SARS-CoV-2 is the etiologic agent of COVID-19. There is currently no effective means of preventing infections by SARS-CoV-2, except through restriction of population movement and contact. An understanding of the origin, evolution and biochemistry (molecular biology) of SARS-CoV-2 is a prerequisite to its control. Mutations in the phosphorylation sites of SARS-CoV-2 encoded nucleocapsid protein isolated from various populations and locations, are described. Mutations occurred in the phosphorylation sites, all located within a stretch which forms a phosphorylation dependent interaction site, including C-TAK1 phosphorylation sites for 14-3-3. The consequences of these mutations are discussed and a structure-based model for the role of protein 14-3-3 in the sequestration and inhibition of SARS-CoV-2 nucleocapsid protein’s function is presented. It is proposed that the phosphorylation of SARS-CoV-2 nucleocapsid protein and its sequestration by Protein 14-3-3 is a cellular response mechanism for the control and inhibition of the replication, transcription and packaging of the SARS-CoV-2 genome.

Phosphorylation of serines 197 and 206 of SARS-COV-2 Nucleocapsid protein (NCp) enhanced the stability and binding efficiency and sequestration of NCp to Protein 14-3-3 by increasing the Stability Energy (ΔGstability energy) and Binding Energy (ΔΔGbinding energy) from ~545 Kcal/mol to ~616 Kcal/mol, and from 108 Kcal/mol to ~228 Kcal/mol respectively. The calculated Binding Energy Difference (ΔΔGbinding energy difference) between dephospho-NCp-14-3-3 complex and phospho-NCp-13-3-3 complex was ~72 Kcal/mol. Phosphorylations of serines 186, 197, 202 and 206, and threonines 198 and 205 NCp also caused an increase in the Stability Energy (ΔGstability energy) and Binding Energy (ΔΔGbinding energy) from ~545 Kcal/mol to ~617, 616, 583, 580, 574, 564 and 566 Kcal/mol and from ~108 Kcal/mol to ~228, 216, 184, 188, 184, 174 and 112 Kcal/mol respectively. Phosphorylation of NCp on serines 197 and 206 caused a decrease in Stability Energy and Binding Energy from ~698 Kcal/mol to 688 Kcal/mol, and from ~91 Kcal/mol to ~82 Kcal/mol for the dimerization of NCp. These results support the existence of a phosphorylation dependent cellular mechanism to bind and sequester NCp.

Mutations in several phosphorylation sites within the phosphorylation rich domain of SARS-COV-2 Nucleocapsid protein (NCp), including serines 186, 197 and 202, and adjacent arginine 203 and glycine 204 have been described and have been proposed to prevent the binding and sequestration of NCp by Protein 14-3-3. Structure modeling and thermodynamic calculation show that mutations of phosphorylation sites, phospho-serines 186, 197 and 202 to phenylalanine, leucine and asparagine, and phosphorylation recognition sites, arginine/glycine 203/204 to lysine/arginine or lysine/threonine resulted in signifi cant destabilization of the NCp-14-3-3 complex by causing a decrease in Stability Energy (ΔGstability energy) and Binding Energy (ΔΔGbinding energy). These results evidenced that mutations in NCp underlie a mechanism to bypass sequestration by Protein 14-3-3 which would result in enhanced dimerization of NCp, and replication, transcription and packaging of the SARS-COV-2 genome

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