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.

星状病毒是一种小型、无包膜、正链RNA病毒（+ssRNA）， 在鸟类和动物种群中。人类星状病毒（HAstV）感染在历史上被认为是主要的病原体。 非细菌性胃肠炎的病因。然而，近年来，已经在以下病例中发现了不同的HAstV： 脑炎尽管它们普遍存在，但关于病毒感染机制的信息有限。 已知该病毒产生感染所需的两种非结构多聚蛋白，其被切割成 由宿主信号肽酶和病毒编码的丝氨酸蛋白酶（Pro）的功能亚基。但具体 尚未发现多蛋白中的Pro切割位点。因此，我们不知道具体的序列 病毒蛋白质，这使得很难分配功能，这些蛋白质。同样，缺乏信息 和工具使得研究病毒与宿主的相互作用具有挑战性。因此，很少有调节HAstV的宿主蛋白 感染已被确认。此外，与所有+ssRNA病毒一样，HAstV感染导致显著的 重塑细胞内膜以形成复制细胞器（RO），其浓缩病毒宿主因子 需要感染。然而，这些关键结构的膜的来源还不清楚。 总的来说，我们对HAstV生物学的所有方面的知识都存在重大差距，并且需要工具， 将使我们能够建立一个基础，以扩大我们对调节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， 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，和（3）病毒蛋白操纵宿主细胞器。我们取得了重大 我们的目标是使这些项目能够顺利完成。我们开发了一个多蛋白质库 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的病毒蛋白酶活性报告基因，该基因已被证明对肠道病毒有效 和黄病毒，我们将适应HAstV研究细胞内Pro活性。我们已经开发了工具， 研究活细胞中病毒非结构蛋白的微环境，并进行CRISPR筛选， 前病毒蛋白和抗病毒蛋白。最后，我们有创新的工具和策略来可视化主机的操作， 细胞器对病毒蛋白表达或感染的影响，使用活细胞的长期延时成像。总的来说， 拟议的项目旨在通过以下措施， 分子病毒学和病毒与宿主相互作用的研究。