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

SARS-CoV-2必须对其mrna进行盖帽和甲基化，以确保其稳定性、可翻译性和避免

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.