Poster abstracts
Poster number 11 submitted by Chun-Ta Dustin Chiu
RNA 2’-O-Methylation Modulates SARS-CoV-2 Replication
Chun-Ta Chiu (Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA), Chuan He (Department of Chemistry, The University of Chicago, Chicago, IL, USA), Jianrong Li (Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA)
Abstract:
Post-transcriptional RNA modifications are essential regulators of RNA metabolism and protein translation. Among over 170 known RNA modifications, ribose 2’-O-methylation (Nm) is uniquely prevalent across all RNA species and is critically associated with cancers, neurological disorders, and infectious diseases. However, the roles of Nm in virus replication and innate immune responses are poorly understood. Here, we developed a high-throughput, base resolution, and quantitative sequencing technique (Nm-Mut-Seq) to map transcriptome-wide Nm sites in viral and host RNAs isolated from primary human bronchial epithelial (HBE) cultures and Vero E6 cells. Using this technique, we found for the first time that SARS-CoV-2 RNA is not only methylated at ribose 2’-O position of 5’ cap structure (cap Nm) but also contains a wide spread of internal Nm sites, occurring in the body of RNA molecules. Subsequently, we screened a panel of host RNA 2’-O methyltransferases (2’-O MTases) that are essential for SARS-CoV-2 replication and found that depletion of two host RNA 2’-O MTases, cap methyltransferase 1 (CMTR1) and FtsJ RNA methyltransferase homolog 2 (FTSJ2), dramatically reduced replication of SARS-CoV-2 WA1 and Omicron variants. Interestingly, knockdown of CMTR1 or FTSJ2 significantly increased the expression of the RNA sensors RIG-I and MDA5, and IRF3 phosphorylation, leading to an enhanced type I interferon (IFN) response. This suggests that depletion of host 2’-O MTases leads to enhanced innate immune responses which in turn suppress virus replication. To understand the biological functions of the Nm sites in SARS-CoV-2 RNA, we developed a yeast-based reverse genetics system and generated a panel of recombinant SARS-CoV-2 lacking cap Nm and/or internal Nm sites. We found that these Nm-deficient SARS-CoV-2 mutants were defective in virus replication and gene expression, induced higher type I IFN, and were more sensitive to the type I IFN treatment compared to the parental virus. Collectively, these data demonstrate that Nm machinery modulates SARS-CoV-2 replication, gene expression, and innate immune responses.
Keywords: 2-O-methylation, SARS-CoV-2, RNA epitranscriptomic modification