Mechanisms of astrovirus infection

星状病毒感染机制

• 批准号: 10712313
• 负责人: Nicholas J Lennemann
• 金额: $ 36.54万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-05 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10712313
• 关键词: Animals; Astrovirus; Biology; Birds; CRISPR screen; Cells; Cellular biology; Complementary DNA; Disease; Encephalitis; Enterovirus; Flavivirus; Foundations; Gastroenteritis; Goals; Human; Human Biology; Image; Infection; Integration Host Factors; Intracellular Membranes; Investigation; Knowledge; Libraries; Membrane; Molecular Virology; Nature; Organelles; Peptide Hydrolases; Polyproteins; Population; Prevalence; Production; Proteins; RNA Viruses; Regulation; Reporter; Research; Serine Protease; Site; Source; Structure; Viral; Viral Nonstructural Proteins; Viral Proteins; Virus; Virus Diseases; Visualization; Work; Zoonoses; design; innovation; interdisciplinary approach; protein expression; signal peptidase; tool; virus host interaction; virus infection mechanism

Astroviruses are small, non-enveloped, positive-sense single-stranded RNA viruses (+ssRNA) that are prevalent in bird and animal populations. Human astrovirus (HAstV) infection has historically been known as a leading cause of non-bacterial gastroenteritis. However, in recent years, divergent HAstVs have been found in cases of encephalitis. Despite their prevalence, there is limited information regarding the mechanisms of virus infection. The virus is known to produce two nonstructural polyproteins required for infection, which are cleaved into functional subunits by host signal peptidase and the virus-encoded serine protease (Pro). However, the specific sites of Pro cleavage in the polyprotein have not been discovered. Thus, we do not know the specific sequences of viral proteins, which has made it difficult to assign functions to these proteins. Similarly, lack of information and tools has made studying virus-host interactions challenging. Thus, few host proteins that regulate HAstV infection have been identified. Furthermore, like all +ssRNA viruses, HAstV infection leads to dramatic remodeling of intracellular membranes to form replication organelles (ROs), which concentrate viral host factors required for infection. However, the source of the membranes for these critical structures is not well understood. Overall, there are major gaps in our knowledge of all aspects of HAstV biology and there is a need for tools that will allow us to build a foundation to expand our research on fundamental mechanisms that regulate HAstV infection. The goal of this proposal is to utilize tools we have developed for +ssRNA viruses and HAstV to understand the mechanisms of (1) viral nonstructural polyprotein processing and Pro activity, (2) host protein regulation of virus infection, and (3) viral protein manipulation of host organelles. We have made significant progress to be well-suited to accomplish these proposed projects. We have developed a library of polyprotein expression constructs and a cDNA infectious clone to investigate Pro-dependent cleavage. Additionally, we have successfully developed a versatile viral protease activity reporter that has been shown to work for enteroviruses and flaviviruses, which we will adapt for HAstV to study intracellular Pro activity. We have generated tools to study the microenvironment of the viral nonstructural proteins in living cells and to perform a CRISPR screen for pro- and anti-viral proteins. Lastly, we have innovative tools and strategies to visualize the manipulation of host organelles upon viral protein expression or infection using long-term, time-lapse imaging of living cells. Overall, the proposed projects are designed to significantly advance the field of cell biology of HAstV infection through investigation of the molecular virology and virus-host interactions.

星状病毒是普遍存在的小型、无包膜、正义单链 RNA 病毒 (+ssRNA) 在鸟类和动物种群中。人类星状病毒（HAstV）感染历来被认为是主要的 非细菌性胃肠炎的病因。然而，近年来，在以下情况中发现了不同的 HAstV： 脑炎。尽管病毒流行，但有关病毒感染机制的信息有限。 已知该病毒会产生感染所需的两种非结构性多蛋白，它们被切割成 由宿主信号肽酶和病毒编码的丝氨酸蛋白酶（Pro）组成的功能亚基。不过，具体 尚未发现多蛋白中 Pro 裂解位点。因此，我们不知道具体的顺序 病毒蛋白，这使得很难为这些蛋白分配功能。同样，缺乏信息 和工具使得研究病毒与宿主的相互作用变得具有挑战性。因此，调节 HAstV 的宿主蛋白很少 已确定感染。此外，像所有 +ssRNA 病毒一样，HAstV 感染会导致严重的 重塑细胞内膜以形成复制细胞器 (RO)，集中病毒宿主因子 感染所需。然而，这些关键结构的膜的来源尚不清楚。 总体而言，我们对 HAstV 生物学各个方面的知识都存在重大差距，因此需要能够 将使我们能够建立一个基础来扩大我们对调节 HAstV 的基本机制的研究 感染。该提案的目标是利用我们为 +ssRNA 病毒和 HAstV 开发的工具来 了解 (1) 病毒非结构多蛋白加工和 Pro 活性的机制，(2) 宿主蛋白 病毒感染的调节，以及（3）宿主细胞器的病毒蛋白操纵。我们已经做出了重大的 进展非常适合完成这些拟议项目。我们开发了一个多蛋白库 表达构建体和 cDNA 感染性克隆以研究 Pro 依赖性切割。此外，我们还有 成功开发了一种多功能病毒蛋白酶活性报告基因，已被证明适用于肠道病毒 和黄病毒，我们将对其进行改编以用于 HAstV 以研究细胞内 Pro 活性。我们已经生成了工具 研究活细胞中病毒非结构蛋白的微环境并进行 CRISPR 筛选 促病毒蛋白和抗病毒蛋白。最后，我们拥有创新的工具和策略来可视化主机的操作 使用活细胞的长期延时成像来观察病毒蛋白表达或感染时的细胞器。全面的， 拟议的项目旨在通过以下方式显着推进 HAstV 感染的细胞生物学领域 分子病毒学和病毒与宿主相互作用的研究。