Poster abstracts
Poster number 17 submitted by Dina Bayachou
Investigating the effect of phosphomimetic mutations of SARS-CoV-2 nucleocapsid protein on viral RNA binding
Dina Bayachou (Department of Chemistry and Biochemistry, The Ohio State University ), Megan Sullivan (Department of Chemistry and Biochemistry, The Ohio State University ), Loulia Rouzina (Department of Chemistry and Biochemistry, The Ohio State University ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University )
Abstract:
SARS-CoV-2, the causative agent of COVID-19, is a positive-sense single-stranded betacoronavirus. Significant progress has been made in understanding the SARS-CoV-2 lifecycle. The Nucleocapsid protein (Np) is responsible for viral RNA (vRNA) packaging, plays a vital role in replication and transcription and is associated with viral replication-transcription complexes (RTCs). Np contains a conserved Ser-Arg (SR) rich disordered linker between its two structured domains, which is a known site of phosphorylation. Phosphorylation of Np by multiple host kinases is thought to regulate its function during infection. The 5’ untranslated region (UTR) of vRNA has conserved secondary structures that are necessary for vRNA replication. Phosphorylated Np has been proposed to participate in viral transcription while non-phosphorylated Np packages gRNA into new virions. However, little is known about Np binding specificity to nucleic acids, and how it is modulated by phosphorylation. We used RNA constructs derived from the SARS-CoV-2 5’UTR containing either stem loops (SLs) 1-4, SL5, or SL1-5, as well as HIV-1 derived RNAs. We also prepared phosphomimetic Np mutants with either 3 or 6 Ser/Thr to Asp mutations in the SR linker. Direct binding experiments using fluorescence anisotropy (FA) showed that phosphomimetic mutants and non-phosphorylated Np have high-affinity binding (low nM) to all RNAs tested. FA salt-titration binding assays were used to compare salt dependence and binding specificity among wild-type and mutant proteins. The non-phosphorylated Np showed no statistically significant specificity for SARS-CoV-2 derived RNAs versus HIV-1 RNAs, while phosphomimetic Np mutants showed greater specificity for SARS-CoV-2 RNAs. The location of the phosphomimetic mutations determined whether binding was primarily electrostatic or hydrophobic in nature. Overall, this data supports the conclusion that phosphorylation of SARS-CoV-2 Np may modulate RNA interactions during infection.
Keywords: SARS-CoV-2, Nucleocapsid protein, 5 untranslated region