Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

Kristina V Tugaeva, Andrey A Sysoev, Anna A Kapitonova, Jake L R Smith, Phillip Zhu, Richard B Cooley, Alfred A Antson, Nikolai N Sluchanko

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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, 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 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, 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.

Original languageEnglish
Article number167891
Number of pages17
JournalJournal of Molecular Biology
Issue number2
Early online date5 Dec 2022
Publication statusPublished - 30 Jan 2023

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