Regulation of Rotavirus Replication

项目摘要 我们的长期目标是了解轮状病毒(RV)如何利用自噬等细胞途径 膜、钙稳态和脂滴(LD)的形成促进它们的复制和原因 疾病。尽管引入了疫苗，轮状病毒仍然是重要的人类病原体。房车的几个方面 复制是独特的，但与其他病毒广泛相关，如细胞质细胞器(病毒质，VI) 由病毒和细胞蛋白以及LDS形成，形成有效的病毒复制和 成熟。乳酸脱氢酶是一种动态的、多功能的细胞内细胞器，参与脂质的储存和代谢 以及信号转导、膜转运以及免疫和炎症反应的调节。 LDS在几种病毒和细胞内细菌感染中起着重要作用，在许多方面都很重要 健康和疾病(新陈代谢、糖尿病、肥胖症、心脏病)。然而，这一机制的信息 脂肪堆积和这些病原体之间的相互作用，以及疾病远未完成。我们的建议 对LDS和VIS的研究建立在我们最近工作的基础上。而病毒蛋白NSP2和NSP5已知是 VI的形成所需要的，这两种蛋白质如何相互联系的分子机制 与其他病毒和细胞蛋白一样，形成VI/LDS的LD组分仍有待阐明。我们 发现了两种形式的NSP2与不同的NSP5亚型相互作用：一种分散的(DNSP2)形式相互作用 与低磷酸化的NSP5和先前识别的VI(VNSP2)形式相互作用 磷酸化的NSP5。我们阐明了一种新的依赖磷酸化的VI形成机制，其中 无处不在的细胞蛋白激酶CK1α通过以下方式部分控制RV Vis的组装 使NSP2磷酸化以触发NSP2八聚体-八聚体晶格的形成。我们还发现，NSP2是一种 并预测NSP2可能磷酸化其他病毒或细胞蛋白以用于VI组装和RV 复制。我们假设轮状病毒与细胞蛋白在特定的微域中的相互作用 内质网成核并诱导病毒复制所必需的VI/LDS，影响病毒的组成 LD相关蛋白，并导致以前未知的轮状病毒诱导的致病机制。我们 提出实验，回答三个问题。(1)NSP4、NSP2和专业微域如何 内质网导致VI/LDS成核？(2)磷酸化如何协调VI的形成和转化 DNSP2到vNSP2启动VI/LD形成和随后的VI/LD成熟？(3)DGAT1是如何 降解导致LD的形成以及PLIN1和PLIN3 LDS在RV感染和 发病机制？这些研究意义重大，因为病毒对宿主信号和代谢的扰动 涉及LDS的通路对多种病原体至关重要。因为RV在小鼠的肠道细胞中复制 肠道是体内脂肪吸收的主要部位，了解轮状病毒感染对LD生物学的影响 有可能揭示病毒感染对宿主新陈代谢的影响的新见解。

Project Summary Our long-term goal is to understand how rotavirus (RV) exploits cellular pathways such as autophagy membranes, calcium homeostasis, and lipid droplet (LD) formation to enhance their replication and cause disease. RVs remain significant human pathogens in spite of the introduction of vaccines. Several aspects of RV replication are unique yet broadly relevant to other viruses, such as cytoplasmic organelles (viroplasms, VI) formed by both viral and cellular proteins and LDs that form a physical platform for efficient viral replication and maturation. LDs are dynamic, multi-functional intracellular organelles involved in lipid storage and metabolism as well as in signal transduction, membrane trafficking and modulation of immune and inflammatory responses. LDs play essential roles in several viral and intracellular bacterial infections and are important in many aspects of health and disease (metabolism, diabetes, obesity, heart disease). However, mechanistic information of the interplay between lipid accumulation and these pathogens, and disease is far from complete. Our proposed studies on LDs and VIs build on our recent work. While the viral proteins NSP2 and NSP5 are known to be required for VI formation, the molecular mechanisms of how these two proteins associate with each other as well as with other viral and cellular proteins and LD components to form VI/LDs remain to be elucidated. We discovered two forms of NSP2 that interact with different isoforms of NSP5: a dispersed (dNSP2) form interacts with hypo-phosphorylated NSP5 and a previously recognized VI (vNSP2) form interacts with hyper- phosphorylated NSP5. We elucidated a novel phosphorylation-dependent mechanism for VI formation, in which the ubiquitous, constitutively active cellular protein kinase CK1α partially controls the assembly of RV VIs by phosphorylating NSP2 to trigger NSP2 octamer-octamer lattice formation. We also discovered that NSP2 is an autokinase and predict that NSP2 may phosphorylate other viral or cellular proteins for VI assembly and RV replication. We hypothesize that interactions of RV and cellular proteins in specialized microdomains of the endoplasmic reticulum nucleate and induce VI/LDs essential for virus replication, affect the composition of the LD-associated proteins and result in previously unrecognized mechanisms of RV-induced pathogenesis. We propose experiments to answer three questions. (1) How do NSP4, NSP2 and specialized microdomains in the ER lead to nucleation of VI/LDs? (2) How does phosphorylation orchestrate VI formation and the conversion of dNSP2 to vNSP2 to initiate VI/LD formation and subsequent VI/LD maturation? (3) How does DGAT1 degradation lead to LD formation and what are the specific roles of PLIN1 and PLIN3 LDs in RV infection and pathogenesis? These studies are significant because viral perturbations of host signaling and metabolic pathways that involve LDs are critical for multiple pathogens. Because RVs replicate in enterocytes in the small intestine, the major site of fat absorption in the body, understanding the effects of RV infection on LD biology has the potential to reveal new insights into the consequences of virus infection on host metabolism.