Defining mechanisms of PKR activation and evasion during Adenovirus infection

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。蛋白激酶RNA激活（PKR）是病毒产生的双链RNA （dsRNA）的关键传感器，对人类病原体的保护至关重要。激活的PKR停止全局蛋白转译以限制病毒感染。由于许多DNA病毒能拮抗PKR，人们推测它们产生dsRNA。事实上，PKR在缺乏PKR抑制剂的突变病毒感染时被激活。有人认为，病毒基因组的两条紧致DNA链的转录产生dsRNA。然而，这种情况发生的直接证据有限。这一建议解决了我们对DNA病毒感染期间关键抗病毒传感器PKR如何被激活的理解空白。以重要的临床病原体人腺病毒（AdV）为模型，我们在感染野生型（WT）或突变型病毒（缺乏表征良好的PKR抑制剂VA RNA (f:..VA)）时无法检测到dsRNA。然而，感染泛素连接酶缺陷(f：…E4) AdV突变体产生了大量由加工不良的病毒转录物组成的核dsRNA，尽管有足够的VA RNA表达，但却激活了PKR。在病毒连接酶的底物中，有一种剪接体蛋白hnRNPC，旨在促进病毒晚期mrna的有效剪接。敲低hnRNPC可减少dsRNA的积累和PKR的激活。同样，我的初步数据揭示了宿主蛋白NF90在f期调控PKR激活中的新作用。弗吉尼亚州的感染。总之，这些数据表明，在AdV感染期间，PKR可能独立于其典型激活子dsRNA而被激活。该提案的目的是确定hnRNPC和NF90在f期间PKR激活中的作用。弗吉尼亚州的感染。我建议通过co-IP验证这些蛋白与PKR之间的相互作用。这两种蛋白的表达和定位将在一段时间内被跟踪。VA感染和改变与PKR激活的时间相关。每个蛋白的敲低（KO）将被用来检测PKR在f期间的激活。VA感染和检查病毒后期mRNA积累或剪接缺陷的挽救。CLIP-qPCR将用于探测与VA RNA和病毒mrna的结合。我还将研究使用HPG标记新生蛋白对病毒mrna蛋白质翻译的影响。本研究的结果将描述NF90在AdV感染期间的抗病毒功能，扩展我们对VA RNA的亲病毒作用的理解，并重新定义我们对f: v期间PKR激活的理解。VA感染对其他复制核的DNA病毒有更广泛的影响。这项工作将在Weitzman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。在Weitzman实验室进行这些研究，我将获得蛋白质组学、RNA生物学、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我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。

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.