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

SARS-CoV-2 must cap and methylate its mRNAs to ensure their stability, translatability, and avoiddetection 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 molecularmechanisms 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 thisimportant area of coronavirus molecular biology that will be addressed in this proposal. First, the identity of theguanylyltransferase (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 molecularand biochemical approaches to address this glaring gap in our understanding of SARS-CoV-2 mRNA cappingmechanisms, laying the foundation for the development of capping-targeted antivirals. Second, while RNAcapping is a regulated process and uncapped RNAs play an influential role in the biology of other positivesense 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 arole of regulated capping and non-coding viral transcripts in SARS-CoV-2 infections. Finally, every cellularmRNA that begins with a terminal adenosine has that residue 2'O methylated at the ribose ring as well as N6methylated on the adenosine base (m6Am). The strong conservation of this m6Am modification indicates itsimportance in cell biology, an assertation recently confirmed with data suggesting that the modificationincreases translatability and facilitates recognition of the transcript as `self'. Interestingly, SARS-CoV-2 andother coronaviruses all initiate their transcripts with an A residue, but it is not known whether that A residuecontains an m6A modification. In the final part of this project, we will determine the m6A modification status ofthe 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 intothe molecular biology of SARS-CoV-2 and open up avenues for the development of broad-spectrum anti-coronaviral therapeutics.