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. 2010 Feb 10;84(9):4840–4844. doi: 10.1128/JVI.01911-09

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

Qiujia Shao 1, Yudi Wang 1,2, James E K Hildreth 1, Bindong Liu 1,*
PMCID: PMC2863731  PMID: 20147392

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.

Human immunodeficiency virus type 1 (HIV-1) encodes the viral infectivity factor (Vif) to induce proteasomal degradation of APOBEC3G (A3G) (4, 17, 19, 21, 23, 27), a potent host restriction factor of HIV-1 (20). A functional Cul5-Vif-APOBEC3 ubiquitin ligase complex is required for Vif to induce APOBEC3 degradation (15, 22, 28, 29). A3G polyubiquitination has been shown in vivo and in vitro (4, 5, 12, 17, 19, 21, 27). HIV-1 Vif is also ubiquitinated and degraded by the proteasomal pathway (1, 7, 14, 18, 19). Dang et al. mutated all 20 lysines in A3G to arginine and found that lysine-free A3G (A3G20K/R) was still degraded in a Vif-dependent manner; however, they could not detect the polyubiquitination of A3G20K/R (5). The authors argued that polyubiquitination and degradation of HIV-1 Vif are essential for A3G degradation. Here we show evidence that polyubiquitination of A3G, and not that of HIV-1 Vif, is essential for the degradation of A3G.

It has been reported that Vif from other lentiviruses, such as rhesus macaque simian immunodeficiency virus 251 (SIVmac), could also subvert the antiviral function of human A3G through the Cullin5 E3 complex (8, 15, 16, 26). To determine if SIVmac Vif is also codegraded with A3G, we first compared the stability of SIVmac Vif to that of HIV-1 Vif in human 293T cells. Expression vectors for HIV-1 Vif, SIVmac Vif, and tantalus monkey SIV (SIVtan) Vif were transfected into 293T cells. Twenty-four hours posttransfection, the transfected 293T cells were treated with the proteasome inhibitor MG132 (2.5 μM) overnight. Subsequently, the cells were harvested for Western blot analysis. After MG132 treatment, HIV-1 Vif expression dramatically increased (Fig. 1A, lane 5 versus lane 6), while SIVmac Vif (Fig. 1A, lane 1 versus lane 2) and SIVtan Vif (Fig. 1A, lane 3 versus lane 4) expression levels only slightly increased. Next, we used the protein synthesis inhibitor cycloheximide (CHX) to study the half-lives of HIV-1 Vif, SIVmac Vif, and SIVtan Vif. Twenty-four hours after different Vifs were transfected into 293T cells, we treated the cells with CHX (100 μg/ml); nearly 70% of HIV-1 Vif but only 30% of SIVmac Vif and SIVtan Vif were degraded within 120 min (Fig. 1B and C). To determine whether the Cullin5 E3 complex mediates degradation of different Vif proteins, HIV-1 Vif, SIVmac Vif, and SIVtan Vif were cotransfected with either empty vector or a Cullin5 dominant negative mutant, Cul5ΔNedd8 (27), into 293T cells. Because HIV-1 Vif is regulated by Cullin5 E3 ligase, HIV-1 Vif expression levels increased in the presence of Cul5ΔNedd8, as expected (Fig. 1D, lane 2 versus lane 1). By contrast, SIVmac Vif and SIVtan Vif expression levels did not dramatically increase when the function of the Cullin5 E3 complex was blocked by Cul5ΔNedd8 coexpression (Fig. 1D, lanes 4 and 6), indicating that SIVmac Vif and SIVtan Vif are more stable than HIV-1 Vif in 293T cells.

FIG. 1.

FIG. 1.

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SIVmac Vif is more stable than HIV-1 Vif. (A) c-Myc-tagged SIVmac Vif, SIVtan Vif, and HIV-1 Vif were transfected into 293T cells. Twenty-four hours posttransfection, MG132 (2.5 μM) was used to treat the cells for 16 h. An equivalent volume of dimethyl sulfoxide (DMSO) was used to treat cells as a negative control. Vif expression was analyzed by Western blotting, using an anti-c-Myc antibody. Actin staining was used as a loading control. (B) HIV-1 Vif, SIVtan Vif, and SIVmac Vif were transfected into 293T cells. Twenty-four hours posttransfection, cycloheximide ([CHX] 100 μg/ml) was used to inhibit protein translation. Samples were harvested at the indicated time points. Vif expression was visualized by anti-c-Myc antibody in a Western blot. (C) Relative expression levels of Vif were calculated by quantifying the results shown in panel B. Vif expression at minute zero before treatment was set to 1. (D) HIV-1 Vif, SIVmac Vif, SIVtan Vif, and Cul5ΔNedd8 were transfected as indicated. Western blotting was performed, using anti-c-Myc antibody to detect both Cul5ΔNedd8 and Vif proteins. The results are representative of at least three independent experiments.

HIV-1 Vif has been shown to induce degradation of lysine-free A3G (A3G20K/R) and to overcome its anti-HIV function (5). We wanted to test if other lentiviral Vif proteins, such as those of SIV, can induce A3G20K/R degradation and overcome its antiviral function. An HIV-1 Vif-deficient proviral construct (HXB2ΔVif) was cotransfected with A3G or A3G20K/R (from Y. H. Zheng, Michigan State University) and either HIV-1 Vif or SIVmac Vif into 293T cells. Forty-eight hours later, supernatants were harvested for determining infectivity by a multinuclear activation of a galactosidase indicator (MAGI) assay. The viral particles were normalized by standard HIV-1 p24 enzyme-linked immunosorbent assay (ELISA). Both A3G and A3G20K/R dramatically decreased the infectivity of Vif-deficient HIV-1 (Fig. 2A, lanes 2 and 5). However, the infectivity of Vif-deficient HIV-1 was restored in the presence of HIV-1 Vif and SIVmac Vif provided in trans (Fig. 2A, lanes 3, 4, 6, and 7). These data indicate that SIVmac Vif is able to overcome the antiviral function of both A3G and A3G20K/R as efficiently as HIV-1 Vif.

FIG. 2.

FIG. 2.

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SIVmac Vif degrades A3G and A3G20K/R without being degraded by the Cullin5 E3 complex. (A) HIV-1 Vif-deficient proviral construct B2NΔVif was cotransfected with A3G, A3G20K/R, HIV-1 Vif, or SIVmac Vif as indicated. Viral supernatants were collected and tested by MAGI assay for infectivity. HIV-1 p24 ELISAs were used to normalize viral loading. Error bars represent the standard deviations of the results of three independent experiments. (B) A3G or A3G20K/R was cotransfected with SIVmac Vif, HIV-1 Vif, and Cul5ΔNedd8 as indicated. c-Myc (Cul5ΔNedd8 and Vif), V5 (A3G and A3G20K/R), and actin antibodies were used to analyze protein expression by Western blotting. (C) Relative expression levels of Vif were calculated by analyzing the results shown in panel B. The expression of untreated Vif was set to 1. The results are representative of at least three independent experiments.

We then wanted to determine if SIVmac Vif could overcome A3G20K/R by a degradation mechanism. We transfected A3G, A3G20K/R, SIVmac Vif, HIV-1 Vif, and Cul5ΔNedd8 into 293T cells. Both HIV-1 Vif and SIVmac Vif induced degradation of A3G and A3G20K/R (Fig. 2B, lanes 2, 4, 7, and 9). The degradation of A3G and A3G20K/R was blocked by Cul5ΔNedd8 coexpression (Fig. 2B, lanes 3, 5, 8, and 10). Since HIV-1 Vif is also degraded by the Cullin5 complex (14, 18), we observed an increase in HIV-1 Vif expression of 2.5- to 3-fold in the presence of Cul5ΔNedd8 (Fig. 2B, lane 3 versus lane 2 and lane 8 versus lane 7, Western blot; Fig. 2C, densitometry calculation). Surprisingly, when Cul5ΔNedd8 was cotransfected with SIVmac Vif, although A3G and A3G20K/R expression significantly increased, there was almost no change in the expression of SIVmac Vif (Fig. 2B, lane 5 versus lane 4 and lane 10 versus lane 9, Western blot; Fig. 2C, densitometry calculation). These data suggest that SIVmac Vif degrades A3G and A3G20K/R through the Cullin5 proteasomal degradation pathway without being degraded itself and argues against the Vif and A3G codegradation model proposed by Dang et al. (5).

We previously showed that A3G and APOBEC3F expression increases HIV-1 Vif stability (14) and wanted to test if expression of A3G20K/R also increases the stability of HIV-1 Vif. Different amounts of A3G or A3G20K/R DNA were cotransfected with HIV-1 Vif in 293T cells, and Vif expression was analyzed by Western blotting. A3G and A3G20K/R expression increased HIV-1 Vif expression in a dose-dependent manner (Fig. 3A and B). CHX was also used to study the half-life of HIV-1 Vif when A3G or A3G20K/R was coexpressed. Twenty-four hours posttransfection, CHX was used to treat the transfected cells to stop translation. HIV-1 Vif was rapidly degraded by 80% in 120 min without A3G expression, while approximately 20 to 30% of Vif was degraded in 120 min when A3G or A3G20K/R was coexpressed (Fig. 3C and D). With these findings taken together, we concluded that HIV-1 Vif is more stable when A3G or A3G20K/R is coexpressed. Dang et al. proposed a model that polyubiquitinated HIV-1 Vif functions as a vehicle to transport A3G to proteasomes for degradation. According to this model, expression of A3G or A3G20K/R would have no effect on the half-life of Vif or even shorten its half-life by engaging more Vif molecules in the degradation pathway. As shown in Fig. 3, our data indicate that expression of A3G or A3G20K/R stabilizes HIV-1 Vif. HIV-1 Vif appears to be analogous to F-box protein within the Cullin5 complex (14, 18). Our result, together with the results from Galan et al. (9) and Li et al. (13), favors the “substrate shield model” proposed by R. J. Deshaies (6). According to this model, binding of the substrate shields the F-box protein from autoubiquitination, and overexpression of the substrate is thus predicted to stabilize the corresponding F-box protein. Although SIV Vif shares some critical domains with HIV-1 Vif, such as the BC-box motif and the HCCH motif, which are essential for functional Cullin5 ubiquitin complex formation, there is only around 25% homology between these two proteins. This structural difference may prevent SIVmac Vif from autoubiquitination in human cells, so that SIVmac Vif could induce A3G degradation without being degraded. It has also been reported recently that MDM2, a human E3 ligase, induces polyubiquitination and degradation of HIV-1 Vif and reversely increases A3G levels (11). This might be another unique feature for the rapid degradation of HIV-1 Vif in human cells.

FIG. 3.

FIG. 3.

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HIV-1 Vif is more stable when A3G or A3G20K/R is coexpressed. (A) Different doses (1 μg, 2 μg, and 4 μg) of A3G and A3G20K/R (indicated by black triangles) were cotransfected with HIV-1 Vif. pcDNA3.1 empty vector was transfected as a control lacking A3G. V5 (A3G and A3G20K/R) and c-Myc (HIV-1 Vif) antibodies were used to visualize protein expression levels in Western blots. (B) Relative expression levels of Vif were analyzed by quantifying the intensity of the Western blot. Expression for a control lacking A3G was set to 1. (C) HIV-1 Vif was cotransfected with pcDNA3.1, A3G, and A3G20K/R. Twenty-four hours after transfection, CHX (100 μg/ml) was used to monitor the half-life of HIV-1 Vif. (D) Relative expression levels of Vif were calculated by analyzing the intensity of the Western blot. Vif expression at minute zero before treatment was set to 1. The results are representative of at least three independent experiments.

To determine if HIV-1 Vif could induce the polyubiquitination of A3G20K/R, we transfected A3G, A3G20K/R, hemagglutinin (HA)-tagged ubiquitin, HIV-1 Vif, and c-Myc-tagged Cullin5 mutant expression vectors as indicated in Fig. 4. As suggested by Iwatani et al. (10), A3G20K/R has two lysine residues in the C-terminal tag region. We removed the two lysine residues by deleting the lysine- containing region (to make a construct termed A3G20K/RΔ2K) to avoid the influence of the two lysines on A3G20K/R polyubiquitination. Polyubiquitination of A3G29K/R2ΔK was also evaluated as indicated in Fig. 4. Cells were treated for 16 h with 2.5 μM MG132 at 24 h posttransfection and were lysed in lysis buffer as described previously (27). Powdered urea was added into each cell lysate to a final concentration of 8 M. Ni-NTA agarose (Qiagen) was used to immunoprecipitate APOBEC under denaturing and native hybrid conditions according to instructions in the Invitrogen Ni-NTA purification system manual. Polyubiquitinated forms of APOBEC were visualized by staining HA-tagged ubiquitin molecules. Ubiquitinated A3G, A3G20K/R, and A3G20K/RΔ2K were not detected in cells lacking Vif expression (Fig. 4A, lanes 1, 4, and 7). However, significant levels of ubiquitinated A3G and A3G20K/R were detected in the presence of HIV-1 Vif (Fig. 4A, lanes 2 and 5). Polyubiquitination of A3G20K/RΔ2K was detected to a lesser extent (Fig. 4A, lane 8), suggesting that lysine residues are the dominant sites for A3G polyubiquitination. HIV-1 Vif-induced ubiquitination of A3G, A3G20K/R, and A3G20K/RΔ2K was inhibited by Cul5ΔNedd8 (Fig. 4A, lanes 3, 6, and 9). When this membrane was stained by anti-V5-tagged antibody to visualize A3G, only unmodified A3G (not the polyubiquitinated form of A3G) was detected (Fig. 4B), indicating that only a very small amount of A3G was polyubiquitinated and that the amount is too small to be detected by anti-V5-tagged antibody. In a previous study, Dang et al. reported in vivo polyubiquitination of A3G, but not A3G20K/R (5). There are three major differences between our in vivo polyubiquitination assay and that of Dang et al. First, in our assay we used MG132, a proteasome inhibitor, to block 26S proteasome function to reduce the degradation of polyubiquitinated APOBEC. In the absence of a proteasome inhibitor, 26S proteasome will quickly turn over polyubiquitinated proteins so that the chance of detecting polyubiquitinated forms of proteins will be decreased. Second, we cotransfected wild-type ubiquitin with APOBEC instead of the Ubiquitin48A (Ub48A) mutant. As mentioned by Dang et al., the Ub48A mutant interrupts Lys-48-linked ubiquitin chain initiation (5). Therefore, using Ub48A instead of wild-type ubiquitin decreases the formation of polyubiquitinated forms of APOBEC. Third, they immunoprecipitated Ub48A-FLAG in their assay. Since ubiquitinated forms of APOBEC represent only a small portion of the pool of ubiquitinated proteins in the cells, the chance of detecting polyubiquitinated forms of APOBEC after immunoprecipitation of Ub48A-FLAG would be significantly less than with direct immunoprecipitation of A3G. It has been reported that lysine is not the only target for polyubiquitination. Cysteine, serine, threonine, or the N-terminal of the protein could be alternative sites for polyubiquitination (2, 3, 24). Further study is warranted to determine the polyubiquitination site of lysine-free A3G.

FIG. 4.

FIG. 4.

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HIV-1 Vif induces A3G20K/R polyubiquitination. A3G, A3G20K/R, and A3G20K/RΔ2K were transfected with HA-tagged ubiquitin (Ub-HA), HIV-1 Vif, and Cul5ΔNedd8 as indicated. Twenty-four hours posttransfection, MG132 (2.5 μM) was used to treat cells for 16 h. A3G, A3G20K/R, and A3G20K/RΔ2K were immunoprecipitated by Ni-NTA agarose affinity gel (Qiagen) under denaturing and native hybrid conditions. (A) Ubiquitinated forms of A3G, A3G20K/R, and A3G20K/RΔ2K were visualized by anti-HA antibody. (B) A3G, A3G20K/R, and A3G20K/RΔ2K were stained by anti-V5 antibody. The results are representative of at least three independent experiments. WB, Western blotting.

Our data show that SIVmac Vif degrades A3G and A3G20K/R through the Cullin5 proteasomal degradation pathway without the degradation of SIVmac Vif itself (Fig. 1 and 2). 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 and others (9, 13, 25, 30) for F-box proteins. In the presence of A3G, HIV-1 Vif was shielded from degradation and stabilized (Fig. 3). Most importantly, we demonstrated that HIV-1 Vif induced the polyubiquitination of A3G20K/R and A3G20K/RΔ2K, supporting the idea that HIV-1 Vif induces degradation of lysine-free A3G, an idea that is still debated (5, 10). Together, our data support the notion that polyubiquitination of A3G, not that of HIV-1 Vif, is crucial for the degradation of A3G. With increasing attention being paid to the mechanisms of interactions between HIV-1 Vif and A3G, fully understanding how HIV-1 Vif induces the polyubiquitination and degradation of A3G will have a significant impact on novel anti-HIV drug design.

Acknowledgments

We thank X.-F. Yu and Y. H. Zheng for reagents. We thank P. T. Sarkis, M. Linde, and J. Elzey for assistance in editing the manuscript. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program: TZM-bl from J. C. Kappes, X. Wu, and Tranzyme, Inc., and anti-Vif antibody from D. Gabuzda. The HA-ubiquitin plasmid was obtained from Ted Dawson through Addgene.

This work was partially supported by NIH grants 5U54RR019192, 5G12RR003032, and 5P30AI054999.

Footnotes

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Published ahead of print on 10 February 2010.

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