Font Size: a A A

Molecular Mechanism Of BVDV Non-structural Protein Npro-mediated Ubiquitination Degradation Of ELF4

Posted on:2021-04-12Degree:MasterType:Thesis
Country:ChinaCandidate:L WangFull Text:PDF
GTID:2370330602467816Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea virus?BVDV?is a single-stranded positive-strand RNA virus that belongs to the Flaviviridae and pestivirus genus.It mainly causes respiratory symptoms in cattle,diarrhea,mucosal erosion,abortion or birth defects Such symptoms can also cause leukopenia and immunosuppression.Npro protein is the first protein encoded in ORF.It is highly conserved among pestiviruses and has protease activity.However,the mechanism of proteolysis and involvement in regulating immunosuppression cannot yet be fully explained.It is reported that the BVDV Npro protein degrades IRF3 through the proteasome pathway,and E1 ubiquitin activating enzyme plays a role;the transcription factor ELF4 can form a dimer and activate the type I IFN promoter,induce the production of type I IFN,and participate in the body's resistance Virus innate immune response.Our previous research proved that: BVDV non-structural protein Npro degrades Bo ELF4 through the proteasome pathway and negatively regulates the expression of type I interferon;however,the mechanism of Npro protein degradation of ELF4 protein is not fully understood.Therefore,this study explored the molecular mechanism of BVDV Npro protein degradation of ELF4 through ubiquitination,and screened proteins that interact with Npro protein.In order to understand the function of Npro protein and the mechanism of immune escape after BVDV infection,Effective immune prevention and control measures provide theoretical basis.First,the eukaryotic expression plasmid of pc DNA3.1-Npro was constructed,and the target gene was amplified by PCR.After the gel was recovered,the target gene and the expression vector were double-digested,followed by ligation and transformation,the correct recombinant plasmid pc DNA3.1-Npro was transfected into 293 T cells,and verification by Western blot.The results showed that an obvious target band appeared at 23 KDa,which was consistent with the expected size,prove the successful construction of BVDV Npro protein eukaryotic expression vector.Secondly,the bioinformatics software was used to predict the interaction between Npro and ELF4 and potential ubiquitination sites;and the Co-IP method was used to verify the interaction between the two proteins.The results showed that BVDV Npro protein co-precipitated with transcription factor ELF4;BVDV Npro protein degraded ELF4 by ubiquitination,and degraded ELF4 by K48 polyubiquitination.The above results indicate that the BVDV Npro protein interactswith the transcription factor ELF4,and the BVDV Npro protein degrades ELF4 by polyubiquitination at position K48.Finally,the pc DNA3.1-Npro eukaryotic expression plasmid was transfected into MDBK cells,verified by the Co-IP method,and sent to mass spectrometry for GO analysis of the identification results.After that,we screened 25 candidate proteins from the mass spectrometry results,focusing on the verification of Hsp70 protein.As a result,the pc DNA3.0-Hsp70 eukaryotic expression plasmid was successfully constructed;Co-IP results showed that BVDV Npro protein co-precipitated with Hsp70 protein;Hsp70 protein can degrade Bo ELF4,and when adding Npro plasmid,the degradation of ELF4 is more obvious.The above results indicate that there is an interaction between Hsp70 protein and Npro protein,and the two may cooperate to degrade ELF4.In summary,this study demonstrated that BVDV Npro protein interacts with ELF4 and degrades ELF4 through the ubiquitination pathway;BVDV Npro protein interacts with Hsp70 and synergistically degrades ELF4,the mechanism of Hsp70 in synergistic degradation of ELF4 needs further study.This study provides a theoretical basis for further understanding the immune escape mechanism of BVDV-infected organisms and establishing effective immune control measures.
Keywords/Search Tags:bovine viral diarrhea virus, Npro protein, transcription factor ELF4, heat shock protein 70, ubiquitination
PDF Full Text Request
Related items