Mechanism Of Human TRA2A Regulating Influenza Virus Host Adaptation

Aquatic birds are natural hosts of Influenza A virus(IAV).It is very frequent that influenza virus spillover to mammals species,but very limited IAVs are able to adapt to and maintain in mammals.When the influenza virus accumulates enough adaptive mutations or replacements and is infected for the first time in humans,it is likely to cause influenza pandemics,as has been the case in 1918,1957,1968,1977 and 2009.At present,mutations in the HA receptor binding domain and mutations in the polymerase PB2 627 K of the avian influenza virus are considered to be the key mutations in the adaptation of avian influenza to mammals.The host factor ANP32 A is a key factor that limits the transmission of avian influenza to mammals.However,the mechanisms that cause cross-species infections remain not completely understood.In this study,we revealed that a splicing perspective the specific mechanism by which a human host factor TRA2A(transformer 2 alpha)regulated influenza virus host adaptability.The details are as follows:1.TRA2 A interacted with v RNPFirst,coIP and Western blot experiments showed that TRA2 A interacted with the viral ribonucleoprotein(v RNP)complex of the influenza virus.Further indirect immunofluorescence(IFA)and in situ hybridization(FISH)assays confirmed that the virus v RNA and NP co-localized with TRA2 A in the nucleus.We next determined which specific component of v RNP binds to TRA2 A.CoIP assays showed that TRA2 A interacted with PB2 and NP,and IFA revealed that they co-localized in the nucleus,indicating that TRA2 A interacts with v RNP,but only interacts with PB2 and NP in the nucleus.2.TRA2 A promoted human influenza virus replication and inhibited avian influenza virus replication.Based on the interaction between TRA2 A and v RNP,we speculate that TRA2 A is involved in the life cycle of the influenza virus.We then investigated the effects of overexpression and knockdown of TRA2 A on human influenza PR8 and avian influenza YS virus replication.Results showed that overexpression TRA2 A protein on A549 cells promoted human influenza virus replication at 24 and 36 hours post infection(hpi),but inhibited avian influenza virus replication.Knockdown TRA2 A showed completely opposite results.3.TRA2 A regulated m RNAs splicing of different genes in human virus and avian virus.At the early stage of influenza virus infection,knockdown TRA2 A significantly reduced the expression level of human influenza virus protein,but significantly increased the expression level of avian influenza virus protein.Interestingly,we found that knockdown TRA2 A significantly increased the ratio of avian influenza virus protein YS-M2/M1 and the ratio of human influenza virus protein PR8-NEP/NS1.However,the ratio of YS-NEP/NS1 and PR8-M2/M1 remained unchanged.These results suggested that TRA2 A may inhibit YS-M and PR8-NS m RNA splicing.To confirm that,we found that TRA2 A inhibited splicing of YS-M and PR8-NS m RNA,but did not affect splicing of YS-NS and PR8-M m RNA by silencing the TRA2 A gene in A549 cells.4.TRA2 A bound to different m RNAs of human and avian influenza viruses.We speculated that TRA2 A regulated its splicing by binding to viral m RNA.We confirmed that TRA2 A bound to the intron splicing silencer(ISS)motif(intronic on PR8-NS sites 233-242 of human influenza virus and intronic on YS-M sites 333-343 of avian influenza virus)by RNA pull-down assays,but couldnot bind to PR8-M and YS-NS m RNA.The mutant PR8-NS and YS-M ISS motifs significantly reduced the binding of TRA2 A,suggesting that TRA2 A may inhibit m RNA splicing by binding to ISS.5.Mutation at M-334 in ISS motif altered virus splicing,replication and pathogenicity.The ISS motif of M1 in avian influenza virus and human influenza virus were compared.Although their nucleotide sequences were different,their amino acid sequences were the same.The M-334 site-mutated virus was rescued by applying the reverse genetic manipulation technique of influenza virus.We found that compared with the wild-type PR8 virus,PR8-M-334 G single muated virus reduced replication and its M m RNA splicing.RIP experiments also shown that its viral M m RNA increased the binding to TRA2 A.However,YS-M-334 C lost ISS mutant virus promoted replication and M m RNA splcing compared with the wild-type virus,while the binding with TRA2 A was decreased.Pathogenicity experiments showed that the pathogenicity of PR8 mutant virus was reduced and the pathogenicity of YS mutant virus was enhanced compared to the wild-type virus.Inhibition of M splicing results in less M2.M2 is closely related to virus budding.We found that whether knockdown TRA2 A or mutated virus can significantly change virus budding.6.Mutations at NS-234/236 of the ISS region alter virus splicing and replication.We also rescued the NS-234/236 mutated virus and found that PR8-NS-234/236 G loses the ISS-type mutant increased virus replication and inhibited NS m RNA splicing.However,YS-NS-234/236 A obtained ISS-type mutation also inhibited the virus replication but promoted NS m RNA splicing.As NS splicing enhancement produced more NEP protein,we investigated the effect of NEP protein on viral polymerase activity and found that human influenza virus NEP promoted its polymerase activity,and avian influenza virus NEP inhibited the polymerase activity.7.TRA2 A regulated viral RNA splicing.In order to further determine that the difference in RNA splicing because of TRA2 A regulation,rather than by affecting virus replication and then caused different splicing,we studied the effect of TRA2 A on mutant viruses and found that the effect was limitted.These results showed that TRA2 A regulated virus replication and pathogenicity by regulating splicing.This study revealed that the human host factor TRA2 A inhibited the splicing of avian influenza virus M m RNA and human influenza virus NS m RNA by binding to the ISS of influenza virus m RNA,thereby inhibiting the replication and pathogenicity of avian influenza virus and promoting the replication and pathogenicity of human influenza virus.and provided a new perspective for the cross-species transmission of avian influenza virus.