Investigation Of The Effection Of IER3 Gene Regulae Bovine Viral Diarrhea Virus Replication

Bovine viral diarrhea/Mucosal disease(BVD/MD)is an acute contact infectious disease,mainly caused by Bovine viral diarrhea virus(BVDV)infection,which can cause Persistent calf infection and diarrhea mucosal disease,among which the fatality rate of mucosal disease is as high as 100%,posing a serious threat to large-scale breeding and the safety of biological products.Our group used BVDV to infect Cajal interstitial cells(Interstitial cells of cajal,ICC)in the early stage,and performed Illumina high-throughput transcriptomics sequencing.The results showed that 539 genes,including IER3,HEBP1 and SERPINF2,were up-regulated and 268 mRNAs were up-regulated after ICC cells were infected.Gene mRNA is down-regulated,but whether these genes can affect the replication of BVDV and the specific mechanism of how it affects the replication of BVDV have not yet been reported.Early response gene 3(Immediate early response 3,IER3)is a key regulator in the immune response and plays a key role in the proliferation and differentiation of many cells.Studies have found that the IER3 gene is associated with viral infections.Studies have shown that BVDV infection in MDBK cells can induce the expression of IER3,resulting in a decrease in the synthesis of interleukin-8(IL-8),thereby blocking the NF-?B pathway;however,there is no research on the regulation of BVDV by IER3 Report.This study explores the above key issues.The specific test content and results are as follows:1.Research on gene screening and effect of regulating the replication of bovine viral diarrhea virus:CRISPR/Cas9 technology was used to knock out the IER3,MCPIP1,HEBP1,SERPINF2 genes of MDBK cells,and the knockout efficiency was detected by sequencing and Western blot;BVDV infected cells with gene knockout,using immunofluorescence staining and cytopathic effect(Cytopathic effect(CPE),real-time quantitative PCR(qRT-PCR)and virus titer determination methods are used to detect the replication of BVDV and study the influence of genes on BVDV replication.The test results showed that:IER3 KO,MCPIP1 KO,HEBP1 KO and SERPINF2 KO cells were successfully constructed,and the knockout efficiency was 79.31%,70%,72.41%,and 70.37%respectively;the corresponding protein levels in IER3 KO and MCPIP1 KO cells were significantly reduced;The results of immunofluorescence staining,CPE,qRT-PCR and virus titer showed that the expression of red fluorescence in the knock-out cells was significantly weakened;CPE was significantly reduced,and the production time was later than that of the control group;BVDV 5'UTR mRNA expression level and BVDV The titer was significantly reduced;IER3 and HEBP1 showed better inhibition.It shows that knocking out IER3,MCPIP1,HEBP1,SERPINF2 genes can significantly inhibit the intracellular replication of BVDV,and IER3 has the most significant effect.2.Use the TurboID system of proximity labeling technology to screen the proteins that interact with IER3:Establish cells with stable expression of TurboID and IER3-TurboID;Western blot to detect the expression of IER3-TurboID complex protein,optimize the processing conditions of Biotin;After Biotin treatment,extract the total cell protein,use DynabeadsTM MyOneTM Streptavidin T1 magnetic beads And after chromatography,perform protein profile analysis.The results of the experiment showed that the TurboID and IER3-TurboID cells were successfully constructed,and the IER3-TurboID fusion protein was highly expressed in IER3-TurboID cells;the optimal concentration of Biotin was optimized to be 50 ?M,and the optimal processing time was 6 h;bioinformatics analysis It shows that GO functions are mainly enriched in endoplasmic reticulum stress-related pathways;KEGG shows that it is mainly enriched in protein processing in the endoplasmic reticulum;indicating that IER3 may regulate the replication of BVDV through endoplasmic reticulum stress.3.Research on the effect of IER3 gene on the replication of bovine viral diarrhea virus:After BVDV TC strain was infected with MDBK,qRT-PCR was used to detect the transcription level of IER3 mRNA;Western blot was used to detect the expression level of IER3 protein.Using CRISPR/Cas9 technology to construct knock-out cells,using immunofluorescence staining,qRT-PCR,CPE,and virus titer determination methods to study the effect of interaction factors on BVDV replication.The plasmid was constructed and transfected into 293 T cells.After affinity chromatography,Western blot was used to detect the interaction of proteins.The test results showed that BVDV significantly up-regulated the expression of IER3 protein and mRNA in MDBK cells,indicating that BVDV infection induced IER3 expression;cells with key gene knockouts were successfully constructed,and immunofluorescence staining showed HSP90B1 KO,SEC6248 h after infection The amount of green fluorescently labeled dsRNA in KO,PDIA4 KO and UGGT1 KO cells was significantly reduced,while the green fluorescence in PDIA3 KO,EPHA2 KO,STT3B KO,STIM1 KO cells did not change significantly;qRT-PCR,CPE and virus titer detection The results showed that the levels of BVDV 5'UTR mRNA in the four inhibitory cells were reduced,the CPE of the cells was lower than that of the control group,and the virus titer was significantly reduced;indicating that knocking out the HSP90B1,SEC62,PDIA4 and UGGT1 genes can inhibit the replication of BVDV,among which HSP90B1 And SEC62 genes have the most significant inhibitory effects,while PDIA3,STT3B,STIM1 and EPHA2 genes have no effect on the replication of BVDV.It was found that IER3 interacts with PDIA4 and SEC62 proteins,but has no effect with HSP90B1 protein,indicating that IER3 interacts with PDIA4 and SEC62.Function to regulate the replication of BVDV.This experiment uses CRISPR/Cas9 technology to study that IER3,HEBP1,SERPINF2,and MCPIP1 genes have inhibitory effects on the intracellular replication of BVDV.Among them,IER3 has the most significant effect.The protein profile analysis of adjacent marker TurboID technology found that the IER3 interaction factor has a significant effect.The functions are mainly enriched in the endoplasmic reticulum stress pathways,and the genes related to IER3 and enriched in the endoplasmic reticulum stress pathways are analyzed and screened;they are knocked out using CRISPR/Cas9 technology,and the study found that HSP90B1,SEC62,PDIA4 and UGGT1 gene can inhibit the replication of BVDV;IER3 interacts with PDIA4 and SEC62 proteins.The research results will provide new drug targets for anti-BVDV research.