| Institution: | 1. A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia;2. York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom;3. Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA |
| Abstract: | 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, present in 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. |
