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Research On The Mechanism Of DDIT3 Gene In Promoting Bovine Viral Diarrhea Virus Replication By Inhibiting MAVS

Posted on:2022-03-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:S WangFull Text:PDF
GTID:1480306335472234Subject:Cell biology
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea-mucosal disease(BVD-MD)is a close contact infectious disease of cattle caused by bovine viral diarrhea virus(BVDV)infection.The main clinical symptoms of BVD-MD include mucosal disease,diarrhea,and respiratory and reproductive system disorders.It is worth noting that BVDV can suppress the host immune system and reduce immunity,leading to mixed infections with other pathogenic microorganisms,which seriously threatens the development of the cattle breeding industry in China.At present,the prevention and control of BVD-MD is mainly based on vaccine immunization;however,BVDV,a single-stranded,positive-stranded RNA virus,is characterized by an extremely high mutation rate,multiple circulating strains and ubiquitous distribution,leading to poor prevention and control effects.While focusing on vaccine development,conducting research on host-virus interactions and exploring the molecular mechanisms by which host genes affect virus replication can provide new ideas for the comprehensive prevention and control of BVD-MD.Therefore,in this study,we used high-throughput sequencing technology,which demonstrated that the host gene DNA damage-inducible transcript 3(DDIT3)was differentially expressed after BVDV infection of bovine kidney cells(Madin-Darby bovine kidney(MDBK)cells),and investigated the biological functions and molecular regulatory mechanisms of DDIT3 during BVDV infection.DDIT3,also known as CHOP,C/EBPzeta and GADD153,is a member of the C/EBP transcription factor family and is a 29 kDa protein that plays an important regulatory role in the cellular stress response.DDIT3 gene expression is upregulated in cells under adverse conditions such as DNA damage,endoplasmic reticulum stress and starvation.During viral infection,DDIT3 induces apoptosis or cellular autophagy,but whether it affects viral replication by regulating the natural immune response is unclear.Preliminary analysis revealed that DDIT3 can downregulate the expression of mitochondrial antiviral signaling protein(MAVS,also known as VISA,CARDIF or IPS-1),inhibit natural immunity and promote BVDV replication.MAVS is mainly distributed on mitochondria,and host anti MAVS is a key protein in the natural immune signaling pathway of RNA viruses and a common target molecule for viral antagonism of the natural immune response.However,it is not clear how DDIT3 interacts with natural immunity-related molecules such as MAVS and how this affects the interaction of DDIT3 with BVDV.The present study focused on the relationship between DDIT3 and the natural immune response induced by BVDV infection,and the following results were obtained.(1)The DDIT3 gene promotes the replication of BVDV in MDBK cells.RT-qPCR and Western blot experiments confirmed that both the mRNA and protein levels of DDIT3 were significantly increased during BVDV infection.MDBK cell lines with DDIT3 overexpression or knockdown were established by infection with packaged lentivirus,and the transcription of viral genes,one-step growth curves and viral titers were evaluated.The results showed that DDIT3 could promote BVDV replication.Subsequently,experiments with inhibitors of apoptosis or cellular autophagy demonstrated that DDIT3-induced apoptosis did not affect BVDV replication,suggesting that there are other mechanisms by which DDIT3 promotes viral replication in addition to inducing cellular autophagy.(2)The DDIT3 gene targets MAVS to negatively regulate the natural immune response.To detect the effect of DDIT3 on the antiviral innate immunity,RT-qPCR experiments showed that overexpression of DDIT3 was found to inhibit the transcription of IFN-? and downstream interferon-stimulated genes(ISGs)in MDBK cells.Treatment of MDBK cells with bovine type I interferon could not induce DDIT3 expression,as confirmed by Western blot experiments.RT-qPCR and dual luciferase reporter assays demonstrated that DDIT3 targets MAVS,an important junction protein in the RIG-I signaling pathway,to block antiviral signaling.Furthermore,the cells were treated with proteasome or lysosome inhibitors,and the results of Western blot experiments confirmed that DDIT3 induced by BVDV infection could promote the degradation of MAVS via the ubiquitin proteasome pathway.(3)DDIT3 regulates the NF-?B-OTUDl-Smurfl pathway to promote MAVS degradation.To further investigate the role of MAVS degradation by the proteasome pathway,this study used transcriptome sequencing technology to screen host genes differentially expressed during BVDV infection after overexpression of the DDIT3 gene.The results showed that DDIT3 could promote the upregulated expression of the deubiquitinating enzyme OTU deubiquitinase 1(OTUD1).The inhibitory effect of OTUD1 on type ? interferon antiviral response during BVDV infection of MDBK cells was demonstrated by RT-qPCR and Western blot experiments of stable cell lines generated by lentivirus infection.Immunoprecipitation,Western blot and laser confocal microscopy assays of OTUD1 mutants lacking enzyme activity demonstrated that the deubiquitinating enzyme activity of OTUD1 could remove the ubiquitination of the E3 ubiquitin ligase Smurf1 and increase the protein level of Smurfl,while BVDV infection could promote the interaction between Smurfl and MAVS to degrade MAVS via the proteasome pathway.Western blot and RT-qPCR experiments of cells treated with pyrrolidine dithiocarbamate(PDTC)showed that the DDIT3 gene suppressed the natural immune response against BVDV by activating NF-?B to promote the upregulated expression of OTUD1.(4)Knockdown of the DDIT3 gene in C57BL/6J mice promotes anti-BVDV natural immunity.To investigate the effect of the DDIT3 gene on the anti-BVDV natural immune response in vivo,animal experiments were performed using DDIT3-knockout C57BL/6J mice.In this study,we found by HE staining,indirect immunofluorescence assay,ELISA,RT-qPCR and Western blot that compared with wild-type mice,BVDV-infected DDIT3-knockout mice showed suppressed viral replication in susceptible organs,significantly reduced pathological damage,enhanced transcript levels of IFN-? and ISGs,and significantly increased serum levels of IFN-?.Further studies revealed that both the mRNA and protein levels of OTUD1 were significantly reduced,while the protein levels of MAVS were significantly increased,after DDIT3 knockdown in mice.These results suggest that knockdown of the DDIT3 gene can promote the natural immune response against BVDV infection in mice.In summary,this study demonstrated that the DDIT3 gene induced the gene expression of the deubiquitinating enzyme OTUD1 by enhancing the activation of NF-?B,which in turn increased Smurf1 protein levels to degrade MAVS,thus blocking the antiviral response during BVDV infection,and finally inhibiting the expression of IFN-?and downstream ISGs to promote the replication of BVDV.The results of this study reveal for the first time the molecular mechanism by which DDIT3 modulates viral replication by regulating the host natural immune response which provides novel insight into BVDV immunosuppression and target molecules for the development of antiviral drugs.
Keywords/Search Tags:Bovine viral diarrhea virus, DDIT3, OTUD1, MAVS, Antiviral innate immunity
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