Poster abstracts
Poster number 139 submitted by Megan Sullivan
Probing the impact of SARS-CoV-2 nucleocapsid protein phosphorylation on its aggregation and RNA chaperone activities
Megan Sullivan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Dina Bayachou, Ioulia Rouzina, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
The causative agent of COVID-19, SARS-CoV-2, is a positive-sense single-stranded betacoronavirus. The viral nucleocapsid protein (Np) plays a vital role in RNA replication and packaging, and phosphorylation of Np within its disordered Ser-Arg (SR)-rich linker has been proposed to modulate these functions. Coronaviruses use nested sub-genomic mRNAs (sgmRNAs) for expression of structural and accessory genes. Np is believed to facilitate viral sgmRNA synthesis by chaperoning the viral replication-transcription complex (RTC) “jump” from transcription regulatory sequences (TRS) at the 5’ end of a gene to the 5’UTR, although the mechanism of this process is not well understood. Phosphorylated Np is known to associate with the viral RTC and form liquid-like phase-separated condensates, while packaged Np is mostly non-phosphorylated and condenses the genomic RNA. We hypothesize that phosphorylated Np acts as a more dynamic RNA remodeler or destabilizer, while non-phosphorylated Np is primarily an RNA condenser and annealer. To test this hypothesis, we designed fluorescence-based molecular beacons using complementary oligonucleotides derived from both HIV-1 TAR microhelices and SARS-CoV-2 TRS systems. Annealing rates measured to date showed that non-phosphorylated Np is a more robust annealer under the conditions tested than phosphomimetic Np mutants (3 or 6 Ser/Thr to Asp changes) or HIV-1 nucleocapsid protein (NCp7). Measurements using a dual-labeled DNA hairpin alone show that phosphomimetic SARS-CoV-2 Np promotes more robust hairpin unfolding than non-phosphorylated Np or HIV-1 NCp7. The phosphomimetic Np and HIV-1 NCp7 demonstrate more apparent nucleic acid aggregation at a fixed protein concentration than non-phosphorylated Np. Mass photometry measurements are underway to elucidate RNA binding stoichiometry differences between phosphomimetic and non-phosphorylated Np. To date, our data support the hypothesis that non-phosphorylated SARS-CoV-2 Np packages genomic RNA by acting primarily as an RNA annealer, while phosphorylated Np aids in transcription through its destabilizing activity.
Keywords: RNA chaperone, SARS-CoV-2, phosphorylation