PDF(Pubmed)
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,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.
摘要：
SARS-CoV-2核蛋白的磷酸化募集人胞质14-3-3蛋白，在许多病毒的复制中起着公认的作用。在这里，我们使用遗传密码扩展来证明14-3-3结合是由SARS-CoV-2核蛋白在以Ser197和Thr205为中心的两个假重复序列之一的磷酸化触发的。根据荧光各向异性测量，pT205-motif,出现在SARS-CoV-2中，但不在SARS-CoV中，所有七个人14-3-3同种型均优于pS197基序，其共同显示不可预见的pT205/pS197肽结合选择性层次。晶体结构表明pS197和pT205是互斥的14-3-3结合位点，而SAXS和在完整的蛋白质-蛋白质复合物上获得的生化数据表明，14-3-3结合阻塞了核蛋白的富含Ser/Arg的区域，抑制它的去磷酸化。这个富含Ser/Arg的区域极易发生突变，以Omicron和Delta变体为例，我们的数据表明，14-3-3/核蛋白相互作用的强度可以与病毒的复制适应性有关。
