Glycogen Synthase Kinase-3 Interaction Domain Enhances Phosphorylation of SARS-CoV-2 Nucleocapsid Protein

Abstract

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis

“…5A). In accord, treatment of cells with kenpaullone, a pharmacological inhibitor of glycogen synthase kinase 3β (GSK-3β), which is a primary kinase for coronaviral N phosphorylation (24,25,66,(69)(70)(71), reduced N ISGylation in a dose-dependent manner (Fig. 5B).…”

The hyperphosphorylation of SARS-CoV-2s nucleocapsid protein by GSK-3 involves a complex and redundant priming mechanism

“…One more mechanism to strengthen the binding between N and GSK-3 may involve a short segment of N that bears sequence similarity to the GSK-3 interacting domains (GID) of AXIN1 and FRAT1 (Yun et al, 2022). Mutation of the hydrophobic motif (L223E, L227E) partially suppresses N hyperphosphorylation, increases its sensitivity to CHIR-99021, but has no effect on the response to priming kinase inhibitor (Fig.…”

“…( G ) A schematic model illustrating multiple contacts between N and GSK-3, which include the interaction of GSK-3 with several phosphorylated S/T(s) and the docking of a GSK-3 interacting domain (GID) slightly downstream of the SR domain (Yun et al, 2022). Priming phosphorylation at each contact site (e.g., Ser-206) is probably catalyzed by more than one kinase.…”

“…( H ) Sequence similarity among the GIDs of N, AXIN1 and FRAT1 (Yun et al, 2022). Mutations of the shared (L/F)xx(L/A)xxRL motif partially inhibit but do not eliminate N hyperphosphorylation.…”

“…(H) Sequence similarity among the GIDs of N, AXIN1 and FRAT1 (Yun et al, 2022). Enzastaurin is an orally available PKC inhibitor but also potently inhibits GSK-3 (Kotliarova et al, 2008;Liu et al, 2021).…”