Screening Of H9N2 AIV Proteins That Down-Regulate The Expression Of FcRY And Preliminary Study On Their Regulatory Effect In HD11 Chicken Macrophage Cell Line

The H9N2 subtype avian influenza virus infection primarily induces respiratory disorders,sinusitis,as well as vitellogenic peritonitis in poultry.Furthermore,the virus can infect humans across species,hereby endangering both the sound growth of the worldwide poultry industry and human health.IgY is a major immunoglobulin in poultry,and FcRY is a transport receptor for IgY,which can bidirectionally transcytose IgY across polarized epithelial cells,playing a crucial role in poultry immunity.Our previous studies have shown that H9N2 subtype avian influenza virus infected HD11 chicken macrophage cell line can down regulate the expression of FcRY,while the UV inactivated virus can up regulate the expression of FcRY,thus presumably the viral protein is involved in the regulation of FcRY expression in HD11 cells.1.Screening of H9N2 AIV proteins that down-regulate FcRY expression in HD11cellsRNA from influenza virus was extracted,and c DNA was obtained by reverse transcription,which was used as template with specific primers for the influenza virus protein gene for PCR amplification,after recovery,digestion,ligation of the amplified product was performed with the eukaryotic expression vector p CAGGS-HA.After enzymatic identification and verification by sequencing,the plasmid was removed for endotoxin extraction and stored at the measured concentration.11 successful constructed viral protein particles(NS1,M1,M2,NP,HA,NA,PB1-F2,NS2,PA,PB1,PB2)were transfected into HD11 cells for 24 hours and 36 hours,respectively.The expression of viral proteins in HD11 cells was confirmed by Western blot.Then,The expression levels of FcRY at the transcriptional and protein was detected by fluorescence quantitative RT-PCR and Western blot.The results showed that NS1 and M1 proteins could reduce the expression of FcRY,NP,NA and PB2 proteins could up-regulate the expression of FcRY,and PB1-F2 could up-regulate the expression of FcRY at 24 hours.Both PA and PB1 up-regulated FcRY expression at 36 hours,HA up-regulated FcRY expression at 24 hours,and down-regulated FcRY expression at 36 hours,while M2 and NS2 had no effect.2.Effects of NS1 and M1 proteins on MAPK and NF-κB signaling pathways after transfection of HD11 cellsNS1 or M1 protein pellets were transfected into HD11 cells for 24 hours,and the key proteins involved in the MAPK,NF-κB signaling pathway were determined by Western blot.Compared with the control group,the results showed that the NS1 protein promoted the phosphorylation of two key proteins in the MAPK pathway,p38 and JNK,and inhibited the phosphorylation of ERK,and the M1 protein promoted the phosphorylation of JNK and ERK,indicating that the NS1 protein can activate the p38 / MAPK and JNK / MAPK pathways,and inhibit the ERK / MAPK pathway,and the M1 protein can activate the JNK/ MAPK pathway,ERK / MAPK pathway;After stimulation of HD11 cells with NS1 or M1 proteins,we found that all could inhibit p65 phosphorylation when compared with the control cells,indicating that both NS1 and M1 proteins could inhibit the NF-κB signaling pathway.3.FcRY core promoter construction and screeningNetwork Promoter Prediction(NNPP)promoter software was used to predict the transcription start site of FcRY,and 1232 bp upstream of ATG was set as the transcription start site of FcRY after analysis.Then the construction of FcRY truncated promoter fluorescent reporter plasmid was carried out to screen the core promoter region of FcRY using dual luciferase gene reporter assay,and the core promoter region of FcRY was found to be in the range of 52～1247 bp.4.Relationship between chicken FcRY promoter and transcription factors p65 and AP-1Transcription factors chicken p65/NF-κB and AP-1/ JNK eukaryotic overexpression plasmids were carried out to analyze the effects of overexpression of chicken p65 and AP-1 on FcRY promoter activity in HD11 cells using a dual luciferase reporter assay.It was found that transcription factors p65 and AP-1 all reduced the promoter activity of FcRY.In conclusion,this study found that the NS1 and M1 proteins of H9N2 subtype avian influenza virus could down-regulate the expression of FcRY in HD11 cells;the NS1 protein can activate the p38 / MAPK and JNK / MAPK pathways,and inhibit the ERK / MAPK pathway,and the M1 protein can activate the JNK / MAPK pathway,ERK / MAPK pathway;both NS1 and M1 proteins could inhibit the NF-κB signaling pathway.the core promoter region of FcRY was found to be in the range of 52～1247 bp;transcription factors p65 and AP-1 all reduced the promoter activity of FcRY.