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.

星体病毒是小的，不发达的，正义的单链RNA病毒（+ssRNA），很普遍 在鸟类和动物种群中。人类星座病毒（HASTV）感染历史上一直被称为领先 非细菌胃肠炎的原因。但是，近年来，在 脑炎。尽管存在率，但有关病毒感染机制的信息有限。 已知该病毒会产生两种感染所需的非结构多蛋白，它们被裂开 通过宿主信号肽酶和病毒编码的丝氨酸蛋白酶（PRO）的功能亚基。但是，具体 尚未发现多蛋白中的乳化位点。因此，我们不知道特定序列 病毒蛋白，这使得很难为这些蛋白质分配功能。同样，缺乏信息 工具使研究病毒宿主相互作用具有挑战性。因此，很少有调节HASTV的宿主蛋白 已经确定了感染。此外，像所有 + +ssRNA病毒一样，HASTV感染导致戏剧性 重塑细胞内膜形成复制细胞器（ROS），该细胞器浓缩病毒宿主因子 感染所必需的。但是，这些关键结构的膜的来源尚不清楚。 总体而言，我们对HASTV生物学各个方面的了解都有很大的差距，并且需要使用工具 将使我们能够建立基础，以扩大对规范HASTV的基本机制的研究 感染。该提案的目的是利用我们为 +ssRNA病毒开发的工具，然后hastv 了解（1）病毒非结构性多蛋白处理和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。我们已经很重要 进步非常适合完成这些提议的项目。我们已经开发了一个多蛋白库 表达构建体和cDNA感染克隆，以研究促依赖性裂解。另外，我们还有 成功地开发了一种多功能病毒蛋白酶活性报道，该报道已被证明可用于肠病毒 和黄病毒，我们将适应HASTV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，并执行CRISPR屏幕 亲病毒蛋白。最后，我们有创新的工具和策略来可视化主机的操纵 病毒蛋白表达或感染的细胞器，使用活细胞的长期延时成像。全面的， 拟议的项目旨在通过 分子病毒学和病毒宿主相互作用的研究。