Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry 英文参考文献.docVIP

MappingthePhosphoproteomeofInfluenzaAandB VirusesbyMassSpectrometry EdwardC.Hutchinson,EleanorM.Denham,BenjaminThomas,DavidC.Trudgian¤a,SvenjaS.Hester, GabrielaRidlova¤b,AshleyYork,LaurenTurrell,ErvinFodor* SirWilliamDunnSchoolofPathology,UniversityofOxford,Oxford,UnitedKingdom Abstract Proteinphosphorylationisacommonpost-translationalmodificationineukaryoticcellsandhasawiderangeoffunctional effects. Here, we used mass spectrometry to search for phosphorylated residues in all theproteins of influenza A andB 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 ubiquitinationofviralproteinswasalsodetected.Phosphorylationwasdetectedinthepolymeraseproteins(PB2,PB1and PA),glycoproteins(HAandNA),nucleoprotein(NP),matrixprotein(M1),ionchannel(M2),non-structuralprotein(NS1)and nuclearexportprotein(NEP).Manyofthephosphorylationsitesdetectedwereconservedbetweeninfluenzavirusgenera, indicatingthefundamentalimportanceofphosphorylationforallinfluenzaviruses.Theirstructuralcontextindicatesroles forphosphorylationinregulatingviralentryandexit(HAandNA);nuclearlocalisation(PB2,M1,NP,NS1and,throughNP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reversegeneticsweshowthatforNPofinfluenzaAvirusesphosphorylationsitesintheN-terminalNLSareimportantfor 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,constitutivephosphorylationofthesesitesisnotoptimal.Takentogether,theconservation,structural contextandfunctionalsignificanceofphosphorylationsitesimpliesakeyroleforphosphorylationininfluenzabiology.By identifyingphosphorylationsitesthroughoutth