Post-translational modifications of coronavirus proteins: roles and function

Fung, To Sing; Liu, Ding Xiang

Phosphorylation sites and the corresponding protein kinases have been identified for some coronaviruses. For the Alphacoronavirus TGEV, four phosphorylation sites have been identified in the N protein, namely S9, S156, S254 and S256 [126] . Using mass spectroscopy, two clusters of phosphorylation sites were identified in IBV, namely amino acid residues S190/S192 and T378/S379 [127] . Importantly, although both phosphorylated and nonphosphorylated IBV N protein bound to viral RNA with the same affinity, phosphorylated N protein bound to viral RNA with higher affinity than nonviral RNA, compared with the nonphosphorylated IBV N protein [127] . This suggests that N phosphorylation may facilitate the differential recognition of viral RNA. Consistently, using a reverse genetic system based on Vaccinia virus, Spencer et al. showed that IBV N protein was essential for the recovery of recombinant IBV, and that phosphorylated IBV N protein was more efficient than partially or nonphosphorylated N protein [128] . Phosphorylation at T378 and S379 of IBV N protein was shown to be dependent on ATR, a kinase activated during IBV replication [119] . However, recombinant IBV harboring alanine substitutions at all four putative phosphorylation sites (S190A/S192A/T378A/S379A) could still be recovered and grew at a similar future science group www.futuremedicine.com growth rate as wild-type IBV, suggesting that ATR-dependent phosphorylation of N protein is not essential for IBV replication in vitro [119] . As for betacoronavirus, the N protein of SARS-CoV can be phosphorylated by multiple host kinases, including cyclin-dependent kinase, glycogen synthase kinase, mitogen-activated protein kinase and casein kinase II [129] . Using mass spectrometry analysis, six phosphorylation sites (S162, S170, T177, S389, S424 and T428) on the MHV-A59 N protein were identified [130] . Phosphorylation of the N protein of SARS-CoV and MHV-JHM by the host protein GSK-3 was precisely mapped to S197 and S177 in the serine arginine-rich region, respectively [131] . Moreover, inhibition of GSK-3 by kenpaullone significantly reduced the phosphorylation level of N protein, as well as the supernatant virus titer and cytopathic effects on VeroE6 cell-infected SARS-CoV [131] . Therefore, phosphorylation of the N protein appears to be essential for the replication of some Betacoronaviruses. In fact, a recent study showed that phosphorylation of the MHV-JHM N protein by GSK-3 allowed the recruitment of RNA helicase DDX1 to facilitate template read-through, enabling the synthesis of genomic RNA and longer sgRNAs [132] . On the other hand, when N protein was not phosphorylated, template switching was favored during transcription, leading to the preferential generation of shorter sgRNAs but not genomic RNA or longer sgRNAs [132] . Therefore, the phosphorylation status of MHV-JHM N protein acts as a switch to regulate the process of genome replication/transcription.