Regulation of Rotavirus Replication

项目摘要 我们的长期目标是了解轮状病毒（RV）如何利用细胞途径，如自噬 膜，钙稳态和脂滴（LD）的形成，以增强其复制，并导致 疾病尽管引入了疫苗，RV仍然是重要的人类病原体。RV的几个方面 复制是独特的，但广泛相关的其他病毒，如细胞质细胞器（病毒素，VI） 由病毒和细胞蛋白以及LD形成，其形成用于有效病毒复制的物理平台， 成熟LDs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 以及在信号转导、膜运输和免疫及炎症反应的调节中。 LD在几种病毒和细胞内细菌感染中起着重要作用，并且在许多方面都很重要 健康和疾病（新陈代谢，糖尿病，肥胖，心脏病）。然而， 脂质积累和这些病原体之间的相互作用，以及疾病还远未完成。我们提出的 对LD和维斯的研究建立在我们最近的工作基础上。虽然已知病毒蛋白NSP 2和NSP 5是 VI形成所需的分子机制，以及这两种蛋白质如何相互关联的分子机制 与其它病毒和细胞蛋白质和LD组分形成VI/LD一样，仍有待阐明。我们 发现了两种形式的NSP 2与不同的NSP 5亚型相互作用：分散的（dNSP 2）形式相互作用 与低磷酸化的NSP 5和先前识别的VI（vNSP 2）形式相互作用， 磷酸化NSP 5。我们阐明了VI形成的一种新的磷酸化依赖机制，其中 普遍存在的组成型活性细胞蛋白激酶CK 1 α通过以下途径部分控制RV维斯的组装 磷酸化NSP 2以触发NSP 2八聚体-八聚体晶格形成。我们还发现NSP 2是一种 NSP 2可能磷酸化其他病毒或细胞蛋白，用于VI组装和RV 复制的我们假设RV和细胞蛋白在特定微区的相互作用， 内质网成核并诱导病毒复制所必需的VI/LD，影响细胞的组成 LD相关蛋白，并导致以前未认识到的RV诱导的发病机制。我们 提出实验来回答三个问题。(1)NSP 4，NSP 2和专门的微域如何在 ER导致VI/LD的成核？(2)磷酸化是如何协调VI的形成和 dNSP 2转化为vNSP 2以启动VI/LD形成和随后的VI/LD成熟？(3)DGAT 1如何 降解导致LD形成，PLIN 1和PLIN 3 LD在RV感染和 发病机制？这些研究意义重大，因为病毒干扰宿主信号传导和代谢 涉及LD的途径对多种病原体至关重要。因为RV在小肠上皮细胞中复制， 肠道是体内脂肪吸收的主要部位，了解RV感染对LD生物学的影响 有可能揭示病毒感染对宿主代谢的后果的新见解。

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.