Characterzation Of Polyubiquitination In APOBEC3 Degredation Induced By HIV-1 Vif

Apolipoprotein B mRNA-editingicatalytie Polypeptide like 3G(APOBEC3G or A3G)and related cytidine deaminases such as apolipoprotein B mRNA- editing catalytic polypeptide-like3F (APOBEC, orA3F)are potent inhibitors of retroviruses.Vif is a small protein encoded by all lentiviruses except equine infectious anemia virus. The primary function of HIV-1 Vif is to neutralize A3G antiviral function. In the absence of human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif), APOBEC3G and APOBEC3F are incorporated into HIV-1 virions and hypermutate the viral genomic DNA by theircytidine deaminase activity.Vif protein protects HIV from the anti-viral activity of A3G.Vif utilizes a viral-specific BC-box (SLQY/FLA) to recruit ElonginsB and C and a novel Zn2+-binding HCCH motif to recruitCullin5 (Cu15) to form an E3 ubiquitin ligase targeting A3G forpolyubiquitination and subsequent proteasomal degradation.Thus, intra-cellular levels of A3G are reduced,which blocks their packaging into virions and thus preservesviral infectivity.There is also limited evidence that Vif may also help to exclude A3G from virions by amore direct mechanism,The N-terminal region in Vif has been implicated in interactingwith the N-terminal region of A3G.We know that Proteasomal degradation of APOBEC3G is a critical step for HIV-1 replication. However, the necessity of polyubiquitination of APOBEC3G in this process is still controversial. In this study,we show thatSIVmac Vif degrades A3G through the Cullin5 proteasomal degradationpathway without the degradation of SIVmac Vif itself.This result argues against the model of codegradation of HIV-1 Vif and A3G.When the A3G substrate was expressed, HIV-1 Vif became more stable, supporting the model that HIV-1 Vif functions as an adaptor protein. In the absence of A3G, HIV-1 Vif undergoes ubiquitination within the Cullin5 complex in an autocatalytic manner, as proposed by Zhou et al. for F-box proteins.In the presence of A3G,HIV-1 Vif was shielded from degradation and stabilized.Although SIVmac Vif is more stable than HIV-1 Vif in human cells, SIVmac Vif induces degradation of APBOEC3G as efficiently as HIV-1 Vif; over-expression of APOBEC3G or APOBEC3F stabilized HIV-1 Vif, indicating APOBEC3G degradation is independent of the degradation of Vif. Furthermore, the in vivo polyubiquitination assay showed that lysine free APOBEC3G was also polyubiquitinated. Our data suggest that polyubiquitination of APOBEC3G, not HIV-1 Vif, is crucial for APOBEC3G degradation.As introduced, the polyubiquitylation of A3G was thought to be essential for proteasomal turnover. Accordingly, we tried to map the specific site for A3G polyubiquitylation. Our rationale was that if such a site exists, we should be able to block A3G polyubiquitylation by removal of this site and generate an A3G protein insensitive to Vif. Because polyubiquitylation usually occurs on lysine residues, we first mutated each lysine in A3G to arginine to see if the protein becomes stable in the presence of HIV-1 Vif. However, the Lys-free A3G protein was still sensitive to Vif, Thus, removal of all lysines did not convert A3G to Vif resistance.It had been shown that cysteine, serine, threonine, or N-terminus of a protein can serve as alternative sites for polyubiquitination, In our study, we examine whether the N-terminus of A3G was a target for polyubiquitination. Our investigations revealed that when an HA tag was fused to the N-terminus of A3G20K/R, its protein expression level dramatically increased, HA-A3G20K/R was resistant to HIV-1 Vif-induced proteasomal degradation. We observed that the Vif protein level when coexpressed with HA-A3G20K/R was higher than that when coexpressed with A3G, HA-A3G, or A3G20K/R, although we transfected the same amount of Vif expression plasmid in each instance. This piece of data is consistent with our previous finding that increased expression of A3G will stabilize Vif protein expression.HA-A3G20K/R molecules were packaged into wild type HIV-1 particles, the N-terminus of the HA-tagged lysine-deficient A3G was resistant to HIV-1 Vif-induced degradation, decreased HIV-1 infectivity, and inhibited wild-type HIV-1 reverse transcription.The pulse-chase assay showed that HA-A3G20K/R had a longer half-life than A3G20K/R. We also showed that the N-terminus of A3G was a target for polyubiquitination. The findings further reinforced the idea that polyubiquitination of A3G is essential for its degradation and that the N-terminus of A3G is an essential ubiquitin target for this process induced by HIV-1 Vif. but we found that the N-terminal HA tag did not affect the formation Cullin 5-Vif-APOBEC complex in the affinity precipitation assay, suggesting that the N-terminal HA tag affected a downstream degradation process. A functional Cullin 5-Vif-APOBECcomplex is required for HIV-1 Vif to induce the degradation ofAPOBEC, but we found that the N-terminalHA tag did not affect the formation of the Cullin 5-Vif-APOBEC complex in the affinity precipitation assay, suggesting that the N-terminal HA tag affected a downstream degradation process. In fact, we found that HIV-1 Vif induced less polyubiquitination of HA-A3G20K/R than that of A3G20K/R, suggesting that the HA tag blocked ubiquitinationat the N terminus of A3G.Although N-terminal polyubiquitination of A3G is essential for its proteasomal degradation, blocking the N-terminal ubiquitination alone is not sufficient to inhibit A3G turnover by Vif since we showed that HA-A3G is still sensitive to Vif induced degradation. The fact that HA-A3G20K/R is resistant to Vif induced degradation suggests that internal lysine residues in A3G also play an important role in A3G degradation induced by Vif.Our observations not only enhance our knowledge of the mechanism of degradation of an important host restriction factor, they may also contribute to the development of novel anti-AIDS pharmacologics.