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.

宿主细胞已经进化了一系列传感器，以检测病原体相关的分子模式并激活防御反应。蛋白激酶RNA激活（PKR）是病毒产生的双链RNA（DSRNA）的关键传感器，对于保护人类病原体至关重要。活化的PKR停止了全局蛋白转化以限制病毒感染。由于许多DNA病毒拮抗PKR，因此已经假定它们会产生dsRNA。实际上，在缺乏PKR抑制剂的突变病毒感染后，PKR被激活。已经提出，来自紧凑型DNA病毒基因组的两个链的转录都会产生dsRNA。但是，直接证据有限。该提案解决了我们对在DNA病毒感染过程中如何激活关键抗病毒传感器PKR的差距。使用重要的临床病原体人腺病毒（ADV）作为我们的模型，我们无法检测到缺乏特征良好的PKR抑制剂VA RNA（F：.. VA）的野生型（WT）或突变病毒感染期间DSRNA。然而，尽管足够的VA RNA表达足够，但泛素连接酶缺陷型（F：.. E4）Add突变体产生了丰富的核DSRNA，并激活了PKR。病毒连接酶的底物中有剪接蛋白HNRNPC，旨在促进病毒晚期mRNA的有效剪接。 HNRNPC的敲低降低了DSRNA的积累和PKR激活。同样，我的初步数据揭示了宿主蛋白NF90在调节F：.. VA感染过程中的PKR激活中的新作用。总之，这些数据表明PKR可以在ADV感染过程中独立于其规范激活剂DSRNA而被激活。该提案的目的是定义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验证这些蛋白质与PKR之间的相互作用。两种蛋白质的表达和定位将在F：.. VA感染的时间过程中跟踪，并且变化与PKR激活的时机相关。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积累或剪接中缺陷中的救助。夹子QPCR将用于探测与VA RNA和病毒mRNA结合。我还将使用新生蛋白的HPG标记来检查对病毒mRNA蛋白质翻译的影响。该提案的结果将描述NF90在ADV感染期间的抗病毒功能，扩展我们对VA RNA的促病毒作用的理解，并重新定义我们对F：.. VA感染期间PKR激活的理解，对其他对其他核重复DNA病毒的影响。这项工作将在Weitzman Lab和费城儿童医院和宾夕法尼亚大学的综合研究社区提供的合作和跨学科培训环境中进行。我在Weitzman Lab中进行这些研究的独特位置，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动手培训。该培训奖将使我有望为未来的研究研究病毒如何克服宿主抗病毒反应。