Polymorphism And Function Analysis Of PA-X Gene In H7N9 Avian Influenza Virus

Avian Influenza virus(AIV)belongs to the Orthomyxoviridae family.It is a negative-strand segmented RNA virus,can infect birds,mammals,and even humans.Thus,it is pathogens that treat the health of poultry and human being.Its genome has 8 fragments,of which the PA gene encodes four proteins:PA,PA-N155,PA-N182 and PA-X.The PA-X protein was discovered by Jagger et al.in 2012 and is a fusion protein encoded by the+1 reading frame generated by the PA ribosomal frameshift.As a newly discovered viral protein encoded by the PA gene,the PA-X protein has been shown to affect the biological characteristics and the pathogenicity of influenza virus by affecting the virus life cycles in various ways.Its main functions include,1:modulating the virulence of influenza virus,polymerase activity and virus-induced cell death and cytokine immune response;2:having endonuclease activity,can broadly degrade host mRNA;3:exerting the inhibition role in modulating the expression of the host protein(host-shutoff).Moreover,there are differences in the above functions of PA-X among influenza viruses of different subtypes and different host sources.Since 2013,the new H7N9AIV infection has occurred frequently,posing a huge threat to public health.Previous studies have shown that PA-X plays different roles between different subtypes and different hosts.However,its underlying molecular mechanisms are still unclear.This study firstly based on big data analysis to explore the differences in PA-X gene polymorphism between different subtypes,and then studied the PA-X gene polymorphism function from peptide,protein and virus levels.At present,there is no associated research on the pathogenicity of PA-X protein in terms of H7N9 influenza virus in animal models or in cell lines.Therefore,in this study,we mainly explored the effects of PA-X gene polymorphism on the biological characteristics and pathogenicity of H7N9 influenza virus.Our study will provide a foundation for the analysis of the molecular mechanism of the different role of PA-X protein between different subtypes of influenza virus.1.Analysis of PA-X gene polymorphism and function analysis of PA-X gene polymorphism from protein levelWe first downloaded all the obtainable PA-X sequences of the H5N1,H5N6,H7N9,and H9N2 viruses from the GISAID database.According to the classification of avian or mammalian origin influenza virus,a total of 589 strains of avian H5N1,75 strains of human H5N1,651 strains of avian H5N6,19 strains of human H5N6,917 strains of avian H9N2,9 strains of human H9N2,437 strains of avian H7N9,813 strains of human H7N9 were analyzed.The analysis results of the PA-X sequence by biological software comparison revealed some regular site mutations.For example,in the amino acid positions 37A,611,101D,193N,195R,199R,and 2281,the H7N9 and H9N2 PA-X sequences have high mutation rates,while H5N1 and H5N6 PA-X have low mutation rates at these sites,especially the human source H5N1.At the 6 amino acid positions of 27D,58G,1291,208Q,2130G,and 215P,the mutation rate of H5N1 PA-X was high,while the mutation rate of H9N2 and H7N9 PA-X was very low.At the three amino acid positions of 20A,142K and 251K,the avian H5N6 mutation rate was high,while H5N1,H9N2 and H7N9 had low mutation rates at these three points.Next,we focused on the analysis of 1359 H7N9 strains from year 2013 to 2017.Based on the results of the analysis and in combination with the published article on PA-X functional regions,seven potential functional peptides were screened and named pPA-X-1,pPA-X-2,pPA-X-3,pPA-X-4,pPA-X-5,pPA-X-6,pPA-X-7,respectively.Then,a series of functional studies were carried out on the synthesized peptides.The results of cell death experiments showed that the peptide had no significant effect on the cell apoptosis and necrosis.The results of polypeptide inhibition of host protein expression assays confirmed that pPA-X-1,pPA-X-4,and pPA-X-7 significantly inhibited the expression of firefly luciferase and Renilla luciferase.In addition,pPA-X-5 significantly inhibited the expression of firefly luciferase.The results of the peptide pathogenicity study in mice showed that all the mice in the intranasal group showed different degrees of weight loss compared with the mock PBS group.Moreover,when comparing the pPA-X-5 with parental pPA-X-3 group,we found that pPA-X-5 significantly decreased the body weight of the mice(p<0.05).2.Function analysis of PA-X gene polymorphism from protein levelIn order to verify the results of the peptide level,we constructed a eukaryotic expression vector expressing the PA-X gene(pCAGGS-GD15-PA-X-Flag)of H7N9 GD15 strain A/Chicken/Guangdong/GD 15/2016(referred to as GD15).According to the difference of polypeptide sites,the corresponding amino acid site mutations were introduced into the pCAGGS-GD15-PA-X-Flag plasmids,and the recombinant expression plasmids were named as:pCAGGS-PAX-2,pCAGGS-PAX-4,pCAGGS-PAX-5,pCAGGS-PAX-6,pCAGGS-PAX-7,respectively.From the results of inhibition of GFP fluorescence by each recombinant PA-X plasmid,we found that all the expressed PA-X proteins plasmid significantly inhibited the expression of GFP(due to some problems in the process of construction of pCAGGS-PA-X-6 and pCAGGS-PAX-7 expression plasmids,and relevant experiments are currently underway).Among them,the ability of each PA-X protein to inhibit GFP expression from strong to weak is:pCAGGS-PAX-2>pCAGGS-PAX-4>pCAGGS-PAX>pCAGGS-PAX-5.Moreover,the results of inhibition of the expression of Renilla luciferase showed that each recombinant PA-X protein significantly inhibited the expression of Renilla luciferase.However,the inhibition ability of each group was different,and the ability of inhibition of Renilla luciferase reporter gene from high to low was:pCAGGS-PAX-2>pCAGGS-PAX-4>pCAGGS-PAX>pCAGGS-PAX-5,highly consistent with peptide level results.Therefore,together,the results of peptide level and protein level indicate that pCAGGS-PAX-4 and pCAGGS-PAX-5 significantly affect the effect of PA-X gene on inhibiting host protein and are newly discovered PA-X host-shutoff functional regions3.Function analysis of the PA-X gene polymorphism from virus levelTo further validate the results of protein levels,we reverted the PA point mutations in the virus to verify the effect of PA-X gene polymorphism on viral biological characteristics and pathogenicity.According to the difference in the modification sites,the recombinant viruses were named:GD15-PA,GD15-PA-2,GD15-PA-4,GD15-PA-5,GD15-PA-6,GD15-PA-7,respectively.The preliminary biological characteristics of the rescued 6 recombinant viruses showed that GD15-PA-4,GD15-PA-5 and GD15-PA-7 have significantly higher TCID50 on MDCK than the parental virus GD15-PA.The results of pathogenic experiments in mice showed that the pathogenicity of GD15-PA-4 and GD15-PA-5 are significantly higher than GD15-PA(the MLD50 of GD15-PA was above 6.5 log10EIDso,while the MLD50 of GD15-PA-4 and GD15-PA-5 were both 5.51og10EID50).The results of replication curve of the recombinant virus in MDCK and in various tissues of the mice indicated that the replication ability of GD15-PA-4 and GD15-PA-5 were significantly higher than the parental virus of GD15-PA.In addition,the polymerase activity results also showed that the polymerase activity of the GD15-PA-4 and GD15-PA-5 recombinant virus was significantly higher than that of the parental virus.The results of cytokine expression in mouse lung tissue also showed that GD15-PA-4 and GD15-PA-5 induced higher levels of cytokines than parental viruses.Taken together,we believe that the above results together explain why lung injury in GD15-PA-4 and GD15-PA-5-infected mice was more severe than the parental virus infected-mouse.Collectively,this study firstly screened out the potential PA-X protein functional regions based on big data analysis;then further verified these potential PA-X protein functional regions from peptide level,protein level and virus level,respectively.Finally,new functional regions and key amino acid sites affecting the host-shutoff function of PA-X protein were discovered,and the effects of these functional regions on the biological characteristics and pathogenicity of H7N9 influenza virus were also explored.This study laid the foundation for revealing the differences in the function of PA-X protein in different subtypes of influenza virus and also the molecular mechanism by which PA-X protein regulates differentially in terms of pathogenicity in various subtypes of influenza virus.Currently,we are validating the selected functional regions in different subtypes of influenza virus to explore the underlying molecular mechanisms by which PA-X plays different roles between different subtypes and different hosts.