Mechanisms and functional implications of SARS-CoV-2 mRNA capping and modification.

SARS-CoV-2必须对其mRNAs进行封顶和甲基化，以确保其稳定性、可译性和避免 由宿主先天免疫机制检测为非自身转录本。因此，RNA封顶的过程， 对SARS-CoV-2感染的成功至关重要。它也代表着分子的一个关键贡献者 其发病机制也是抗病毒治疗发展的一个非常吸引人的靶点。 然而，有三个关键的知识差距减缓了我们对这一点的理解 本提案将涉及冠状病毒分子生物学的重要领域。首先，身份是 鸟苷酰转移酶(GTase)，病毒RNA封端机制的核心，将GTP转移到5‘ 新生的文字记录的结尾，是未知的。我们将使用互补分子的双管齐下的策略 和生物化学方法来解决我们对SARS-CoV-2mRNA封顶理解中的这一明显差距 机制，为开发封顶靶向抗病毒药物奠定了基础。第二，虽然RNA 封顶是一个受调控的过程，无封顶的RNA在其他阳性的生物学中起着重要的作用 对于RNA病毒感染，目前尚不清楚RNA封顶在冠状病毒中是受调节的还是默认的事件。 我们将确定非封顶RNA是否由SARS-CoV-2产生，以便为 调控的封端和非编码病毒转录本在SARS-CoV-2感染中的作用。最后，每个细胞 以末端腺苷开始的mRNA在核糖环上有2‘O甲基化残基以及N6 腺苷碱基甲基化(M6Am)。这种m6Am修饰的强烈保守性表明它 在细胞生物学中的重要性，这一断言最近得到了数据的证实，表明这种修饰 提高可译性，并有助于将文字稿识别为“自我”。有趣的是，SARS-CoV-2和 其他冠状病毒都以A残基启动转录本，但尚不清楚该A残基是否 包含M6A修改。在本项目的最后部分，我们将确定M6A的修改状态 SARS-CoV-2mRNA5‘A末端残基及其修饰(或缺失)的作用 在冠状病毒转录本的生物学中发挥作用。总的来说，这些研究将提供重要的新见解 SARS-CoV-2的分子生物学研究为广谱抗病毒的开发开辟了道路 冠状病毒疗法。

SARS-CoV-2 must cap and methylate its mRNAs to ensure their stability, translatability, and avoid detection by host innate immune mechanism as non-self transcripts. The process of RNA capping, therefore, is pivotal to the success of a SARS-CoV-2 infection. It also represents a key contributor to the molecular mechanisms of pathogenesis as well as a very attractive target for the development of antiviral therapeutics. However, there are three key knowledge gaps that have slowed progress in our understanding of this important area of coronavirus molecular biology that will be addressed in this proposal. First, the identity of the guanylyltransferase (GTase), the centerpiece of the viral RNA capping machinery that transfers GTP to the 5' end of the nascent transcript, is unknown. We will use a two-pronged strategy of complementary molecular and biochemical approaches to address this glaring gap in our understanding of SARS-CoV-2 mRNA capping mechanisms, laying the foundation for the development of capping-targeted antivirals. Second, while RNA capping is a regulated process and uncapped RNAs play an influential role in the biology of other positive sense RNA viral infections, it is not known if RNA capping is a regulated or a default event in coronaviruses. We will determine if uncapped RNAs are produced by SARS-CoV-2 in order to establish the foundation for a role of regulated capping and non-coding viral transcripts in SARS-CoV-2 infections. Finally, every cellular mRNA that begins with a terminal adenosine has that residue 2'O methylated at the ribose ring as well as N6 methylated on the adenosine base (m6Am). The strong conservation of this m6Am modification indicates its importance in cell biology, an assertation recently confirmed with data suggesting that the modification increases translatability and facilitates recognition of the transcript as `self'. Interestingly, SARS-CoV-2 and other coronaviruses all initiate their transcripts with an A residue, but it is not known whether that A residue contains an m6A modification. In the final part of this project, we will determine the m6A modification status of the terminal 5' A residue of SARS-CoV-2 mRNAs and investigate the role that the modification (or lack thereof) plays in the biology of coronaviral transcripts. Collectively these studies will provide important new insights into the molecular biology of SARS-CoV-2 and open up avenues for the development of broad-spectrum anti- coronaviral therapeutics.