Abstract 1070 A Tale of Two RNAs: How Nonstructural Protein 1 of SARS Coronavirus Selectively Suppresses Host mRNA Translation

Nonstructural protein 1 (nsp1) of recently identified zoonotic coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS-CoV) acts as the host shutoff protein that stalls host mRNA translation and triggers their decay, thereby suppressing host gene expression. In contrast, viral RNA selectively escapes this host shutoff mechanism and continues the synthesis of viral proteins due to the presence of a specific stem-loop (SL1) structure in the viral leader sequence. The recent structure analysis of nsp1 complexed with the 40S ribosome suggests that the C-terminal region of nsp1 binds the 40S ribosome at the mRNA binding site, precluding host mRNAs from binding and hence stalling host mRNA translation. In contrast, the interaction between nsp1 and viral leader sequence is predicted to overcome nsp1-mediated translation suppression for the viral RNA. However, the exact nature of the interaction between nsp1 and viral RNA that enables viral RNA translation remains unresolved. Understanding this mechanism will significantly alleviate our understanding of the similar host shutoff mechanisms in other viruses and uncover new anti-viral targets to control the spread of coronaviruses. Research in our laboratory is based on the working hypothesis that the selective protection and preferential translation of viral RNA over cellular mRNA is based on nsp1's ability to interact with RNA in both the nucleus and cytoplasm. The objective of this project is to characterize the interaction of nsp1 and viral RNA and understand the role of nsp1 in nuclear pre-mRNA processing. To dissect this mechanism our laboratory investigates the binding of nsp1 to viral RNA and host proteins using a diverse range of biochemical techniques including gel-shift experiments. Using purified nsp1 and biotinylated viral RNA sequence, we have characterized the binding between nsp1 of SARS-CoV-1 and SARS-CoV-2 with their respective RNAs. We are also investigating the role of nsp1 in nuclear pre-mRNA processing. We have also performed nuclear RNA sequencing from human embryonic kidney (HEK 293) cells in the presence of nsp1 to demonstrate defects in pre-mRNA processing in the presence of nsp1. We identified about a thousand transcripts carrying pre-mRNA splicing and polyadenylation defects. Our results conclude that nsp1 plays a more versatile role in suppressing gene expression in addition to suppression of translation. This research project was conducted by undergraduate researchers at a Primarily Undergraduate University (PUI). The research is partially supported by the DRP grant from the National Institutes of Health National Institute of General Medical Sciences (P20GM103499) and R15 grant from the National Institute of Allergy and Infectious Diseases (1R15AI178603-01).