Modulation of RNA modifications by RNA viruses

我们最近已经证明，具有代表性的单链正义RNA(ss(+)RNA)病毒的基因组具有大量的转录后修饰(PTM)，并且宿主细胞的表位转录谱在感染后发生了显着变化。与此同时，其他研究小组也报告了N6-甲基腺苷(M6A)--这些表位转录标记之一--影响HIV-1RNA的核输出、丙型肝炎病毒RNA的包装和寨卡病毒基因的表达。因此，我们推测，在这些病毒基因组上观察到的大量PTM可能构成了介导病毒基因表达和病毒与宿主相互作用的普遍调控机制的基础。在初步研究中，我们发现二甲基胞嘧啶变异体m5Cm和m44C一直存在于ss(+)RNA病毒的基因组和细胞RNA中，而在模拟感染细胞的RNA中不存在。在这项研究中，我们将以丙型肝炎病毒为模型，以m5Cm/m44C为例，研究pTMS在ss(+)RNA病毒中的功能，剖析pTMS在病毒-宿主相互作用中的意义，并阐明其对病毒和细胞基因表达的影响。更具体地说，我们将应用反义亲和捕获和质谱分析来确定m5Cm/m44C和其他PTM在丙型肝炎病毒RNA基因组上的序列位置。这些信息将指导反向遗传学方法，目的是评估PTM在选定的修饰位点的功能。接下来，我们将对假定的二甲基胞嘧啶编写器酶进行耗尽/过表达，以确定其对丙型肝炎病毒翻译、RNA复制/翻转和病毒粒子组装的影响。最后，我们将通过对二甲基胞嘧啶写入器酶调控的mRNA进行RNA-SEQ分析，通过反义亲和捕获分离选定的靶标，并将捕获的材料提交给PTM分析，从而鉴定受m5Cm/m44C调控的细胞RNA。这些数据将使我们能够评估受二甲基胞嘧啶和二甲基胞嘧啶编写器酶调控的mRNAs对丙型肝炎病毒基因表达的影响。这个项目将改变我们对病毒RNA功能的看法，从单纯的翻译和复制模板转变为感染周期的关键调控因素。除了病毒学领域，该项目产生的知识将有助于建立一个一般框架，以了解PTMS在基因调控中的作用，这是人们高度期待但仍严重缺乏的。这些知识将通过揭示表位转录组在涉及RNA的许多其他过程中的功能，从而巩固该项目的更广泛影响，这将有助于增加我们对与RNA故障相关的无数健康状况的理解。

We have recently demonstrated that the genomes of representative single-stranded positive-sense RNA (ss(+) RNA) viruses are decorated with a large number of post-transcriptional modifications (PTMs) and that the epitranscriptomic profiles of host cells undergo significant variations upon infection. At the same time, other groups have reported that N6-methyladenosine (m6A) –one of these epitranscriptomic marks– affects nuclear export of HIV-1 RNA, packaging of hepatitis C viral RNA, and Zika virus gene expression. We therefore hypothesize that the numerous PTMs observed on these viral genomes may constitute the basis for pervasive regulatory mechanisms mediating viral gene expression and virus-host interactions. In preliminary studies, we have found that the dimethyl cytosine variants m5Cm and m44C were consistently present on the genomes of ss(+) RNA viruses and on cellular RNAs but were absent from the RNA of mock-infected cells. In this proposal we will use hepatitis C virus (HCV) as a model and m5Cm/m44C as a case-study to investigate the functions of PTMs in ss(+) RNA viruses, dissect the implications of PTMs in virus-host interactions, and elucidate the effects on viral and cellular gene expression. More specifically, we will apply antisense affinity capture and mass spectrometry (MS) analysis to identify the sequence position of m5Cm/m44C and other PTMs on HCV RNA genome. This information will guide a reverse genetics approach aimed at evaluating the function of the PTM at the selected modification sites. Next, we will perform depletion/overexpression of putative dimethyl cytosine writer enzyme to determine the effects on HCV translation, RNA replication/turnover, and virion assembly. Last, we will identify the cellular RNAs that are modulated by m5Cm/m44C by performing RNA-seq analysis of mRNAs modulated by dimethyl cytosine writer enzyme, isolating selected targets by antisense affinity capture, and submitting the captured material to PTM analysis. These data will enable us to evaluate the impact of mRNAs modulated by dimethyl cytosine and the dimethyl cytosine writer enzyme on HCV gene expression. This project will transform our view of the functions of viral RNA from mere templates for translation and replication to pivotal regulators of the infectious cycle. Beyond the virology field, the knowledge generated by this project will help establish a general framework for understanding the role of PTMs in gene regulation, which is highly anticipated but still sorely missing. This knowledge will cement the broader impact of this project by shedding new light onto the functions of the epitranscriptome in many other processes involving RNA, which will help increase our understanding of the myriad health conditions linked to RNA malfunction.