Multiple phosphorylable sites in the Zaire Ebolavirus nucleoprotein evidenced by high resolution tandem mass spectrometry.

The 739-amino-acid nucleoprotein (NP) of Zaire Ebolavirus (ZEBOV) plays a key role in Ebola virion formation and replication. A stable HEK-293 cell line capable of producing an N-ter 6His-tagged recombinant form of NP - ZEBOV was created. Production of this protein was triggered in batch culture using microcarriers. Because NP Ebola phosphorylation has been shown to occur but localization of the modified residues remained to be established, the phosphorylation status of recombinant NP - ZEBOV was investigated through extensive characterization by high-resolution tandem mass spectrometry. The NP - ZEBOV sequence may well be covered by the use of multiple proteases. NP was found to be phosphorylated in two different amino acid stretches: [561-594] and [636-653]. Furthermore, residues Thr(563), Ser(581), Ser(587) and Ser(647) were accurately identified as phosphorylated sites. These data highlight how high resolution tandem mass spectrometry is a method of choice for characterizing post-translational modifications of viral proteins. Because these four phosphorylable sites are conserved among Ebolavirus and Marburgvirus NPs, their modification may play a modulatory role in viral RNA synthesis.