Selection Of RBX In E3Complex For HIV-1Vif-mediated Degradation Of A3G

Vif, a23KD viral accessory protein of HIV-1, is required for production ofinfectious virus in a cell with type-specific manner.Viruses lacking a vif gene areseverely restricted in the ability of viral replication in non-permissive celltypes． The heterokary fused with permissive and nonpermissive cell lines exhibitethe phenotype of nonpermissive cells, the study suggestes that there is a host factornamed CEM15/A3G inhibiting the replication of virus. APOBEC3G widelyexpressed in nonpermissive cells is a cytidine deaminase, which can induce C-to-Umutations on the (-) strand DNA. At the same time, A3G can inhibit the reversetranscription and integration for the genes of virus.Vif can neutralize the cytidine deaminase of A3G by the cellularubiquitin-dependent proteasome machinery.The E3ubiquitin ligase complexes areconsist of CBFβ, Cullin5, ElonginC, ElonginB and RBX. Several domains in Vifwhich are critical for recruitment of E3complex have been identified.TheN-terminal of Vif recognizes CBFβ, the SLQ motif in the BC box is binding toElongin C, the HCCH motif located upstream of the BC box was found to mediatethe interaction with Cullin5.The skeleton protein Cullin5, as a regulatory subunit,can activate E3complex after combined with cofactor CBFβ. The selection forRing-protein of E3complex, which has two isoforms, needs to be resolved.This thesis focus on the selection of Ring-protein for CRL-Vif-CBFβE3complex. We investigate the interactions of Cul5and RBX1/RBX2by usingimmunoprecipitation assay and the critical motif mutation methods. Based on theinteraction, we discussed the selection of RBX on degradation of A3G in cell systemand viral system through RNA interference and he critical motif mutation. Inaddition, we investigate the ubiquitinylation of A3G with solubility analysis, proteinco-purification and in vitro ubiquitinylation assays, et al.The interaction of Cul5and two isoforms of RBX show that Cul5can interactwith RBX1/RBX2whether there is tag or not on target protein. Besides, theinteraction of two isoforms RBX with Cul5is competitive.In the result of mechanism on A3G degradation, we find that two isoforms ofRBX can be assembled in E3complex for A3G degradation. In addition, RBX2playmore important role in this process.In vitro ubiquitinylation assay, we find that two isoforms of RBX are both the components of ubiquitinylation on A3G.All in all, both two isoforms of RBX can interact with the skeleton protein ofCRL-Vif-CBFβE3Ligases. Moreover they can induce A3G ubiquitinylation equally.Since the two isoforms of RBX have competitive interaction with Cul5, the RBX2has the selective advantage in Vif induced the degradation of A3G in vitro.Our work extend the understanding of the function and mechanism on theCRL-Vif-CBFβE3Ligases of Vif induced the degradation of A3G. And provide thepossibility and new target for HIV therapy.