Defining mechanisms of PKR activation and evasion during Adenovirus infection

腺病毒感染期间 PKR 激活和逃避的定义机制

• 批准号: 10535732
• 负责人: Robert Theodore Steinbock
• 金额: $ 3.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-06 至 2025-09-05
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535732
• 关键词: Address; Adenovirus Infections; Adenoviruses; Antiviral Response; Award; Binding; Biochemical; Bioinformatics; Biology; Cells; Clinical; Communities; Cyclic AMP-Dependent Protein Kinases; DNA Virus Infections; DNA Viruses; Data; Defect; Double-Stranded RNA; Environment; Genetic; Genetic Transcription; Infection; Label; Ligase; Messenger RNA; Modeling; Molecular; Nuclear; PRKR gene; Pattern; Pediatric Hospitals; Pennsylvania; Philadelphia; Play; Positioning Attribute; Process; Protein Kinase; Proteins; Proteomics; RNA; RNA Splicing; Research; Resolution; Role; Sentinel; Spliceosomes; Time; Training; Transcript; Translations; Universities; Viral; Viral Genome; Virus; Virus Diseases; Work; clinically significant; defense response; human pathogen; image processing; inhibitor; knock-down; microscopic imaging; mutant; novel; nuclear factor of activated T-cells, 90 kD; pathogen; protein activation; protein kinase inhibitor; research study; sensor; ubiquitin ligase

Host cells have evolved an array of sensors to detect pathogen-associated molecular patterns and activate defense responses. Protein Kinase RNA-activated (PKR) is a key sensor of double-stranded RNA (dsRNA) produced by viruses and is vital for protection against human pathogens. Activated PKR halts global protein translation to limit virus infection. Since many DNA viruses antagonize PKR, it has been presumed that they produce dsRNA. Indeed, PKR is activated upon infection with mutant viruses lacking a PKR inhibitor. It has been suggested that transcription from both strands of compact DNA virus genomes generates dsRNA. However, there is limited direct evidence this occurs. This proposal addresses gaps in our understanding of how the critical antiviral sensor PKR is activated during DNA virus infection. Using the important clinical pathogen human adenovirus (AdV) as our model, we could not detect dsRNA during infection with wildtype (WT) or mutant virus lacking the well-characterized PKR inhibitor VA RNA (f:..VA). However, infection with ubiquitin ligase-deficient (f:..E4) AdV mutants produced abundant nuclear dsRNA composed of poorly processed viral transcripts and activated PKR despite adequate VA RNA expression. Among the substrates of the viral ligase is a spliceosome protein hnRNPC, targeted to promote efficient splicing of viral late mRNAs. Knockdown of hnRNPC reduced dsRNA accumulation and PKR activation. Similarly, my preliminary data reveal a novel role for the host protein NF90 in regulating PKR activation during f:..VA infection. Together, these data suggest PKR may be activated independently of its canonical activator dsRNA during AdV infection. The objective of the proposal is to define the role that hnRNPC and NF90 play in PKR activation during f:..VA infection. I propose to validate the interaction between these proteins and PKR by co-IP. Expression and localization of both proteins will be tracked over a time course of f:..VA infection and changes correlated with the timing of PKR activation. Knockdown (KO) of each protein will be used to examine activation of PKR during f:..VA infection and to check for rescue from defects in viral late mRNA accumulation or splicing. CLIP-qPCR will be used to probe for binding to VA RNA and viral mRNAs. I will also examine impacts on protein translation of viral mRNAs using HPG labeling of nascent proteins. Results of this proposal will delineate the antiviral function of NF90 during AdV infection, expand our understanding of VA RNA's pro-viral roles, and redefine our understanding of PKR activation during f:..VA infection with broader implications for other nuclear-replicating DNA viruses. This work will take place in the collaborative and interdisciplinary training environment provided by the Weitzman lab and the integrated research communities of both the Children's Hospital of Philadelphia and the University of Pennsylvania. I am uniquely positioned to perform these studies in the Weitzman lab where I will gain hands-on training in proteomics, RNA biology, and high-resolution microscopy and image processing. This training award will leave me poised for my future research studying how viruses overcome host antiviral responses.

宿主细胞已经进化出一系列传感器来检测病原体相关的分子模式并激活防御反应。 RNA 激活蛋白激酶 (PKR) 是病毒产生的双链 RNA (dsRNA) 的关键传感器，对于预防人类病原体至关重要。激活的 PKR 会停止全局蛋白质翻译以限制病毒感染。由于许多 DNA 病毒拮抗 PKR，因此推测它们产生 dsRNA。事实上，PKR 在感染缺乏 PKR 抑制剂的突变病毒时被激活。有人提出，紧凑 DNA 病毒基因组两条链的转录都会产生 dsRNA。然而，这种情况发生的直接证据有限。该提案解决了我们对 DNA 病毒感染期间关键抗病毒传感器 PKR 如何激活的理解上的空白。使用重要的临床病原体人类腺病毒 (AdV) 作为我们的模型，我们在感染野生型 (WT) 或缺乏充分表征的 PKR 抑制剂 VA RNA (f:..VA) 的突变病毒时无法检测到 dsRNA。然而，泛素连接酶缺陷型 (f:..E4) AdV 突变体的感染产生了丰富的核 dsRNA，其由加工不良的病毒转录本组成，并且尽管有足够的 VA RNA 表达，但仍激活了 PKR。病毒连接酶的底物之一是剪接体蛋白 hnRNPC，其目标是促进病毒晚期 mRNA 的有效剪接。 hnRNPC 的敲低减少了 dsRNA 积累和 PKR 激活。同样，我的初步数据揭示了宿主蛋白 NF90 在 f:..VA 感染期间调节 PKR 激活中的新作用。总之，这些数据表明 PKR 在 AdV 感染期间可能独立于其经典激活剂 dsRNA 被激活。该提案的目的是确定 hnRNPC 和 NF90 在 f:..VA 感染期间 PKR 激活中发挥的作用。我建议通过 co-IP 验证这些蛋白质和 PKR 之间的相互作用。将在 f:..VA 感染的时间过程中跟踪这两种蛋白的表达和定位，以及与 PKR 激活时间相关的变化。每种蛋白质的敲低 (KO) 将用于检查 f:..VA 感染期间 PKR 的激活，并检查病毒晚期 mRNA 积累或剪接缺陷的修复情况。 CLIP-qPCR 将用于探测与 VA RNA 和病毒 mRNA 的结合。我还将使用新生蛋白的 HPG 标记来研究对病毒 mRNA 蛋白翻译的影响。该提案的结果将描述 AdV 感染期间 NF90 的抗病毒功能，扩大我们对 VA RNA 促病毒作用的理解，并重新定义我们对 f:..VA 感染期间 PKR 激活的理解，对其他核复制 DNA 病毒具有更广泛的影响。这项工作将在韦茨曼实验室以及费城儿童医院和宾夕法尼亚大学的综合研究社区提供的协作和跨学科培训环境中进行。我拥有在 Weitzman 实验室进行这些研究的独特优势，在那里我将获得蛋白质组学、RNA 生物学以及高分辨率显微镜和图像处理方面的实践培训。这个培训奖将使我为未来的研究做好准备，研究病毒如何克服宿主的抗病毒反应。