Effect Of PA-X On The Pathogenicity Of H7N9 Avian Influenza Virus In Mice And Identification Of Its "Host-shutoff" Function Area

Since the first outbreak of the H7N9 avian influenza virus in 2013,there have been at least five waves of human infection in China.Comparing with the previous four waves,the fifth waves not only caused huge economic losses but also resulted in a human influenza pandemic.At present,the H7N9 AIVs epidemic both in birds and humans has been effectively controlled,while its pathogenesis still deserves further study.Initially,the influenza A virus was identified with eight gene fragments,including PB1,PB2,PA,HA,NP,NA,M1 and NS1,which encoded ten viral proteins.However,along with the further research for the influenza virus,the novel viral proteins were constantly discovered and identified,including PB1-N40,PB1-F2,PA-X,M42,NS3,PA-N155 and PA-N182.The PA-X is a fusion protein that contains the N-terminal endonuclease domain of PA,and a short C-terminal sequence(either 61 or 41 codons)from an overlapping ORF(’X-ORF’)in segment 3 that is accessed by+1 ribosomal frameshifting.Since the discovery of PA-X protein,its function has been extensively studied in different subtypes of influenza virus,and in various host animal models,such as in mice,ducks,chickens,dogs and pigs.Normally,the main function of PA-X protein is to inhibit the overall host gene expression during influenza virus infection("host-shutoff"),which is critical in regulating the host innate immune response.It is interesting that although the host-shutoff activity of PA-X protein existed in all subtypes of influenza A virus,the virulence and replication capability of influenza virus regulated by "host-shutoff" are different or even opposite among various subtypes of influenza virus.Importantly,the effect of PA-X on the pathogenicity of H7N9 AIVs is unknow.In order to further explore PA-X protein whether regulate the virus virulence and virus-induced innate immune response,we have successfully constructed the mutated highly pathogenic viruses GD15PAX-3 and GD15PAX-5 and the mutated low pathogenic viruses r15PAX-3 and r15PAX-5 with decreased expression of PA-X by genetically modifying the ribosomal frameshift region of PA gene using the highly pathogenic strain A/chicken/Guangdong/GD15/2017(GD15PA)and the low pathogenic strain r15PA.Then we systematically explored the differences of these recombinants in virus replication,polymerase activity,virulence,and innate immune response in mice.We also determined the function area and the key amino acids that associated with the "host-shutoff" activity of the H7N9 influenza virus PA-X and further explored the impact of these amino acid sites on the pathogenicity of viruses in mice.Lastly,we evaluated whether these key amino acids also affect the "host-shutoff" of the H5N1 subtype AIVs.Our study will further expand the understanding of the complexity of the PA-X protein in regulating influenza virus pathogenicity and discover novel amino acid sites that determined the "host-shutoff" activity of PA-X protein.1.The effect of PA-X on the pathogenicity of H7N9 virus in miceTo explore the effect of PA-X on the pathogenicity of H7N9 virus in mice,we constructed a series of PA-X-deficiency viruses(GD15PAX-3,GD15PAX-5,r15PAX-3 and r15PAX-5)based on the highly pathogenicity H7N9 AIVs strain A/Chicken/Guangdong/GD15/2017(GD15PA)and its attenuated mutant strain r15PA whose HA cleavage site was deleted.Then we systematically compared their differences in viral replication,polymerase activity,"host-shutoff" ability,pathogenicity in mice and virus-induced innate immune response,respectively.The results showed that the replication ability of recombinant viruses GD15PAX-3 and GD15PAX-5 were significantly enhanced after 24h and 36h post infection compared with that of GD15PA.In addition,the replication ability of the recombinant virus r15PAX-3 was significantly increased after 36h,48h and 60h post infection than r15PA.However,the replication of r15PAX-5 was significantly decreased compared with its parental strain r15PA.The polymerase activity results showed that loss of PA-X expression significantly enhanced viral polymerase activity.In addition,the ability of recombinant viruses to inhibit the expression of GFP and Renilla luciferase reporter gene(pRL-TK)were significantly attenuated,indicating that H7N9 AIVs PA-X has "host-shutoff" activity.The pathogenicity results in mice showed that there were no significant differencesbetween GD15PA/r15PA and their corresponding PA-X-deficiency recombinant viruses.However,the replication ability of the recombinant viruses GD15PAX-3 and r15PAX-5 in mice were significantly enhanced than the parental GD15PA and r15PA virus,respectively.Then we systematically compared the results of histopathological changes in the mouse lung caused by these recombinant viruses.And we found that compared with the mock control,the virus-infected mouse lung showed some typical histopathological changes,such as mild alveolar hemorrhage,infiltration of a small number of lymphocytes in perivascular,bronchial,and alveolar,lung edema and alveolar expansion.However,by statistical analysis of the lung lesion score,we found that there was no significant difference among the recombinant viruses-infected mice and the parental virus at each time point.Through the analysis of the results of the innate immune response induced by virus infection in the mouse lungs,we found that the recombinant virus GD15PAX-3 showed significantly lower expression level of MIP-1β when compared to the GD15PA WT virus at 3 d p.i..In addition,the r15PAX-3 and r15PAX-5 recombinant viruses showed significantly higher expression level of MIP-1α and CXCL11.Most importantly,as for the overall expression level of all the tested genes on total three days(designed as ld+3d+5d),GD15PA/r15PA WT viruses and GD15PAX-3/r15PAX-5 were comparable,while GD15PAX-5 and r15PAX-3 group were slightly higher than that of GD15PA/r15PA and GD15PAX-3/r15PAX-5 virus.Therefore,our study showed that the PA-X protein could regulate the replication of the H7N9 AIVs.However,PA-X has minor effect on the virulence of H7N9 AIVs in mice.In addition,like other subtypes of influenza viruses,the H7N9 AIVs PA-X has "host-shutoff"activity,indicating that the regulation of the PA-X may be subtype or strain-specifical.Therefore,our study may shed some light on the intricate impact of the PA-X in regulating viral virulence of different subtype of influenza A viruses.2.Identification of the "host-shutoff" functional region of PA-X and the pathogenicity of H7N9 virus in miceIn the previous chapter,we have confirmed that the PA-X protein of H7N9 AIVs has"host-shutoff" activity.In order to further explore the possible key functional areas of the PA-X protein,we then constructed a series of mutant plasmids based on the N-terminal and C-terminal potential functional region of the PA-X protein,namely pCAGGS-PAX-N1、pCAGGS-PAX-N2、pCAGGS-PAX-C1 and pCAGGS-PAX-C2.Based on the five reporter gene plasmid(pIFN-β-Fluc、pISRE-Fluc、GLUC、pRL-TK and GFP),we further verify the "host-shutoff" activity of these different recombinant plasmids.The results showed that the "host-shutoff" activity of pCAGGS-PAX-N1,pCAGGS-PAX-C1,pCAGGS-PAX-C2 showed significant differences than compared with the parental plasmid pCAGGS-PAX.Subsequently,we constructed a series of single-point mutations and combined mutations plasmids to compare their "host-shutoff" activity.We also rescued the corresponding recombinant viruses of each mutation.The results showed that when the amino acids at the N-terminal of PA-X were 941(isoleucine,I)or 100V(valine,V),the"host-shutoff" activity of the corresponding plasmids pCAGGS-PAX or pCAGGS-PAX-N6 were significantly enhanced.When the amino acids at the C-terminal of PA-X were 194L(Leucine,L),198R(Arginine,R)and 198R(Arginine,R),the "host-shutoff" activity of the corresponding plasmids pCAGGS-PAX-C1,pCAGGS-PAX-C3 and pCAGGS-PAX-C4 were also significantly enhanced.Moreover,the replication and virulence in mice of the corresponding recombinant viruses GD15PAX-N6,GD15PAX-C3 and GD15PAX-C6 were significantly enhanced.Therefore,these results showed that 94I,100V,194L,198R,203R amino acids play an important role in regulating the "host-shutoff" activity.Among these amino acids,the 100V,198R,203R amino acids also play important roles in regulating virus replication and virulence in mice.Then we constructed a series of combined mutations plasmids(94,96,97,100,115,194,198,203)to explore the "host-shutoff" activity.The results showed that when amino acids were 941/100V,the "host-shutoff" of the corresponding mutant plasmids pCAGGS-PAX-N6 was significantly enhanced.At the same time,the replication in vitro and virulence in mice were significantly enhanced of the corresponding recombinant virus GD15PAX-N6;when amino acids were 194P/198R/203R,the "host-shutoff" of the corresponding mutant plasmids pCAGGS-PAX-C2 was significantly enhanced.At the same time,the replication in vitro and virulence in mice were significantly enhanced of the corresponding recombinant virus GD15PAX-C2.In addition,the recombinant viruses of GD15PAX-N3,GD15PAX-N6,GD15PAX-C2,GD15PAX-C3,GD15PAX-C4 and GD15PAX-C6 induced more extensive viral infections and more serious pathological damage to the mouse lungs than that of the parental virus GD15PA.By the analysis of the results of the innate immune response induced by virus infection in the lungs of mice,the expression of CXCL9 produced by the host stimulated by recombinant virus GD15PAX-N3 was significantly lower than that of the parental virus GD15PA.The expression of TNF-αand MIP-1β stimulated by recombinant virus GD15PAX-C4 was significantly lower than that of recombinant virus GD15PA.In addition,GD15PAX-N3 infection group caused an overall higher innate immune response than the parental virus GD15PA.GD15PAX-C2,GD15PAX-C3 and GD15PAX-C4 infected groups caused an overall lower innate immune response than that of the parental virus.Therefore,these results showed that the combined mutation 94I/100V or 194P/198R/203R which significantly enhanced the "host-shutoff"activity of the PA-X protein,also significantly enhanced the replication and virulence of H7N9 AIVs.Subsequently,we determined whether the key amino acids(94I,100V,194L,198R,203R)that affect the "host-shutoff" activity of the H7N9 AIVs PA-X exert similar effect on H5N1 AIVs.We then constructed the parent plasmid pCAGGS-CK10-PAX and a series of corresponding mutant plasmids.Four reporter gene,including pIFN-β-Fluc、pISRE-Fluc、GLUC and pRL-TK were included for validation.The results showed that 941 and 100V played an important role in regulating the "host-shutoff’ activity of H5 AIVs PA-X protein.However,the amino acids 198R and 203R have no effect in regulating the "host-shutoff"activity of H5 AIVs PA-X protein.Interestingly,the effect of amino acid 194L on"host-shutoff" of the PA-X protein was quite opposite from that of H7N9 virus.In summary,in this study,the novel key amino acid sites(94I,100V,194P,198R,203R)and functional regions(941/100V and 194P/198R/203R)that determine the "host-shutoff" of the H7N9 AIVs PA-X were successfully identified.In addition,the amino acids which enhance the "host-shutoff" of the PA-X protein,including 100V,198R and 203R also elevated the viral replication and virulence in mice.Simultaneously,the function of 941 and 100V amino acids on the H5 AIVs PA-X was quite similar with that of H7 AIVs PA-X.However,the amino acids 198R and 203R have no effect in regulating the "host-shutoff"activity of H5 AIVs PA-X protein,indicating that the function of the PA-X may be subtype or strain-specific.Therefore,our study further enriched the understanding of the complexity of PA-X protein in regulating viral virulence and protein function.