%0 Journal Article
%T Human 14-3-3 Proteins Site-selectively Bind the Mutational Hotspot Region of SARS-CoV-2 Nucleoprotein Modulating its Phosphoregulation.
%A Tugaeva KV
%A Sysoev AA
%A Kapitonova AA
%A Smith JLR
%A Zhu P
%A Cooley RB
%A Antson AA
%A Sluchanko NN
%J J Mol Biol
%V 435
%N 2
%D 01 2023 30
%M 36427566
%F 6.151
%R 10.1016/j.jmb.2022.167891
%X Phosphorylation of SARS-CoV-2 nucleoprotein recruits human cytosolic 14-3-3 proteins playing a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that 14-3-3 binding is triggered by phosphorylation of SARS-CoV-2 nucleoprotein at either of two pseudo-repeats centered at Ser197 and Thr205. According to fluorescence anisotropy measurements, the pT205-motif,presentin SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which collectively display an unforeseen pT205/pS197 peptide binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data obtained on the full protein-protein complex indicate that 14-3-3 binding occludes the Ser/Arg-rich region of the nucleoprotein, inhibiting its dephosphorylation. This Ser/Arg-rich region is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting that the strength of 14-3-3/nucleoprotein interaction can be linked with the replicative fitness of the virus.