Preliminary Exploration Of The Mechanism Of H5 Subtype Avian Influenza Virus PB2 Protein 283/526 Site Mutation Affecting Mammalian Pathogenicity

Since H5N1 highly pathogenic avian influenza virus(HPAIV)has caused huge economic loss in the global poultry industry.H5N1 HPAIV is also an important threat for public health.Since the H5N1 avian influenza viruses(AIVs)were reported to infect people in 2003,the number of infected worldwide people exceeded 850 and the mortality rate was about 50%.The H5 subtype have been considered to be one of the potential subetype of the next human influenza pandemic.AIV mainly produces mutations through point mutations and gene reassortment,which leads to the change of viral pathogenicity or the the host range.Therefore,it is necessary to analyze the pathogenic mechanism of H5 subtype AIV and provide a theoretical basis for the prevention and control of the disease.We previously found that the synergistic effect of amino acid residues 283M and 526R in polymerase basic protein 2(PB2)enhanced the virulence of H5 subtype HPAIV in mice.We further found that the mutation of PB2-M283L/I caused the pathogenicity change of H5 subtype HPAIV in mice,confirming the important role of PB2-283 in the adaptation of AIV to mammals.Meanwhile,immunoprecipitation(IP)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to screen PB2 host interaction proteins.Furthermore,bioinformatics analysis suggested the role of PB2 protein in regulating virus life cycle and pathogenic ability.On this basis,we also analyzed the key host interaction proteins tin the process of mammalian pathogenesis after the infection of I283M and K526R combined mutation strain,which laid a foundation for the subsequent mechanism studies.1.PB2-M283L/I mutation leads to the pathogenicity change of H5 HPAIV in miceNew mutation pattern M/L/I/L/T/V was determined at PB2-283 by analyzing the gene sequence of influenza virus PB2 in the database.Based on the constructed reverse genetics platform of H5 subtype AIV,the PB2-M283L/I mutant virus was constructed,and its biological characteristics and virulence differences in mice were evaluated.The results showed that PB2-M283L mutation enhanced the viral growth capacity and polymerase activity in mammalian cells compared with rWT virus in vitro.Furthermore,PB2-M283L mutation displayed a higher virulence,resulting in a more virus load in different tissues,more severe histopathological lesions,and stronger proinflammatory cytokines expression in mice,however,the PB2-M283I mutation showed a completely opposite phenotype.2.Screening and analysis of host proteins interacting with PB2 of H5 HPAIVTo screen host proteins that interact with PB2,a eukaryotic expression vector expressing FLAG-tagged PB2 protein of H5 HPAIV was constructed.PB2 protein was overexpressed in 293T cells.Protein was identified and analyzed by IP combined with LC-MS/MS technology.The results showed that at least 490 human host proteins that interacted with PB2 of H5 HPAIV were screened.Furthermore,GO and Pathway analysis confirmed that these proteins were related to viral transcription,viral replication,and immune response.In order to verify the reliability of MS data,we randomly selected CHERP protein to perform the co-immunoprecipitation(Co-IP)and proved its interaction with PB2.50 host proteins with great possibility to interacte with PB2 were screened by comparison with other published data,which provided the basis for the next study.At the same time,the differences host proteins interacted with PB2 and PB2-I283M-K526R combined mutation of H5 HPAIV were analyzed.By comparing and analyzing the data of interaction proteins from PB2-283I-526K and PB2-283M-526R,141 kinds of difference interaction proteins were preliminarily screened,and the difference proteins were mainly found concentrating on the RNA transport Pathway.In summary,the PB2-M283L mutation enhanced the pathogenicity of H5 subtype AIV in mice,the PB2-M283I mutation had an opposite phenotype.Meanwhile,PB2 interacting host proteins were identified and screened by Co-IP and LC-MS/MS,and the mechanism of AIV infection in mammals was analyzed from the perspective of protein interaction.