RNA internal 2'-O methylation in SARS-CoV-2

Among more than 180 types of RNA modification, ribose 2'-O methylation (or called Nm, where N stands for any nucleotide) is the second most abundant. In 2'-O methylation, a methyl group is added to the 2' hydroxyl of the ribose moiety of a nucleoside. There are two types of Nm, the cap Nm which is the 2'-O methylation on the 5' mRNA cap structure, and the internal Nm which occurs in the body of an RNA molecule. Cap Nm is extensively studied and its function is known. However, the enzymatic activities and biological functions of internal Nm are currently unknown. Viral RNA modification is a key step in the SARS-CoV-2 replication cycle. In addition to the 5' cap guanine N-7 methylation and ribose 2'-O methylation (cap Nm), SARS-CoV-2 RNA is heavily epigenetically modified. We recently developed a high throughput Nm-Mut-seq technique allowing for mapping internal Nm sites at a single base resolution. Using this technique, we found for the first time that SARS-CoV-2 RNA isolated from well-differentiated primary human bronchial epithelial (HBE) cultures and Vero-E6 cells contains 24 and 11 internal Nm sites, respectively. Thus, the goal of this R21 project is to identify enzymes that install internal Nm sites in SARS-CoV-2 RNA and to explore the biological functions of internal Nm sites in SARS-CoV-2 RNA. In Aim 1, we will determine if any known host RNA 2'-O methyltransferase (MTase) or viral nsp16 protein (the only known viral RNA cap 2'-O MTase) installs Nm on SARS-CoV-2 RNA. As a parallel strategy, we will also use RNA affinity chromatography, mass spectrometry, and bioinformatics to identify this unknown 2'-O MTase. In Aim 2, we will test the hypothesis that internal Nm in SARS-CoV-2 RNA increases RNA stability, enhances mRNA translation, and prevents viral RNA from being recognized by host innate immunity. The Nm sites in viral RNA will be mutated and recombinant SARS-CoV-2 lacking internal Nm will be recovered using a SARS-CoV-2 reverse genetics system. The effects of internal Nm on viral RNA stability, protein translation, and innate immune response will be determined. This project will have an impact because it will not only fill a major gap in our understanding of the biological functions of internal Nm in SARS-CoV-2 RNA but will also facilitate the development of therapeutic agents for SARS-CoV-2 by targeting internal Nm.