SARS-CoV-2 N interactions with RNA and host cell cyclophilin-A

PROJECT SUMMARY Coronaviruses (CoVs) have emerged as formidable human pathogens since their detection within human populations only 50 years ago, underlying the need to rapidly characterize their molecular mechanisms in order to thwart their infections through therapeutics. Seven CoVs that infect humans are known with a range of pathogenicity, which include the more recent virulent CoVs such as MERS, SARS-CoV-1 and SARS-CoV-2. CoVs comprise four structural proteins that includes the envelope (E), membrane (M), spike (S), and nucleocapsid (N) proteins. CoV N proteins perform numerous functions during the viral life-cycle that includes both packaging genomic RNA and manipulating the host cell machinery, making the N protein the most abundantly expressed viral protein during infection. However, the unique and diverse functions of CoV N proteins have made standard structural methods that address its molecular interactions difficult. For example, such difficulties include their promiscuity in RNA binding, engaging multiple binding sites simultaneously, and the presence of inherently dynamic regions thought to be critical for engaging RNA and host proteins. NMR offers a solution to such challenges, as multiple binding modes can be simultaneously characterized both dynamically and structurally and we have previously shown that the inherently dynamic regions within a CoV N protein (SARS-CoV-1) can be studied by NMR. Thus, the exploratory nature of this R21 proposal is to develop strategies aimed at elucidating the molecular details that underlie the SARS-CoV-2 N protein interactions with RNA (Aim 1) and host proteins (Aim 2). Based on our preliminary studies, we hypothesize that specific regions within the N protein have preferred RNA binding sites (Aim 1) and host cell cyclophilin-A binding sites (Aim 2). We will address these aims through the following: Aim 1) Determine how the SARS-CoV-2 N protein targets RNA. Biochemical and biophysical approaches that include NMR will be used to identify the high affinity binding sites of the N protein within sequences derived from genomic RNA and identify the associated dynamic and structural changes that occur upon complex formation. Aim 2) Determine how the SARS-CoV-2 N protein targets host cell cyclophilin-A. NMR will be used to determine whether the N protein targets the cyclophilin-A active site and whether the N protein is a substrate.