The PA-X Protein Restricts The Reassortment Of The Pa Gene Of H9N2 Avian Influenza Virus To Mammalian Influenza Virus

Avian influenza virus not only causes huge economic losses in the poultry industry,but also threatens human health seriously.Most of the influenza viruses causing pandemics in humanbeings are involved in the gene reassortment with avian influenza viruses.The H9N2 avian influenza virus is one of the most widespread avian influenza viruses in the world.The potential threat of gene reassortant between H9N2 avian influenza virus and mammalian influenza virus should not be ignored.To test the polymerase activities under the different conbination with reassortant subunits of PA,PB1,PB2 and NP proteins from different viruses,the eukaryotic expression plasmid were constructed,each of which containes respectively of PB1,PB2,PA or NP genes derived from A2093(A/Chicken/Shanghai/2093/2009,H9N2)and several mammalian influenza viruses.The polymerase activity of different combinations was tested on 293 T cells using mini-replicon systems of the influenza virus.The results showed that the polymerase activity of mammalian influenza viruses was higher than that of A2093.When the PB1 or NP protein of A2093 was combined with other polymerase proteins of mammalian influenza virus respectively,the polymerase activity kept at the high level.However,when PB2 or PA protein of A2093 was combined with other polymerase proteins of mammalian influenza virus,the polymerase activity was decreased significantly.In the other hand,when PB1 or NP of mammalian influenza was combined with other polymerase proteins of A2093,the polymerase activity Kept at the low level.However,when PB2 or PA of mammalian influenza virus was combined with other polymerase proteins of A2093,the polymerase activity increased significantly.The decrease of polymerase activity may be the main reason why the PB2 and PA genes of H9N2 avian influenza virus are difficult to reassortant with mammalian influenza virusIn order to study the effects of polymerase gene reassortment on viral replication further,the reverse genetic systems of A2093 and WSN viruses were used.Each of the rescued viruses containd HA,NA and one(or two)polymerase genes derived from A2093 and other genes derived from WSN.The reassortant viruses containing the PB1 or NP genes of A2093 could replicate well,while the reassortant viruses containing PA or PB2 gene of A2093 showed a decline of replication on MDCK.Animal experiments showed that the reassortant viruses containing PB1 or PA of A2093 had similar titers in the lungs of the mice infected intranasally.However,the virus containing A2093 PB1 gene caused significant weight loss in mice,while the virus containing A2093 PA gene did not show clear weight loss.To explore the mechanism of A2093 PA genes causing the reduced replication of WSN virus on cells,293 T cells were co-transfected with different combinations of plasmids expressing polymerase proteins and tested by westen-bolt.When A2093 PA protein was expressed,the expression of other polymerase proteins were reduced significantly.When co-expression of PA protein and eGFP,it was also found that PA proteins could inhibit the expression of e GFP gene,which was inserted under the downstream of the polymerase II promoter.The PA of A2093 inhibited eGFP expression more significantly than that of mammalian influenza virus.To further explore the mechanism of PA inhibition of protein expression,the expression of PA-X protein was stoped by demaging the frame-shift using point mutation method.Once the PA-X expression was blocked,the inhibition of e GFP expression by PA protein lost.Those results indicated that the inhibition of eGFP expression induced by PA-X,but not PA protein.In addition,the inhibition activities of A2093 PA-X was stronger than that of WSN.PA-X suppresses gene expression mainly by reducing the mRNA level when tested the amount of mRNA of e GFP by realtime PCR method.Then,the PA-X deletion viruses were rescued respectively under the background of H9N2 avian influenza virus or WSN virus using reverse genetic system of influenza viruse.Once the PA-X wass deleted,the replication of A2093 and WSN virus on both MDCK and MLH cells decreased clearly,indiciting that PA-X was important for replication of each virus.Further,under the backbone of WSN virus,the virus containing 2093 PA-X deleted PA gene replicated in MDCK and LMH more efficiently than the virus containing A2093 PA genes which expressing PA-X.It is speculated that the pA-X protein encoded by the H9N2 avian influenza virus PA gene overdegrades the host mRNA and resulted in the limitation of the virus replication in host cells,thus the PA gene of avian influenza virus is difficult to be reconbitaed into mammalian influenza virus.In this study,the restriction effect in gene reassortment of PA gene of influenza virus from different species was found,and the related molecular mechanism was partially elucidated,which will provid theoretical basis for the prevention and control of influenza virus.