Jembrana Disease Virus Vif Antagonizes The Inhibition Of Bovine APOBEC3 Proteins Through Ubiquitin-mediate Protein Degradation

Viral infectivity factor(Vif)is a virus-assisted factor coded by all lentivirus except Equine infectious anemia virus(EIAV).Vif protein plays a very important role in the process of lentivirus infection.The highly conserved Vif proteins of different lentiviruses also indicate that the function of the Vif protein has to be preserved to a large extent during evolution.Therefore,the study of Vif protein can help to further explore the mechanism of different species of lentivirus infection and defense against host defense mechanisms.Vif protein mainly acts on lentiviral virus replication in host cells by antagonizing the Apolipoprotein B m RNA-editing catalytic polypeptide-like protein(APOBEC)family of proteins.The APOBEC protein family is a type of cytosine deaminase that produces lethal mutations by inducing the genome of the virus,resulting in inactivation of the progeny virus and thus playing an antiviral role.The Vif protein and host cell cytokines together form the E3 ubiquitin ligase complex,which degrades the APOBEC protein through polyubiquitination and proteasome pathways to break through the host's defenses,thereby increasing the infectivity of the lentivirus.Since the mechanism of Vif-mediated degradation of APOBEC3 proteins can provide new strategies for anti-lentivirus treatment,the interaction between Vif and APOBEC3 proteins has been extensively studied.At present,the structure of E3 ubiquitin ligase complex of various kinds of lentivirus Vif,such as HIV-1 Vif,SIV Vif,FIV Vif,MVV Vif,and BIV Vif,has been confirmed.The site of mutual binding between the Vif and the host factor may be a therapeutic target for inhibiting viral infection.Therefore,deepening the understanding of Vif-mediated E3 ubiquitin ligase composition and its interaction with APOBEC3 protein has far-reaching significance for the treatment of lentivirus-induced diseases.In this study,the Vif protein development system of bovine immunodeficiency virus JDV that isolated pathogens in 1993 was studied using biochemistry and molecular biology,cell biology,and virology,and it was found for the first time:(1)JDV Vif has antagonistic effects on bovine A3s(bt A3Z3 and bt A3Z2Z3);We determined the interaction between JDV Vif and bovine A3 s.It was determined through degradation experiments that bt A3Z2Z3,bt A3Z2,and bt A3Z3 are three bovine APOBEC3 s proteins that can be degraded by JDV Vif.Among them,bt A3Z2Z3 and bt A3Z3 have clear antiviral function,but their resistance Viral function can be antagonized by JDV Vif.At the same time,we observed that although bt A3Z2 can be degraded by JDV Vif,it has no antiviral function like bt A3Z1.However,bt A3Z1 is resistant to JDV Vif-mediated degradation and there is no restriction on viral replication.This result suggests that bt A3Z1 evolved to escape the limitations of the BIV or JDV virus and may be selected correctly and may perform other important biological functions.(2)Proteasomal degradation pathway is the molecular mechanism of degradation of bovine A3 s by JDV Vif;In previous studies,the researchers observed that lentiviral viruses usually use the ubiquitin proteasomal degradation pathway to induce the degradation of host antiviral factors,which can escape the host's immune defense mechanism.This mechanism of primate lentivirus represented by HIV-1/SIV and non-primate lentiviruses such as BIV/FIV has been confirmed and widely accepted.In order to verify whether the degradation of bovine A3 s by JDV Vif is through the ubiquitin proteasomal degradation pathway,a series of experiments were designed and it was confirmed that the degradation of bovine APOBEC3 s protein mediated by JDV Vif is through the ubiquitin proteasome pathway.(3)JDV Vif-mediated E3 ubiquitin ligase complex consists of ELOB/C,Cul2 and RBX1;Since the degradation of bovine APOBEC3 s protein mediated by JDV Vif is via the ubiquitin-proteasome pathway,what are the members of the JDV Vif recruitment involved in the formation of the E3 ubiquitin ligase complex? By co-immunoprecipitation experiments,we first determined the binding of ELOB/C,Cul2 and JDV Vif,and did not require the participation of CBF-?.And through further verification,the RBX1-UBE2 M participates in the formation of the JDV Vif-CRL E3 ligase.(4)The BC box,Cullin box and HCHC region of JDV Vif are important functional regions for forming the E3 ubiquitin ligase complex.After clarifying the composition of the JDV Vif-CRL E3 ligase,we further explored the functional domains that play an important role when JDV Vif interacts with intracellular factors.After verification,the BC box of JDV Vif is the 149TLQ151 amino acid sequence,and the cullin box is the Y167 xxxx V/X172 region of the gene sequence.The former participates in the binding of JDV Vif to Elongin B and Elongin C,and the latter participates in the binding of JDV Vif to Cullin2.The zinc finger functional domain H95-C113-H115-C133 of JDV Vif is involved in binding to Cullin2.In summary,we studied the mechanism of JDV Vif-mediated degradation of bovine APOBEC3 protein and found that it is similar to BIV Vif andthat hijacked the Cul2-RBX1 complex,but unlike the HIV-1/SIV and FIV Vif proteins,which hijacked Cul5-RBX2 complex.The E3 ubiquitin ligase that induced degradation of the bt A3 s protein formed by JDV Vif was determined to be independent of CBF-?.In summary,our findings increase the understanding of viral hijacking of the host E3 ubiquitin ligase and demonstrate the evolutionary flexibility of the lentivirus.