K119 And K260 Ubiquitination Of Newcastle Disease Virus M Protein Is Essential For Efficient Virus-Like Particle Formation

Newcastle Disease(ND)is an acute and highly contacting infectious disease caused by Newcastle Disease Virus(NDV)in poultry.It is characterized by high fever,difficulty in breathing,diarrhea,nervous disorder,and hemorrhage of mucosa and serosal membrane,with high morbidity and mortality.NDV belongs to Mononegavirales,Paramyxoviridae,Avulavirus.Ubiquitination plays an important role in protein subcellular localization,signaling transduction,and protein degradation.It has been reported that several paramyxovirus M proteins harbor ubiquitination modification which is essential for M protein export from nucleus and virus budding.However,the ubiquitination site of NDV M protein and its effect in virus budding have not been resolved yet.This study focused on characterization of ubiquitination sites of NDV M protein and investigation of its role in virus like particle(VLP)formation.First,we co-expressed the NDV Flag-M protein and HA-Ubiquitin followed by immunoprecipitated and Western blot.Result showed that M protein was attached with HA-ubiquitin.By using sequence alignment and online analysis,we predicted that the potential ubiquitination sites of M protein were lysine K83,K119,K158,K222,K248,and K260.Site-directed mutation of these potential ubiquitination sites was performed to construct M protein mutants,by mutating the Lysine(K)to Alanine(A)or Arginine(R).The ubiquitination signals of mutants K119 A,K222A,K248 A,K260A,K119 R,K222R,K248 R,and K260 R were reduced,suggesting that K119,K222,K248,and K260 were ubiquitinated.The M protein of paramyxoviruses such as Nipah virus,human Parainfluenza virus 5,Sendai virus,and Newcastle disease virus can trigger the formation of VLP and release out of cells.It has been reported that ubiquitination of Nipah virus and human Paramyxovirus 5 M protein is essential for the formation of VLP.However,whether ubiquitination of NDV M protein is involved in virus budding,remained to be elucidated.We expressed the wild type or mutants of M protein and collected cell culture supernatant to check the formation of VLP.Result showed that mutants K119 A,K119R,K260 A,K260R reduced the formation and release of VLP,compared to those of wild type M protein.After translation,M protein enters the nucleus,exports to cytoplasm,migrates to plasma membrane,where it triggers the virus assembly,budding,and release.As K119 is on the conserved nuclear export sequence of paramyoxivirus,and K260 is on the conserved nuclear import sequence,mutation of them may impair the subcellular trafficking of M protein.By using indirect immunofluorescence,we indeed found that K119 R mutant was accumulated on the plasma membrane,whereas K260 R was remained in the nucleus.The subcellular location change of these mutants may impaired the formation of VLP.In all,the ubiquitination modifications on K119 and K260 of M protein are essential for the formation and release of VLP.We further investigated and compared the interaction of M protein/mutants with with host proteins,results showed that K119 R interacts with more host proteins than wide type M protein,suggesting ubiquitination of K119 may mask the potential interaction domain of M protein and help M protein release from plasma membrane.