Double-stranded RNA during DNA virus infection

项目总结 双链RNA是一种早期危险信号，它向宿主发出病毒入侵的警报，并激活几种 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 诱导抗病毒效应分子，降解寡腺苷酸合成酶-核糖核酸酶L(OAS-核糖核酸酶L)系统 单链RNA和导致细胞凋亡的蛋白激酶RNA依赖(PKR)途径使蛋白质停止 综合。这些宿主对dsRNA的反应，以及病毒用来破坏效应器的许多机制 途径，已经在RNA病毒中得到了广泛的研究。然而，对这方面的研究相对较少。 在DNA病毒感染过程中dsRNA的产生，以及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。腺病毒(ADV)具有双链DNA基因组， 在基因突变体的帮助下，已经成为RNA生物学中开创性发现的强大系统。 ADV和其他基因组大小和蛋白质编码能力有限的病毒进化到最大限度地利用基因 通过受调控的转录和利用两条DNA链来表达蛋白质。因此，退火法 通过对称转录DNA病毒基因组产生的互补单链RNA可以 导致dsRNA。虽然已知ADV可以对抗干扰素反应和阻断PKR激活，但尚不清楚 感染是否产生dsRNA以及感染患者如何避开抗病毒dsRNA激活的途径 细胞。缺失早期区域的ADV突变体对于破译病毒的关键功能是有用的 感染。早期E1B和E4基因的缺失会导致不稳定的病毒RNA，这些RNA运输困难， 翻译过来的。E1B55K和E4orf6基因产物形成有效病毒所需的E3泛素连接酶 制作。然而，在我们对底物如何泛素化的理解中还有另一个差距 复合体促进RNA加工和晚期病毒蛋白质合成。我们最近发现感染了 E1B55K或E4缺陷的ADV突变体会产生dsRNA，并聚集在细胞核中， 核糖核酸酶L和PKR反应被激活。我们还发现了一个功能性的E1B55K/E4orf6复合体 在ADV感染期间阻止dsRNA所需的蛋白质，我们已经鉴定出细胞RNA结合蛋白是 被病毒复合体泛素化。这些初步结果导致了我们的总体假设，即 病毒连接酶泛素化细胞RNA处理因子，以防止dsRNA在 感染和克服抗病毒宿主反应。我们的具体目标是1)确定dsRNA(病毒)的来源 或宿主)，确定激活的宿主响应，以及2)定义E1B55K/E4orf6泛素连接酶活性 防止对dsRNA的抗病毒反应。在这种情况下，我们将使用adv作为模型病原体来研究dsRNA如何 反应会影响DNA病毒的感染。我们的长期目标是发现基因的基本原理 通过破译DNA病毒如何操纵RNA生物发生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.