Identification of mouse hepatitis coronavirus A59 nucleocapsid protein phosphorylation sites

The coronavirus nucleocapsid (N) is a multifunctional phosphoprotein that encapsidates the genomic RNA into a helical nucleocapsid within the mature virion. The protein also plays roles in viral RNA transcription and/or replication and possibly viral mRNA translation. Phosphorylation is one of the most common post-translation modifications that plays important regulatory roles in modulating protein functions. It has been speculated for sometime that phosphorylation could play an important role in regulation of coronavirus N protein functions. As a first step toward positioning to address this we have identified the amino acids that are phosphorylated on the mouse hepatitis coronavirus (MHV) A59 N protein. High performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to identify phosphorylated sites on the N protein from both infected cells and purified extracellular virions. A total of six phosphorylated sites (S162, S170, T177, S389, S424 and T428) were identified on the protein from infected cells. The same six sites were also phosphorylated on the extracellular mature virion N protein. This is the first identification of phosphorylated sites for a group II coronavirus N protein.