Abstract 1091 The biochemical mechanism of SARS-CoV-2 nucleocapsid protein hyperphosphorylation

The nucleocapsid (N) protein of coronavirus becomes heavily phosphorylated within its central Ser-Arg (SR) domain by host glycogen synthase kinase-3 (GSK-3) after the virus enters the cell. GSK-3 is a unique Ser/Thr kinase, preferring substrates that are phosphorylated by another Ser/Thr kinase four residues downstream of its own phosphorylation site. The biochemical mechanism of N hyperphosphorylation, and its functional significance, were not completely understood. Here we present mass spectrometry data showing that GSK-3-mediated N phosphorylation is a highly cooperative process involving multiple and redundant priming phosphorylation sites. The R203M and R203K/G204R mutations, commonly found in the delta and omicron SARS-CoV-2 variants, both hinder N hyperphosphorylation by GSK-3. GSK-3 inactivation by small molecule inhibitors reduces the binding affinity of N to the coronavirus's replication-transcription complex, causing a delay of progeny virion release. Supported by NIH grants GM138722 and GM150502.