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，在抵御人类病原体方面起着至关重要的作用。激活的PKR停止全球蛋白质翻译，以限制病毒感染。由于许多DNA病毒对PKR有拮抗作用，因此推测它们能产生dsRNA。事实上，PKR在感染缺乏PKR抑制剂的突变病毒时被激活。有人认为，从紧凑的DNA病毒基因组的两条链上转录产生dsRNA。然而，发生这种情况的直接证据有限。这项建议解决了我们对关键的抗病毒传感器PKR在DNA病毒感染过程中如何被激活的理解上的空白。以临床重要病原体人腺病毒(ADV)为模型，在野生型(WT)或缺乏PKR抑制剂VA RNA(f：..VA)的突变病毒感染过程中，我们无法检测到dsRNA。然而，感染泛素连接酶缺陷(f：..E4)ADV突变体，尽管VA RNA表达充足，但仍能产生由加工不良的病毒转录物组成的丰富的核dsRNA并激活PKR。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mRNAs的有效剪接。HNRNPC基因敲除后dsRNA积聚减少，PKR激活减少。同样，我的初步数据显示，宿主蛋白NF90在调节f：..VA感染期间PKR激活方面发挥了新的作用。综上所述，这些数据表明，在ADV感染期间，PKR可能独立于其典型的激活物dsRNA而被激活。该提案的目的是确定HNRNPC和NF90在f：..VA感染期间PKR激活中所起的作用。我建议通过共IP来验证这些蛋白与PKR之间的相互作用。这两种蛋白的表达和定位将在f：..VA感染的一段时间内被跟踪，以及与PKR激活的时间相关的变化。每种蛋白的敲除(KO)将被用来检查f：..VA感染过程中PKR的激活，并检查从病毒晚期mRNA积累或剪接缺陷中拯救出来的情况。Clip-qPCR将用于探测与VA RNA和病毒mRNAs的结合。我还将使用新生蛋白的HPG标记来检查对病毒mRNAs蛋白翻译的影响。这一建议的结果将描绘出NF90在ADV感染过程中的抗病毒功能，扩大我们对VA RNA亲病毒作用的理解，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。这项工作将在魏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。我在魏茨曼实验室进行这些研究具有独特的优势，在那里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。这一培训奖项将使我为未来的研究做好准备，研究病毒如何战胜宿主的抗病毒反应。

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.