Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

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Abstract
Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses – to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets. Eukaryotic cells regulate the function of many of their proteins through the reversible phosphorylation of serine, threonine or tyrosine residues. It is known that some influenza virus proteins are phosphorylated, but few sites of phosphorylation have been identified. We used mass spectrometry to identify 39 sites of phosphorylation, most of them novel, in proteins from influenza A viruses and an influenza B virus (a separate genus in the orthomyxovirus family) - to the best of our knowledge, this is the first time this has been attempted for all the proteins in a virus. By integrating sequence conservation data and structural information we were able to propose functions for most of these sites, providing a foundation for further studies, and we assessed experimentally the contribution of multiple phosphorylation sites in the influenza A virus nucleoprotein (NP) to viral growth and to transcription and replication of the genome. In addition, by identifying phosphorylation sites that are common to both influenza A and B viruses, we suggest that phosphorylation at these sites is a highly conserved aspect of influenza biology that may provide targets for antiviral therapy.