Polyubiquitination of APOBEC3G Is Essential for Its Degradation by HIV-1 Vif

Abstract

Proteasomal degradation of APOBEC3G is a critical step for human immunodeficiency virus type 1 (HIV-1) replication. However, the necessity for polyubiquitination of APOBEC3G in this process is still controversial. In this study, we showed that although macaque simian immunodeficiency virus (SIVmac) Vif is more stable than HIV-1 Vif in human cells, SIVmac Vif induces degradation of APBOEC3G as efficiently as HIV-1 Vif. Overexpression of APOBEC3G or lysine-free APOBEC3G stabilized HIV-1 Vif, indicating that APOBEC3G degradation is independent of the degradation of Vif. Furthermore, an in vivo polyubiquitination assay showed that lysine-free APOBEC3G was also polyubiquitinated. These data suggest that polyubiquitination of APOBEC3G, not that of HIV-1 Vif, is crucial for APOBEC3G degradation.

N-Terminal Hemagglutinin Tag Renders Lysine-Deficient APOBEC3G Resistant to HIV-1 Vif-Induced Degradation by Reduced Polyubiquitination

“…1A, lanes 2, 4, and 6, respectively), 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 (60).…”

“…As briefly mentioned in the introduction, Iwatani et al showed that lysines 297, 301, 303, and 334 are critical for A3G degradation (31), as mutation of these four residues to arginines dramatically increased A3G stability. Although this observation contradicts our present data and previously published studies by us and the Zheng lab in which lysine-deficient A3G is still sensitive to Vif-induced degradation (19,60), it may be reasonable to speculate that lysines 297, 301, 303, and 334 and the N terminus of A3G are essential ubiquitin sites for Vif to induce A3G degradation. This discrepancy warrants further investigation.…”

“…Mutation of the four lysine residues to arginine could render A3G resistant to Vif-induced degradation, implying that polyubiquitination of A3G is essential for its degradation by HIV-1 Vif. However, we previously reported that lysine-deficient A3G is still ubiquitinated and degraded by HIV-1 Vif (60). In this study, we showed evidence that the N terminus of A3G was a target for polyubiquitination by HIV-1 Vif and that fusing the hemagglutinin (HA) tag to the N terminus of lysine-deficient A3G blocked its degradation.…”

APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization

“…These results indicate that, despite the cross-species comparison, SIV Vif is still using the canonical poly-ubiquitination and proteasome degradation pathway to purge cells of human A3B. These results mirror prior studies on human A3G and A3F, where 20 and 19 lysines were simultaneously converted to arginine, and the K-less variants resisted Vif-mediated degradation (Albin et al, 2013; Dang et al, 2008; Shao et al, 2010). Since A3B may very well restrict the replication of other viruses, including HBV, HPV, and HTLV (Ahasan et al, 2015; Lucifora et al, 2014; Ooms et al, 2012; Starrett et al, 2016; Starrett et al, 2017; Verhalen et al, 2016; Vieira et al, 2014; Warren et al, 2015), it is likely that additional A3B counteraction strategies exist and that the functional K-free construct described here may be useful as a mechanistic probe.…”

Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Targeting the Interaction between Vif and ElonginC

“…3A). To date, there is no consensus on the necessity for polyubiquitination of Vif for A3G degradation (13,51). However, the notion that both hA3G and HIV Vif are ubiquitinated and degraded by the same E3, ElonginB/C-Cullin5, is generally accepted (13,16,31,43).…”