The hyperphosphorylation of SARS-CoV-2s nucleocapsid protein by GSK-3 involves a complex and redundant priming mechanism
Upon entry into host cells, the nucleocapsid (N) protein of coronavirus becomes heavily phosphorylated within its central Ser-Arg domain by glycogen synthase kinase-3 (GSK-3). Most substrates for GSK-3 need to be phosphorylated, or primed, by another Ser/Thr kinase four residues downstream of the GSK-3 phosphorylation site. It was widely assumed that the hyperphosphorylation of SARS-CoV-2s N protein requires independent priming at Ser-188 and Ser-206, which initiates sequential phosphorylation by GSK-3. Here we present biochemical and mass spectrometry data that contradicts this simple model, revealing instead a much more complex and highly cooperative mechanism where redundant priming, as well as exosite docking, enables N proteins efficient phosphorylation by GSK-3. The R203M and R203K/G204R mutations, found in the recent delta and omicron variants of concern, both hinder N hyperphosphorylation by GSK-3. The mechanistic insights generated in this study suggest a novel approach to treat COVID-19 by combining multiple classes of pharmacological agents to inhibit N hyperphosphorylation.