Mechanisms Of ANP32A Regulating Polymerase Activity Of Influenza Virus

The acidic nuclear phosphoprotein 32 family member A(ANP32A)is species-specific difference between avian and mammalian cells,which is responsible for mammalian-restricted avian influenza virus polymerase activity.However,the exact mechanism of ANP32A modulation of polymerase activity remains poorly understood.Chicken ANP32A-X1(chANP32A-X1)had an extra 33 aa insertion than human ANP32A(huANP32A).In this study,another chANP32A-X2 containing an extra 29aa insertion was cloned from DF-1 cells.The overexpression of chANP32A-X1 and-X2 in 293T cells stimulated activity of PB2 627E polymerase with vNA-Luc template,but overexpression of chANP32A-X2 stimulated less PB2 627E polymerase activity than chANP32A-X1.The expression of chANP32A-X1 and-X2 in 293T cells was increased,the activity of PB2627E polymerase of PR8 virus was correspondingly enhanced.However,the high expression of chANP32A-X1 and-X2 had a negative effect on chANP32A-stimulated PB2627E polymerase activity,and even the high expression of chANP32A-X2 could not promote the activity of PR8 PB2 627E polymerase,which revealed that the regulation of chANP32A in PB2 627E polymerase activity is closely related with the amount of expression of chANP32A in mammalian cells.Overexpression of chANP32A or huANP32A in 293T cells could not promote PB2 627K polymerase activity.In DF-1 cells,overexpression of chANP32A and huANP32A could not promote PB2 627E and PB2 627K activity.These indicates the species-specific regulation of avian and mammalian ANP32A on PB2 627E polymerase in mammalian cells.The activity of polymerase reconstructed with cNA-Luc suggested that avian and mammalian ANP32A also differently regulated PB2 627E polymerase activity in mammalian cells.Distinct effects of ANP32A constructs on polymerase activity suggested that the 180VK181 residues within an extra 33 aa insertion are necessary but not sufficient for different regulation of chANP32A-X1 in PB2 627E polymerase activity in mammalian cells.The PB2 E627K mutation of PR8 virus promoted its polymerase activity in mammalian cells,but overexpression of chANP32A-X1 inhibited PB2 627K polymerase activity.Comparatively,after PB2 K627E mutation,PB2 N567D,T598V,A613V and F636L mutation of PR8 virus were performed,respectively.The activity of PB2 627E polymerase containing N567D,T598V,A613V or F636L mutation was significantly increased and chANP32A-X1 showed additive effects,providing further support that species-specific regulation of ANP32A in polymerase activity might be only relevant with the PB2 E627K mutation but not N567D,T598V,A613V or F636L mutation.This study laid a foundation for further understanding the mechanism of ANP32A in regulating the adaptation of AIV polymerase to mammalian cells.Influenza virus polymerases perform the mRNA transcription activity and functions of cRNA and vRNA replication.Avian and mammalian ANP32A differently regulate the mammalian-restricted avian virus polymerase activity,but the specific regulation process which involved in is not clear.In this study,the recombinant virus(PR8-PB2 K627E)with PB2 K627E mutation was rescued by using the 8 plasmid reverse genetic operation system of PR8 virus.Virus replication assays showed that the replication ability of mutant PR8-PB2 K627E virus in 293T cells was lower than that of wild-type PR8 virus.The overexpression of chANP32A-X1 in 293T cells promoted the replication of mutant PR8-PB2 K627E virus.It was further found that overexpression of chANP32A-X1 promoted the mRNA,vRNA and cRNA levels of PR8-PB2 K627E virus.The overexpression of chANP32A-X1 and huANP32A in 293T cells had no significant effect on the mRNA transcription in inhibition of influenza virus replication by cycloheximide(CHX).Rescue of cycloheximide-mediated inhibition of influenza virus replication by polymerase complementation showed that the overexpression of chANP32A-X1,but not huANP32A,promoted the vRNA levels of PR8-PB2 627E virus,while the overexpression of chANP32A-X1 and huANP32A had no different regulatory effects on the mRNA,vRNA and cRNA levels of PR8-PB2 K627E virus.Comparatively,overexpression of chANP32A-X1 and huANP32A had no different effects on mRNA,cRNA and vRNA levels of wild-type PR8 influenza virus.These suggested that overexpression of chANP32A-X1 and huANP32A in 293T cells had different modulation of "cRNA to vRNA" replication of PR8-PB2 K627E virus,and indicated that ANP32A has species-specific regulation on"cRNA to vRNA" replication of mammalian-restricted AIVs.The 3' promoter mutations(A3G+U8C mutations)and 5'promoter mutations(C3U+G8A mutations)of cRNA that are proposed to stabilize the panhandle structure of cRNA promoter enhanced the mammalian-restriction of PB2 627E polymerase.The regulation of chANP32A on PB2627E polymerase activity is closely related to the expression levels of chANP32A.The high expression of chANP32A-X1 in 293T cells could not promote the activity of PR8 PB2 K627E polymerase with wild-type cNA-Luc,but could promote PB2 627E polymerase activity with mutant cRNA(3' promoter A3G+U8C mutation or 5' promoter C3U+G8A mutation),which revealed the promoter mutation of cRNA affected the regulation of ANP32A in PB2 627E polymerase activity.It could be speculated that the regulation of ANP32A on the activity of influenza virus polymerase was related to the RNA promoter.Co-immunoprecipitation showed that overexpression of chANP32A-X1 in293T cells promoted the production of RNPs(PB2 K627E)complex with cNA-Luc,but did not affect the generation of primary cRNP complexes in quantitative terms.We propose a model that chANP32A-X1 regulates mammalian-restricted PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.Influenza virus PB2 627E and 627K polymerase show different interactions with mammalian cells,which is an important reason for the restriction of avian influenza virus(AIV)polymerase(PB2 627E)in mammalian cells.ChANP32A-X1 and huANP32A interact with AIV polymerase and overexpression of chANP32A-X1,but not huANP32A,promotes PB2 627E polymerase activity in 293T cells,suggesting that chANP32A-X1 and huANP32A interacting with PB2 627E polymerase may also change the interactions of PB2627E polymerase with host proteins,which are responsible for different activity of PB2627E polymerase.In this study,chANP32A-Xl and huANP32A were mainly co-localized with PR8 PB2 627E and 627K polymerase complexes in the nucleus by laser confocal scanning experiment.Co-immunoprecipitation(Co-IP)test showed that huANP32A still interacted with PR8 virus polymerase lacking PB2 627 domain.ChANP32A-X1 and huANP32A were respectively co-expressed with PB2 627E polymerase,and PB2 627E was precipitated by immunoprecipitation(IP)test.The results showed PB2 627E polymerase interacted with both chANP32A-X1 and huANP32A.Mass spectrometry identified 670 host proteins interacting with PB2 627E polymerase in IP sample.Among them,when chANP32A-X1 was overexpressed,PB2 627E polymerase specifically bound 48 host proteins and 357 host proteins were specifically combined with PB2 627E polymerase when huANP32A was overexpressed.It revealed that overexpression of chANP32A-X1 and huANP32A led to the different interactions of PB2 627E polymerase with mammalian host proteins.Bioinformatics showed that the interacting proteins were mainly involved in the splicing process in cells,and the hnRNPs family proteins participated in the splicing.The interaction network showed that PB2 627E polymerase differently interacted with many hnRNP family proteins in presence of chANP3 2A-X1 or huANP32A.Mass spectrometry data showed that PB2 627E polymerase specifically combined host hnRNPAB when huANP32A but not chANP32A-X1 was expressed in 293T cells.Laser confocal scanning experiments showed that hnRNPAB and PB2 627E were co-localized in nucleus.Virus polymerase activity assay showed that overexpression of hnRNPAB inhibited the activity of PB2 627E and PB2 627K polymerase,while knock down of hnRNPAB by siRNA promoted PB2 627E and PB2 627K polymerase activity,which indicated that hnRNPAB was a negative regulator of influenza virus polymerase activity.The above results suggested that overexpression of chANP32A-X1 may eliminate or weaken the interaction between hnRNPAB and PB2 627E for increasing PB2 627E polymerase activity.In addition,it was found by mass spectrometry that the host hnRNPD protein bound PR8 PB2 627E polymerase when chANP32A-X1 and huANP32A were expressed,and the interaction between hnRNPD and PB2 627E or 627K protein was confirmed by Co-IP experiment.The overexpression of hnRNPD inhibited the expression of PR8 PB2 protein,but had no significant effect on the transcriptional levels of PB2 mRNA detected by RT-qPCR.RNA binding protein immunoprecipitation assay showed that hnRNPD could bind PB2 mRNA,and the overexpression of hnRNPD inhibited PB2 mRNA nuclear export for reducing PB2 protein translation.The overexpression of hnRNPD inhibited viral replication and polymerase activity while knock down of hnRNPD by siRNA promoted virus replication and polymerase activity.The above results suggested that hnRNPD can inhibit polymerase activity and viral replication by binding PB2 mRNA and inhibiting its nuclear export for the reduced PB2 protein expression.In this study,we understand the different regulation of diffierent ANP32A on the activity of polymerase from the perspective of chANP32A and huANP32A-mediated different interactions of PB2 627E polymerase with mammalian cells.