Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein of 422 amino acids. Similar to other coronavirus N proteins, SARS-CoV N protein is predicted to be phosphorylated and may contain nuclear localization signals, serine/arginine-rich motif, RNA binding domain and regions responsible for selfassociation and homo-oligomerization. In this study, we demonstrate that the protein is posttranslationally modified by covalent attachment to the small ubiquitin-like modifier. The major sumoylation site was mapped to the 62 lysine residue of the N protein. Further expression and characterization of wild type N protein and K62A mutant reveal that sumoylation of the N protein drastically promotes its homo-oligomerization, and plays certain roles in the N protein-mediated interference of host cell division. This is the first report showing that a coronavirus N protein undergoes posttranslational modification by sumoylation, and the functional implication of this modification in the formation of coronavirus ribouncleoprotein complex, virion assembly and virus-host interactions.
Coronavirus encodes an extensively phosphorylated and highly basic nucleocapsid (N) protein. Previous studies have identified Ser190, Ser192, Thr378 and Ser379 as the phosphorylation sites for coronavirus infectious bronchitis virus (IBV) N protein. In this study, we show that phosphorylation at Thr378 and Ser379 sites is dependent on the ataxia-telangiectasia mutated (ATM) and Rad3-related (ATR), a kinase activated during IBV replication. Introduction of Ala substitutions at these two sites individually, in combination of the two and together with other two sites (Ser190 and Ser192) into an infectious IBV clone did not affect recovery of the recombinant viruses containing the mutations. A mutant virus (rIBV-Nm4) carrying the four Ala substitutions grew at a similar, if not better, growth rate as wild type virus. This study reveals a cellular kinase responsible for phosphorylation of a coronavirus N protein at two positions and the functional consequence of this modification on coronavirus replication.
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