Double-stranded RNA during DNA virus infection

项目摘要 双链（ds）RNA是一种早期的危险信号，它警告宿主病毒入侵，并激活几个 限制病毒复制的先天免疫途径。这些途径包括I型和III型干扰素， 诱导抗病毒效应物，降解寡腺苷酸合成酶-核糖核酸酶L（OAS-RNase L）系统 ssRNA并导致凋亡性死亡，以及蛋白激酶RNA依赖性（PKR）途径， 合成.这些宿主对dsRNA的反应，以及病毒用来破坏效应子的许多机制， 已经在RNA病毒中广泛地研究了这些途径。然而，关于这方面的研究相对较少。 DNA病毒感染期间dsRNA的产生，我们对下游的理解存在差距。 抗病毒途径的作用或病毒拮抗作用。腺病毒（AdV）具有双链DNA基因组， 作为一个强大的系统，在RNA生物学的开创性发现，由遗传突变体的可用性的帮助。 AdV和其他基因组大小和蛋白质编码能力有限的病毒已经进化到最大化基因组大小和蛋白质编码能力。 通过调节转录和使用两条DNA链进行蛋白质生产来表达。因此，退火 由DNA病毒基因组对称转录产生的互补单链RNA可以 导致dsRNA。虽然已知AdV对抗IFN应答并阻断PKR活化，但尚不清楚AdV是否能够抑制IFN应答并阻断PKR活化。 感染是否产生dsRNA以及感染者如何逃避抗病毒dsRNA激活的途径， 细胞早期区域缺失的AdV突变体可用于破译AdV所需的关键病毒功能。 感染早期E1 B和E4基因的缺失导致不稳定的病毒RNA，其转运不良， は自动的E1 B55 K和E4 orf 6基因产物形成高效病毒表达所需的E3泛素连接酶。 生产然而，在我们对底物如何被这种蛋白泛素化的理解中，还有另一个空白。 复合物促进RNA加工和晚期病毒蛋白质合成。我们最近发现， 缺乏E1 B55 K或E4的AdV突变体产生在细胞核中积累的dsRNA， RNase L和PKR反应被激活。我们还表明，一个功能性的E1 B55 K/E4 orf 6复合物， 在AdV感染期间需要防止dsRNA，我们已经鉴定了细胞RNA结合蛋白， 被病毒复合体泛素化。这些初步结果使我们得出了一个总体假设， 的病毒连接酶泛素化细胞RNA加工因子，以防止dsRNA的积累， 感染和克服抗病毒宿主反应。我们的具体目标是：1）鉴定dsRNA（病毒）的来源 或宿主），确定激活的宿主应答，和2）定义E1 B55 K/E4 orf 6泛素连接酶活性如何 阻止对dsRNA的抗病毒反应。因此，我们将以腺病毒为模型病原体，研究dsRNA如何影响腺病毒的表达。 反应影响DNA病毒的感染。我们的长期目标是揭示基因的基本原理 通过破译DNA病毒如何操纵RNA生物合成途径和逃避抗病毒防御来表达。

PROJECT SUMMARY Double-stranded (ds) RNA is an early danger signal that alerts the host to viral invasion and activates several innate immune pathways that limit virus replication. These pathways include type I and type III interferons that induce antiviral effectors, the oligoadenylate synthetase-ribonuclease L (OAS-RNase L) system that degrades ssRNA and leads to apoptotic death, and the protein kinase RNA dependent (PKR) pathway that halts protein synthesis. These host responses to dsRNA, and the many mechanisms viruses use to subvert the effector pathways, have been studied extensively in RNA viruses. However, there is a relative dearth of studies on dsRNA production during infection by DNA viruses, and there is a gap in our understanding of downstream effects or viral antagonism of antiviral pathways. Adenovirus (AdV) has a double-stranded DNA genome that has served as a powerful system for seminal discoveries in RNA biology, aided by availability of genetic mutants. AdV and other viruses with limited genome size and protein coding capacity have evolved to maximize gene expression through regulated transcription and use of both DNA strands for protein production. Thus, annealing of complementary single-stranded RNAs produced by symmetrical transcription of DNA virus genomes could lead to dsRNA. Although AdV is known to counter IFN responses and block PKR activation, it is not known whether infection generates dsRNA and how the antiviral dsRNA-activated pathways are evaded in infected cells. AdV mutants with early regions deleted have been useful for deciphering key viral functions required for infection. Deletion of early E1B and E4 genes results in unstable viral RNAs that are poorly transported and translated. The E1B55K and E4orf6 gene products form an E3 ubiquitin ligase required for efficient virus production. However, there is another gap in our understanding of how ubiquitination of substrates by this complex promotes RNA processing and late viral protein synthesis. We recently discovered that infection with AdV mutants that are defective for E1B55K or E4 generates dsRNA that accumulates in the nucleus, and that RNase L and PKR responses are activated. We also showed that a functional E1B55K/E4orf6 complex is required to prevent dsRNA during AdV infection and we have identified cellular RNA binding proteins that are ubiquitinated by the viral complex. These preliminary results have led to our overall hypothesis that the activity of the viral ligase ubiquitinates cellular RNA processing factors to prevent accumulation of dsRNA during infection and overcome antiviral host responses. Our Specific Aims are 1) to identify the source of dsRNA (viral or host), determine host responses activated, and 2) define how the E1B55K/E4orf6 ubiquitin ligase activity prevents antiviral responses to dsRNA. In this way we will use AdV as a model pathogen to study how dsRNA responses impact infection for DNA viruses. Our long-term goal is to uncover fundamental principles of gene expression by deciphering how DNA viruses manipulate RNA biogenesis pathways and evade antiviral defenses.