The Regulation Of Vif Function By Cbf? And Evolutionary Pressure For The Selectivity Of Vif Function

Viral infectivity factor(Vif)is the first accessory protein of human immunodeficiency virus type 1(HIV-1)being revealed with detailed mechanism against host antiviral defense.It specifically induces proteasomal degradation of APBOEC3 proteins such as APOBEC3 G and APOBEC3 F.To achieve this goal,Vif hijacks host cellular factors like Cul5,Elo B,Elo C,Rbx and forms a Cullin-Ring based ubiquitin ligase(also known as E3 complex).Once the E3 complex is assembled,Vif interacts with APOBEC3 proteins and induces ubiquitination of the latter,explaining the phenomenon of Vif promoting infection of HIV-1 progenies from certain types of cells.We later determined that another cellular factor CBF? is also critical for the formation of Vif-induced E3 complex.Without CBF?,Vif interacts with only Elo C and Elo B,but no longer with Cul5,thus fails in assembling E3 complex and leaves APOBEC3 proteins intact.In addition to its impact on molecular virology research,the discovery of CBF? also stabilizes Vif protein in prokaryotic E.coli cells,which leads to the solution of Vif crystallization and structure.CBF?,as a critical regulator of host transcription,interacts with RUNX(CBF?)enhances the affinity of RUNX for DNA,specifically with the core binding sites of various promoters and enhancers,such as the macrophage colony-stimulating factor receptor(MCSFR)promoter.This enhancement would affect the transcription of various genes in human cells,resulting in the regulation of cell differentiation and proliferation.In the current study,we have further examined several internal regions of CBF? that are crucial either for assisting Vif or regulating RUNX activity.By testing both N-and C-terminal truncation mutants of CBF?,we have also identified a minimally functional fragment 15-126 of CBF? that can mediate the Vif-induced degradation of A3 G.CBF? Loop3 mutant is irrelevant for RUNX activity.However,the first 6 amino acids of CBF? Loop3 is essential for Vif-mediated A3 G degradation.Overall,we have demonstrated that different domains of CBF? are required for the protein's Vif-and RUNX-related functions,indicating that the CBF?-Vif interaction and CBF? –RUNX binding require different domains of CBF? and suggesting new possibilities for anti-HIV-1 drug design.Comparing to comprehensive studies on Vif-APOBEC3F/G interaction,only a little information has been reported on Vif-APOBEC3 H binding.APOBEC3 H exists in several haplotypes in human cells,and some of them,such as hap II,are of potency against HIV-1 replication.Interestingly,different from the universal effect against APOBEC3F/G,only some Vifs can induce the depletion of APOBEC3 H.We recently discovered two different positions 39 and 48 on HIV-1 Vif,that mutations on such positions would greatly affect the capability of APOBEC3 H degradation without compromising APOBEC3F/G depletion,indicating a different region on Vif protein is critical for APOBEC3 H interaction and/or depletion.Besides APOBEC3 protein depletion,Vif also functions as cell cycle regulator to arrest host cells at G2/M phage(G2 arrest),which as well requires the presence of CBF? and Vif-induced E3 complex.It is worthy to note that,similar to APOBEC3 H hap II depletion,Vif-induced G2 arrest is another function that only apply to some Vif proteins.Indeed,comparison between HIV-1 HXB2 and NL4-3 Vifs leads to the discovery of position 31,33,36,47 and 50 being critical for Vif-induced G2 arrest,while alteration of all five residues grants Vif HXB2 with potency on G2 arresting.The fact that APOBEC3 H hap II depletion and G2 arrest are the only two known features being selective among Vif proteins suggests there may be a possible relationship between these two functions on Vif.In summary,we identified that different domains of CBF? are required for assisting Vif or regulating RUNX activity.Meanwhile,we further studied the molecular mechanism of CBF?-Vif triggered cell cycle G2/M arrest.At laset,we demonstrated that Vif harnessed A3 H degradation or cell cycle regulation to increase virus survival in the presence of certain pressure.