several genetic screens to identify important components in this process. Our genome-wide RNAi screen with WNV in insect cells validated 18 genes associated with ER biology that promote infection. Our CRISPR/Cas9 gene-editing screen in human cells with WNV also identified 12 ER-associated genes. These screens converged on ER-resident proteins as being critical for WNV infection and included genes associated with ER-translocation and signal peptide processing, ER-associated degradation (ERAD), protein glycosylation, protein folding and lipid metabolism. Indeed, infection of WNV, ZIKV, JEV, DENV, and YFV all required specific subunit components of the host signal peptidase complex (SPCS) for processing of the viral polyprotein, the production of viral glycoproteins and thus generation of nascent virions. The objective of this proposal is to define the molecular mechanisms by which flaviviruses use specific ER-associated host proteins to promote viral translation, polyprotein processing, RNA replication, and/or assembly. Aim 1 will define the mechanism by which the ER translocon promotes polyprotein translation and processing while Aim 2 will dissect the role of ER-associated decay (ERAD) in promoting flavivirus replication. Our long-term goal is to determine the mechanisms by which flaviviruses exploit the ER for their replication, as this will reveal both fundamental aspects of virology as well as new avenues for antiviral therapeutics.