Effects Of Phosphorylation And Acetylation Of PB2 And PA On The Biological Characteristics Of H5N1 Avian Influenza Virus And Its Mechanism Exploration

Influenza A virus(IAV)is an enveloped virus with a single-stranded negative-sense RNA genome.The frequent occurrence of influenza virus has caused great harm to human health and economic loss.The rapid evolution of IAV,the emergence of drugresistant strains,and the limited efficacy of vaccines may lead to IAV pandemic.H5 avian influenza viruses have killed hundreds of people,and although these viruses have not yet gained the ability to transmit efficiently among human,H5 avian influenza viruses remain a potential threat to IAV pandemic.Phosphorylation and acetylation can regulate many biological processes,including transcription,chemotaxis,metabolism,cell signal transduction,stress response,proteolysis,apoptosis,and neuronal development.Phosphorylation and acetylation of influenza virus proteins significantly affect the viral life cycle,interferon-mediated innate immune response and cell cycle,and also can be used as targets for antiviral research.IAV polymerase is involved in viral transcription and replication and is highly conserved among different subtypes,so it is considered a good target for antiviral drugs.However,currently,the study of the phosphorylation and acetylation of influenza virus proteins mainly focuses on the NP,NS1 and M1 proteins,and there are few studies on the PB2 and PA proteins of the influenza virus polymerase subunits.In this study,a strain of H5N1 subtype HPAIV A/chicken/Jiangsu/k0402/2010(CK10)was used as the parental virus,and 84 phosphorylation sites in PB2,PB1,PA,NP,M1,M2,NS1,NEP,PB1-F2 and PA-X were identified.Among these sites,63 phosphorylation sites were newly identified.In addition,PA S225 phosphorylation was also reported in H1N1,but its functional role remains unknown.Furthermore,PB2 S181 phosphorylation was repeatedly identified from different samples in this study.In addition,for the first time,we found that the PB2 protein also can be acetylated,and the site was PB2 K187.In this study,by phosphorylation/dephosphorylation-mimetic mutations of PB2 S181 and PA S225,and acetylation/deacetylation-mimetic mutations of PB2 K187,we systematically explored the roles and related mechanisms of phosphorylation and acetylation in the biological characteristics and pathogenicity of the virus.1.Mass spectrometry screening and identification of potential phosphorylation sites and acetylation sites of H5N1 avian influenza virus PA and PB2 proteinsIn this study,CK10 strain was used to systematically identify the phosphorylation and acetylation sites of PA and PB2 based on IP-based mass spectrometry and purified virus-coupled mass spectrometry.A total of 84 phosphorylation sites distributed among 8 main encoded proteins were identified,including those in PB1,PB2,PA,NP,M1,M2,NS1 and NEP of influenza virus,as well as in the accessory proteins PB1-F2 and PAX.In addition,63 phosphorylation sites were the newly identified.We also identified the first PB2 acetylation site K187.Among these phosphorylation sites,PA S225 phosphorylation was also reported in the H1N1 influenza virus,while PB2 S181 phosphorylation was identified in both IP-based mass spectrometry and purified virusbased mass spectrometry.In addition,we systematically analyzed the functional regions where these sites were located in,and found 7 sites of PA protein(S60,T173,T177,S184,S190,S194,S225,S280,S395,S405,T564,T567,T570)and PB2 S181,K187,S653 which all located in important functional structural regions.In conclusion,this study further enriched the database of phosphorylation and acetylation sites of influenza virus proteins,laying a foundation for subsequent mechanism studies.2.The effect of PB2 S181 phosphorylation and K187 acetylation on viral biological characteristics in H5N1 subtype avian influenza virus and its mechanismIn the first chapter,we found that there is a highly conserved phosphorylation PB2 S181 identified repeatedly and the first identified PB2 acetylation site K187.In this chapter,the CK10 virus was used as the wildtype virus(rPB2-WT),and the PB2 S181 phosphorylation/dephosphorylation-mimetic mutant recombinant viruses(rPB2S181E/rPB2-S181A)and K187 acetylation-mimetic mutant recombinant viruses(rPB2-K187Q)were successfully rescued.Based on the above recombinant viruses,a series of experiments were carried out.Protein structural modeling analysis showed that the PB2 S181 and K187 PTM-mimetic mutations did not significantly alter the structure of the PB2 protein.Viral replication assay revealed that PB2 S181E significantly attenuated the replication ability of wildtype virus in mammalian cells,but had no significant effect on the replication ability of wildtype virus in avian cells.In addition,the replication ability of rPB2-S181A and rPB2-K187Q were both comparable with rPB2-WT in MDCK and DF1 cells.Viral polymerase activity results showed that PB2 S181A significantly enhanced viral polymerase activity,while PB2 S181E,PB2 K187R and PB2 K187Q all significantly inhibited viral polymerase activity.Protein stability experiment results showed that wild-type PB2 protein was more stable than that of PB2 mutant protein.In nuclear accumulation experiments,we found that the PB2 S181E and K187Q significantly inhibited the nuclear import and export of PB2 protein,and PB2 S181A only inhibited the nuclear import of PB2 protein.In the mouse pathogenicity experiment,we found that PB2 S181E significantly attenuated the pathogenicity and replication ability of the virus in mice,weakened the lung injury ability of the virus,and attenuated IAV-induced innate immune response.Moreover,PB2.S181A slightly attenuated the pathogenicity and replication ability of the virus in mice while PB2 K187Q slightly enhanced the virus pathogenicity and replication ability in mice,enhanced viral induced innate immunity-related cytokine expression.In conclusion,PB2 S181E mimetic phosphorylation significantly inhibited the replication ability and viral virulence in mice.Mechanistically,PB2 S181E significantly suppress viral polymerase activity,PB2 protein stability,PB2 nuclear-cytoplasm shutting ability and IAV-induced innate immune responses.Therefore,the antiviral effect of PB2 S181E mimetic phosphorylation provides a new target for the subsequent study of antiviral drugs.3.The effect of PA S225 phosphorylation on viral biological characteristics in H5N1 subtype avian influenza virus and its mechanismIn the first chapter,we found PA S225 is a phosphorylated site.The phosphorylation of PA S225 has been reported in H1N1 subtype strains,but the role of PA S225 phosphorylation in influenza virus replication and its mechanism remained unknown.We used CK10 as the wildtype virus(rPA-WT),and successfully rescued PA S225 phosphorylated/dephosphorylated-mimetic mutant recombinant viruses(rPA-S225A and rPA-S225E).Based on the above recombinant viruses,a series of experiments were carried out.Protein structure simulation analysis showed that both PA S225A and PA S225E significantly altered the structure of PA protein.Viral replication assay revealed that PA S225A significantly inhibited virus replication in mammalian and avian cells,while PA S225E promoted early viral replication in Vero cells and inhibited the late viral replication in DF1 cells.In the viral polymerase activity experiments,we found that PA S225A and S225E could significantly attenuate viral polymerase activity.In nuclear accumulation experiments,we found that PA S225A significantly promoted the nuclear import and export of PA protein,while PA S225E significantly inhibited the nuclear import but promoted the nuclear export of PA protein.Mouse pathogenicity experiments revealed that PA S225A significantly inhibited the replication ability and pathogenicity of the virus in mice,weakened the lung injury-mediated by the virus,and down-regulated the expression of IAV-induced innate immune response-related cytokines.In adition,PA S225E slightly enhanced the virus replication ability in mice and pathogenicity,while had no significant effect on virus-induced lung injury in mice and the expression of IAV-induced innate immune response-related cytokines.In conclusion,PB2 S225A mimetic phosphorylation significantly inhibited the replication ability and viral virulence in mice.Mechanistically,PA S225A significantly suppress viral polymerase activity,enhancing PA nuclear-cytoplasm shuttting ability and IAVinduced innate immune responses.The antiviral effect of PA S225A mimetic dephosphorylation provides a new target site for antiviral drugs research.