The Mechanism Of ANP32 Family Protein In The Replication Of Influenza A Virus And Its Cross-species Transmission

Influenza A virus can infect a variety of hosts,such as birds and mammals,with multiple subtypes and a wide spectrum of infection.The cross-species transmission of Influenza A virus often causes serious harm or even pandemic.Therefore,the molecular mechanism of its replication and cross-species transmission have always been the focus in this field.The RNA polymerase of influenza virus is the material basis for viral transcription and replication in host cells,and plays an important role in cross-species transmission.It needs some host proteins to complete virus replication.At present,little is known about the host-dependent and cross-species adaptation mechanisms of influenza virus polymerase.It has been found that ANP32A,a member of the acidic(leucine-rich)nuclear phosphoprotein 32kDa(ANP32)family,is related to the species-specific polymerase activity of avian influenza virus,but the function of ANP32A and other proteins in the family and the interaction between ANP32 proteins and influenza A virus polymerase are still not clear.In this study,CRISPR/Cas9 technology was used to construct a series of HEK293T knockout cell lines of polymerase-related host proteins,and the knockout cell lines were transfected with H1N1 subtype influenza A/Sichuan/01/2009(H1N1SC09)polymerase reporting system.It was found that the single knockout of ANP32A(AKO)or ANP32B(BKO)in 293T cells did not affect the polymerase activity.Since ANP32A and ANP32B have high similarity in both structure and known functions,it is speculated that they may function similarly,with one protein knocked out alone and the other still functioning.Therefore,we constructed an ANP32A&B double-knocked cell line(DKO).The knockout cell lines AKO,BKO,and DKO were transfected with the polymerase minigenome reporting systems of different human influenza virus subtypes,and the results showed that the polymerase activity of different human influenza subtypes decreased by more than 10,000 times in the DKO cell lines.Similarly,WSN virus infection experiments also showed that the growth and replication of the virus in DKO cell lines decreased by more than 10,000 times.Supplementation of ANP32A and ANP32B alone or simultaneously in DKO cells restored the replication of influenza virus,which confirmed that host factors huANP32A and huANP32B were necessary for the replication of influenza A viruses.In this study,the dependence of influenza viruses from different species(human,avian,horse,pig and dog)on the ANP32 proteins of different species was evaluated,and it was found that the polymerase activity of influenza viruses from different species requires the support of ANP32 proteins,revealing that ANP32A and ANP32B are the decisive host proteins for the polymerase activity of different species influenza A viruses for the first time.Further study found that avian ANP32B 129/130 as a key site of amino acid mutations,leading to the loss of its natural support for influenza virus polymerase activity,proved that avian ANP32A was the only one of ANP32 family proteins in avian that can support Influenza A virus polymerase activity,which makes the avian ANP32A protein to be the only target for designing transgenic anti-flu chicken.At the same time,it was found that the 129/130 site is the key site for all species ANP32A and ANP32B to bind to influenza virus polymerase,and it is also the key functional site for ANP32 proteins in supporting polymerase activity.After mutating this site,all ANP32 proteins lost their ability to bind to influenza virus polymerase and support to the activity of influenza virus polymerase.Pigs have long been considered as the intermediate host for the cross-species transmission of avian influenza virus to humans,but the adaptation mechanism of avian influenza virus RNA polymerase in pigs is not clear.Based on previous results,we further confirmed the ability of the different species ANP32A&B proteins in supporting the polymerase activity of influenza viruses from human,avian,pigs,dogs,horses and other different species,the results suggested that,compared with other mammals ANP32A or ANP32B proteins,pig ANP32A(pgANP32A)protein can specifically support the polymerase activities of avian H7N9 and H9N2 subtype.Immunoprecipitation results suggested that,compared with huANP32A,pgANP32A could specifically combine with avian polymerase complex,thus supporting the activity of avian polymerase.By constructing the pgANP32A knockout PK15 cell line(PK15-AKO),we analyzed avian polymerase activity and influenza virus infection on the PK15-AKO cell line,it was proved that the deletion of pgANP32A resulted in a significant decrease in avian polymerase activity and viral replication titer,suggesting that pgANP32A is a key factor for porcine PK15 cells to specifically support the replication of avian influenza virus.The 106V/156S site of pgANP32A protein was identified as the key site to support the activity of avian-derived polymerase and to affect its binding to avian-derived polymerase complex.This study revealed that ANP32A and ANP32B proteins from different species play a decisive role in the process of influenza virus replication for the first time,clarified that pig ANP32A as a key factor,plays an important role for the transmission of avian influenza virus from avian to pigs,even to human.This study also identified that some key sites are important for ANP3 2 proteins function to support influenza A virus,which provides effective targets for the design of new anti-flu drugs and transgenic anti-flu animals.