Preliminary Exploration Of The Potential Effect Mechanism Of The H5N1 Highly Pathogenic Avian Influenza Virus PA And PA-X Protein On NF-?B Transcriptional Activity

Systemic infections of humans and birds with highly pathogenic avian influenza A viruses of the H5N1 subtype are characterized by a massive overproduction of cytokines known as cytokine storm.The current H5N1 strains have not yet acquired efficient human-to-human transmissibility.It is well known that H5N1 HPAIV poses a serious pandemic threat due to its occasional infection and high mortality in human beings,and sporadic human infections continue to occur in countries where H5N1 viruses are endemic.The PA protein is the third subunit of the polymerase complex of influenza A virus which plays an important role in viral RNA transcription and replication,host adaption in avian species and mammals,general virus-host interaction,and host protein synthesis shutoff.Our previous study has found that the PA gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus.The PA-X protein is a 29-kD frameshifting product that is encoded by the segment 3 of the IAV genome.Jagger et al.has shown that PA-X inhibits the innate immune response,notably the inflammatory and apoptotic response.Our previous study has revealed that PA-X decreases the viral virulence in avian species and mice by inhibiting viral replication and host immune response.Nuclear factor-kappa B(NF-?B)consists of a family of transcription factors that play indispensable roles in mediating inflammation,immune responses to pathogen infection,proliferation,apoptosis,and stimulation of IFN-?/? synthesis.Based on the important function of PA and PA-X in host response,we surmised the PA and PA-X protein may play a critical role in NF-?B signaling transcription while the underlining mechanisms are still largely unknown.In this study,we analyzed the potential role of influenza virus protein PA and PA-X in NF-?B Transcription.1 Differential immune response and the discrepancy of the two highly pathogenic avian influenza H5N1 viruses with distinct pathogenicity in NF-?B transcriptional activityThe highly pathogenic avian influenza(HPAI)H5N1 viruses which were first detected in sick geese in Guangdong Province in 1996 have spread to over 63 countries.Therefore,threats posed by H5N1 viruses clearly highlight the need to learn more about the interplay of influenza viruses and their hosts.Our lab previously suggested that H5N1 HPAIVs with similar genetic backgrounds may exhibit distinct pathogenicity in specific hosts.CK10 was highly pathogenic in mice and mallard ducks,whereas GS10 was low pathogenic.The two viruses had only 30 aa difference in their whole genome.In this study,strong inflammatory response in terms of the expression level of several cytokines was observed in mallard duck peripheral blood mononuclear cells(PBMC)and DEF infected with CK10 while mild response was triggered in those by GS10 infection.Two rermarkable and intense peaks of immune response were induced by CK10 infection within 24 hours(at 8 and 24 hours post infection,respectively)without reducing the virus replication.Our results showed that CK10 elicited a more potent innate immune response than GS10 and CK10 triggered a robust innate immune response by increasing the number and expression levels of activated genes.The aim of this study was to elucidate determinants of this strong response in cells with a focus on the transcription factor NF-?B.This factor is known as a major regulator of inflammatory response;however,its role in influenza virus replication and virus-induced immune responses is controversially discussed.It is interesting to note the diverse functions of NF-?B in IAV infection.On one hand,some studies suggest that IAV-elicited NF-?B activity helps in virus replication and spreading,based on the finding that NF-?B inhibition also impairs IAV propagation.On the other hand,several reports note an antiviral function of NF-?B.So we explored the regulatory mechanism of NF-?B signal transduction pathway against influenza virus infection in vitro.Moreover,CK10 and GS10 which from different virulence phenotype showed different effect on NF-?B activation,the highly pathogenic avian influenza virus CK10 had a stronger activation effect on NF-?B Transcription than GS10.H5N1 Highly Pathogenic Avian Influenza virus can activate the inflammatory or immune response mediated by NF-?B signal pathway.Control experiments with a low pathogenic influenza strain GS10 revealed a much weaker and less NF-?B-dependent host cell response.2 Analyze the potential role of influenza virus PA protein in NF-?B transcriptionOur previous study have demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice and mallard duck.The exchange of the entire PA caused the largely transfer of viral phenotypes,including virus replication,polymerase activity and manipulation of the innate response,between CK10 and GS10.Moreover,PA gene also plays an important role in inhibiting host type IIFN signaling,a single mutation(K351E)in the PA protein contributes to the inhibitory properties and the high virulence of A/PR/8/34 H1N1 virus in mice.Nuclear factor-kappa B(NF-?B)consists of a family of transcription factors that play indispensable roles in mediating inflammation,immune responses to pathogen infection,proliferation,apoptosis,and stimulation of IFN-?/? synthesis.However,the role of PA in NF-?B signaling is unknown.By comparing the NF-?B activity of the highly pathogenic CK10 and low pathogenic GS10 virus,our data clearly demonstrated that the virulence factor PA gene significantly inhibited the NF-?B transcription in vitro.Moreover,compared with the GS 10-PA,the PA of the highly pathogenic CK10 virus displayed significantly higher inhibitory effect in a dose independent manner.However,we also demonstrated that PA inhibited NF-?B activation not characterized by p65 nuclear translocation and phosphorylation.Furthermore,PA also inhibited NF-?B-regulated inflammatory factors,including IL-6,IL-2,Nos-2 and TNF-?.These data indicate that an important role of PA in the inhibitory effect for NF-?B transcription in vitro,which also suggests the multifunctional role of PA in participating in the modulation of host inflammatory response.However,further understanding of the precise mechanism of PA affect host inflammatory responses may help to develop more effective strategies for the control of highly pathogenic avian influenza.3.Identify the critical amino acid(s)in the PA gene which affect NF-?B transcriptionThe PA protein is the third subunit of the polymerase complex of influenza A virus.Compared with the other two polymerase subunits(PB2 and PB1),its precise functions are less defined,especially its potential role in NF-?B transcription.The CK10 and GS10 viruses have eight amino acid differences in PA gene.Thus,we further investigated the potential role of these amino acids in NF-?B transcription.Mutant plasmid of GS10-PA each contained one mutation(N115S,A127V,E627G,K237E,I353R,D101G,Q259P and T404A)from CK10 PA gene was constructed,respectively.The relative luciferase activity of the PA-K237E,PA-I353R was significantly higher than GS10-PA(p<0.05),while the corresponding activity of PA-N115S,A127V,E627G was significantly lower than GS 10-PA(p<0.05).These results indicate that amino acids N115S,A127V and E627G may mainly contribute to the inhibitory effect mediated by PA on NF-?B transcription.Since we have confirmed that amino acid at residue N115S,A127V,E627G,K237E,I353R of PA affect NF-?B transcription,we then further tested the effect of these amino acids on the expression of NF-?B-regulated gene.We found that almost all the tested plasmids showed inhibitory effects on the expression of the four selected NF-KB-regulated gene(IL-6,IL-2,Nos-2 and TNF-a).However,only residue E627G showed significantly lower inhibitory activity in all the tested cytokines.Therefore,we surmised that the activation of these cytokines may also affect by other pathways or other potential factors.Moreover,we also explored the possible crucial amino acid(s)of PA in the effect of NF-?B p65 nuclear translocation,we found that GS10-PAN115S,GS10-PAA127V and GS10-PAK237E showed significantly higher amount of nuclear p65 protein than GS 10-PA.It is notable that the amino acid at position 127 locates within the first nuclear localization sequence(NLS)of the PA protein,which ranges from amino acids 124 to 139 and position 237 locates within the second NLS which ranges from amino acids 186 to 247.In addition,amino acid 115 also locates in the important functional region which mediates the endonuclease activity of the PA protein.These results provided further insights into the inhibition of PA in NF-?B transcription4.Explore the potential role of the novel influenza virus protein PA-X in NF-?B transcriptionRecently,it was reported that segment 3 of influenza A virus(IAV)encodes a second protein,arising from a +1 open reading frame(ORF)embedded within the PA gene.This ORF is accessed via ribosomal frameshifting to produce a fusion protein,denoted PA-X,whose N terminal 191 amino acids are derived from the PA ORF,while the C-terminal sequence,most commonly 61 amino acids long,is derived from the +1 frameshifted 'X-ORF'.PA-X reduces the pathogenicity of H5N1 HPAIV by inhibiting viral replication and host response.Nuclear factor-kappa B(NF-?B)plays a crucial role in inflammation and immune responses.However,the role of PA-X played in the NF-?B transcription is totally unknown.In addition,the other roles that PA-X may play are not known.In this study,we first showed that PA-X plays an important role in the inhibition of NF-?B activation of influenza A virus when stimulated with poly(I:C).However,NF-?B activation inhibitory effect mediated by PA-X was not characterized by p65 nuclear translocation and phosphorylation.Furthermore,PA-X also inhibited NF-?B-regulated inflammatory factors,including IL-6,IL-2,Nos-2 and TNF-a.These data suggest the multifunctional role of PA-X in participating in the modulation of NF-?B signal pathway.However,further understanding of the precise mechanism of PA-X affect host inflammatory responses and associated signal pathway may help to develop more effective strategies for the control of highly pathogenic avian influenza.