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J Virol. Sep 2009; 83(17): 8674–8682.
Published online Jun 17, 2009. doi:  10.1128/JVI.00653-09
PMCID: PMC2738209

A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G [down-pointing small open triangle]

Abstract

The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K22x3K26x3Y30x9YRHHY44) and a cluster of hydrophobic residues (V55xIPLx4-5LxΦx2YWxL72) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, here called A3G) and related APOBEC3 proteins are potent inhibitors of diverse viruses and endogenous retroelements (2, 9, 11, 13, 18, 30, 42, 47, 48, 63, 72). The Vif protein of human immunodeficiency virus type 1 (HIV-1) and related viruses provides a viral defense against A3G and other APOBEC3 proteins, allowing infection and replication to proceed in host cells. In the absence of the Vif protein, however, A3G is packaged into HIV-1 particles through its interaction with viral Gag molecules (1, 7, 12, 27, 41, 51, 75), with the help of cellular and/or viral genomic RNAs (5, 22, 61, 65, 75). Virion-associated A3G induces C-to-U mutations in the newly synthesized viral minus-strand DNA (17, 24, 31, 33, 60, 71, 76) and reduces the accumulation of viral reverse transcripts (3, 16, 21, 28, 36, 54, 70) and the formation of proviral DNA (28, 36) through both deamination-dependent (40, 55) and -independent (4, 43) mechanisms.

HIV-1 Vif overcomes the antiviral activity of APOBEC3 by assembling with the components of the cellular cullin 5 (Cul5)-elongin B-elongin C E3 ubiquitin ligase complex (73) to target A3G for proteasomal degradation (10, 25, 26, 35, 37, 56, 59, 73). Vif molecules of HIV/simian immunodeficiency virus (SIV) interact with Cul5 using a highly conserved Hx5Cx17-18Cx3-5H zinc binding motif (29, 38, 66, 67) and a BC box (SLQxLA motif) to bind to elongin C, which in turn interacts with elongin B and Cul5 (23, 25, 29, 37, 58, 73, 74). HIV-1 Vif may also inhibit A3G function through degradation-independent mechanisms (45).

The interactions of HIV-1 Vif with substrate APOBEC3 proteins are complicated and are confined to its N-terminal region (35, 39, 49, 53, 57, 62). However, distinct regions of Vif are involved in various aspects of APOBEC3 binding and/or suppression. Amino acids 40 to 44 (YRHHY) of HIV-1 Vif are important for binding and suppression of A3G, but not another APOBECS subtype, A3F (39, 49, 69, 77). In contrast, amino acids 11 to 17 and 74 to 79 of HIV-1 Vif are important for A3F interaction and suppression, but not A3G inhibition (19, 39, 49, 53, 57, 62, 69, 77). More recently, we demonstrated that HIV-1 Vif can overcome A3C, A3DE, and A3F through similar mechanisms (77). The suppression of A3C and A3DE by HIV-1 Vif requires regions that are important for A3F suppression, but not the YRHHY region that is required for A3G suppression (46, 77). A cluster of hydrophobic amino acids (VxIPLx4-5LxΦx2YWxL, where Φ denotes L, I, or V; amino acids 55 to 72) in HIV-1 Vif is important for its interaction with both A3G and A3F (19, 46).

Regions important for Vif interactions have been mapped to the amino-terminal domain of A3G (10, 20, 50, 77) and the carboxyl-terminal domain of A3F (50, 77). The carboxyl-terminal domain of A3F alone is sufficient for HIV-1 Vif-mediated binding and degradation (77), and the amino-terminal domain of A3G is sufficient to mediate its interaction with HIV-1 Vif (10, 50, 77). In particular, a DPD (amino acids 128 to 130) motif in A3G is important for the A3G-Vif interaction (20, 50). Whether the amino-terminal domain of A3G alone is sufficient for Vif-mediated degradation is still controversial (15, 50, 77).

In the current study, we examined a previously observed (44) but uncharacterized SLV/Ix4Yx9Y motif in HIV-1 Vif function. We now demonstrate that K22, K26, and Y30, in addition to Y40RHHY44, are important for A3G binding and suppression of HIV-1 Vif. These positively charged amino acids and bulky hydrophilic Tyr are largely dispensable for A3F, A3C, and A3DE suppression, representing a unique A3G interaction interface. Furthermore, we have also observed that the LV part of the motif is more important for A3F, A3C, and A3DE suppression. We conclude that the SLV/Ix4Yx9Y motif and surrounding residues contribute to the neutralization of various APOBEC3 subtypes by HIV-1 Vif.

MATERIALS AND METHODS

Plasmid construction.

The infectious molecular clone of HIV-1 (NL4-3) and the derived Vif mutant (NL4-3ΔVif) constructs were obtained from the AIDS Research Reagents Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). pHIV-1 Vif-myc, pVif L145A, and expression vectors for A3C-hemagglutinin (HA), A3DE-HA, A3G-HA, and A3F-V5 have been previously described (73, 77). The plasmids pVif K22E, pVif S23A, pVif L24S, pVif V25S, pVif K26A, pVif Y30A, pVif R36A, pVif Y40A, and pVif RH 41/42AA were made from pHIV-1Vif-myc by site-directed mutagenesis and confirmed by DNA sequencing.

Cell culture, transfection, and viral infectivity (MAGI) assays; A3G, A3F, A3C, and A3DE expression; and antibodies.

293T and MAGI-CCR5 (AIDS Research Reagents Program) cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum and penicillin-streptomycin (D-10 medium). Transfection was performed with Lipofectamine 2000 (Invitrogen) as instructed by the manufacturer. The viral infectivity was normalized for input virus using p24 measurement, and the MAGI assay was performed as previously described (25, 73). Virus was produced by transfecting 293T cells in a six-well plate with 1.5 μg of NL4-3 ΔVif, 1.5 μg of wild-type (WT) or mutant Vif expression vector, and 0.5 μg of the A3G or A3F expression vector or 1.5 μg of the A3C or A3DE expression vector. Virus was harvested from the supernatant for viral-infectivity assays, and cell lysates were prepared for immunoblotting. Infectivity was assessed at 48 h postinfection and normalized to the input CAp24. The antibodies used were as follows: mouse anti-p24 monoclonal antibody (MAb) (AIDS Research Reagents Program, Division of AIDS, NIAID, NIH; catalog no. 1513) (8), mouse anti-myc MAb (Upstate; catalog no. M05-724), anti-HA MAb (Covance; catalog no. MMS-101R-10000), mouse anti-V5 MAb (Invitrogen; catalog no. R96025), mouse anti-β-tubulin MAb (Covance; catalog no. MMS-410P), and rabbit anti-Vif polyclonal antibody (AIDS Research Reagents Program; catalog no. 2221).

Immunoprecipitation and immunoblot analyses.

A T-25 flask of 293T cells was transfected with 3 μg APOBEC3 plus 3 μg of WT or mutant expression vector as indicated. At 24 h after transfection, the cells were treated with 10 μM proteasome inhibitor MG132 for 16 h. The cells were harvested, washed twice with cold phosphate-buffered saline, lysed in 900 μl of lysis buffer A (50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1% [vol/vol] Triton X-100, and Complete Protease Inhibitor Cocktail tablets) or lysis buffer B (50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 0.1% [vol/vol] Triton X-100, and Complete Protease Inhibitor Cocktail tablets) at 4°C for 30 min, and then centrifuged at 10,000 × g for 15 min. Precleared cell lysates were mixed with anti-myc MAb and incubated with protein G beads at 4°C for 3 h on an end-over-end rocker. Samples were then washed six times with lysis buffer A or lysis buffer B. The beads were eluted with elution buffer (0.1 M glycine-HCl, pH 2.0), and the eluted materials were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the anti-Vif or anti-HA antibody. The blots were imaged using a FujiFilm LAS-1000 Image Station, and protein band densities were analyzed using the spot density analysis software Image Gauge V3.41. The percent decrease in the A3G binding levels was analyzed after normalizing the A3G intracellular level, as determined by immunoblotting. Values for the positive control (wide-type HIV-1 Vif) were set to 100%, and the relative interaction between A3G and mutant HIV-1 Vifs was calculated as a fraction of the positive control.

RESULTS

Conservation of the SLV/Ix4Yx9Y motif among HIV-1, HIV-2, and SIVsmm Vif molecules.

In 1992, Oberste and Gonda observed two relatively conserved motifs (SLQXLA and SLV/Ix4Yx9Y) among 38 lentiviral Vif sequences then available (44). SLQXLA was later determined to be a virus-specific BC box that mediates the elongin B/C interaction (37, 73, 74). The function of the other motif, SLV/Ix4Yx9Y, is still largely unknown. Examination of updated primate lentiviral Vif sequences indicated the highly conserved nature of this SLV/Ix4Yx9Y motif among Vif molecules from various HIV-1 subtypes (M, O, and N groups), HIV-2 subtypes, and the closely related SIVsmm (Fig. (Fig.1).1). Interestingly, K26, which was not mentioned in the previous search, is also conserved (Fig. (Fig.1).1). Amino acids surrounding the SLV/Ix4Yx9Y motif, including amino acids H41HRY44 of HIV-1 Vif, which are important for A3G binding and suppression (39, 49), are not conserved among the HIV-1, HIV-2, SIVmac, and SIVsmm Vif molecules (Fig. (Fig.1).1). Furthermore, the SLV/Ix4Yx9Y motif, or slightly modified versions, is also present in the Vif sequences of other SIV lineages, such as SIVmnd, SIVsyk, and SIVagm (Fig. (Fig.1).1). The S residue is replaced by an H in SIVsyk Vif and a G in SIVagm Vif. The K residue is invariably conserved among all these Vif molecules and is preceded by two hydrophobic residues (ΦΦK) in all of them.

FIG. 1.
Conservation of the SLV/Ix4Yx9Y motif in HIV and SIV Vif molecules. The N-terminal regions of HIV-1, HIV-2, and various SIV Vif sequences were aligned. Identical residues are indicated by dashes. The conserved residues in the SLV/Ix4Yx9Y motif plus an ...

Role of the SLV/Ix4Yx9Y motif in HIV-1 Vif-mediated suppression of A3G.

To determine whether the SLV/Ix4Yx9Y motif is important for Vif function, we generated HIV-1 Vif mutant constructs in which individual residues within and surrounding this motif were modified (Fig. (Fig.2A).2A). Hydrophobic residues were changed to the hydrophilic amino acid Ser (S). Nonhydrophobic residues were changed to Ala (A). K22 was changed to E22 because it is a natural mutation that has been observed in HIV-1-infected individuals (57). The abilities of various Vif mutants to rescue HIV-1ΔVif infectivity in the presence of A3G were compared to that of the WT Vif. 293T cells were transfected with HIV-1ΔVif and an A3G expression vector plus WT or mutant Vif expression vectors, as indicated (Fig. (Fig.2B).2B). Virus was produced from transfected 293T cells and tested for infectivity in a standard MAGI assay as previously described (64). WT Vif suppressed A3G activity and maintained HIV-1ΔVif infectivity (Fig. (Fig.2B),2B), and this level of infectivity in the presence of WT Vif was set to 100% (Fig. (Fig.2B).2B). As expected, A3G dramatically reduced the infectivity of HIV-1ΔVif in the presence of VifL145A (Fig. (Fig.2B),2B), which was unable to interact with Cul5-elongin B/C (74). Residues surrounding the SLV/Ix4Yx9Y motif have different roles against A3G (Fig. (Fig.2B).2B). This analysis revealed, for the first time, the importance of K26 and Y30 in the protective activity of HIV-1 Vif against A3G. VifK26A was as ineffective against A3G as was the VifL145A mutant (Fig. (Fig.2B).2B). Similarly, VifY30A was as ineffective against A3G as was VifY40A or VifRH41/42AA (Fig. (Fig.2B),2B), both of which have been reported to be defective in countering A3G (49).

FIG. 2.
Effects of mutations in the SLV/Ix4Yx9Y motif and surrounding amino acids on Vif activity against A3G. (A) Vif mutant constructs. (B) Effects of WT and mutant Vif proteins on HIV-1ΔVif infectivity in the presence of A3G. HIV-1 (NL4-3ΔVif) ...

Since HIV-1 Vif overcomes A3G by reducing its intracellular expression, we also examined the effects of WT and mutant Vif molecules on A3G intracellular levels (Fig. (Fig.2C).2C). For this purpose, we transfected 293T cells with an A3G expression vector plus a control vector expressing VifL145A-myc (Fig. (Fig.2C,2C, lane 1) or a vector expressing WT Vif-myc (lane 2), VifK22E-myc (lane 3), VifS23A-myc (lane 4), VifL24S-myc (lane 5), VifV25S-myc (lane 6), VifK26A-myc (lane 7), VifY30A-myc (lane 8), VifY40A-myc (lane 9), VifY40A-myc, or VifRH41/42AA-myc (lane 10). In agreement with the infectivity data, we observed that the intracellular level of A3G was efficiently reduced by WT HIV-1 Vif (Fig. (Fig.2C,2C, lane 2) compared to the VifL145A mutant control (lane 1). VifK26A (Fig. (Fig.2C,2C, lane 7) and VifY30A (lane 8) were defective in reducing A3G expression compared to the WT Vif (lane 2), consistent with their impaired abilities to overcome the antiviral activity of A3G. Although VifK22E had already been reported to be defective against A3G, it was not known whether this mutant was also defective in reducing A3G expression (57). We observed that VifK22E was indeed defective in reducing the intracellular expression of A3G (Fig. (Fig.2C,2C, lane 3) compared to WT Vif (lane 2).

Role of the SLV/Ix4Yx9Y motif in HIV-1 Vif-mediated suppression of A3F, A3C, and A3DE.

We next examined the effects on A3G of the Vif mutants that are most defective in their activities against three other known HIV-1 Vif target molecules, A3F, A3C, and A3DE. In order to assess the abilities of WT and mutant Vif molecules to affect the intracellular expression of A3F (Fig. (Fig.3A),3A), we transfected 293T cells with an A3F-V5 expression vector plus the control VifL145A-myc vector (Fig. (Fig.3A,3A, lane 1) or a vector expressing WT Vif-myc (lane 2), VifK22E-myc (lane 3), VifK26A-myc (lane 4), VifY30A-myc (lane 5), VifY40A-myc (lane 6), or VifRH41/42AA-myc (lane 7). The intracellular level of A3F was efficiently reduced by HIV-1 WT Vif (Fig. (Fig.3A,3A, lane 2) compared to VifL145A (lane 1); all the other Vif mutants maintained the ability to reduce the expression of A3F (lanes 3 to 7). VifK22E-myc, VifK26A-myc, VifY30A-myc, VifY40A-myc, and VifRH41/42AA-myc also maintained their abilities to overcome the antiviral activity of A3F (Fig. (Fig.3B3B).

FIG. 3.
Effects on A3F, A3C, and A3DE of Vif mutants found to be defective in A3G inactivation. (A) VifK22E, VifK26A, VifY30A, VifY40A, and VifRH41/42AA are able to reduce the intracellular expression of A3F. 293T cells were cotransfected with NL4-3ΔVif ...

As was observed for A3F, we found that VifK22E-myc, VifK26A-myc, VifY30A-myc, VifY40A-myc, and VifRH41/42AA-myc also maintained their abilities to reduce the intracellular expression of A3C (Fig. (Fig.3C)3C) and A3DE (Fig. (Fig.3E).3E). More importantly, these mutants could also overcome the antiviral activity of A3C (Fig. (Fig.3D)3D) and A3DE (Fig. (Fig.3F).3F). The fact that these mutants are active against A3F, A3C, and A3DE indicates that mutations at these residues do not affect the overall folding of the Vif molecule and that the mutant Vif molecules are competent for functional assembly with Cul5-elongin B/C E3 ubiquitin ligase and interaction with certain substrates.

VifS23A-myc, VifL24S-myc, and VifV25S-myc (unlike VifK22E-myc, VifK26A-myc, VifY30A-myc, VifY40A-myc, and VifRH41/42AA-myc) all maintained a strong ability to neutralize A3G (Fig. (Fig.2).2). VifS23A-myc and VifV25S-myc maintained close to 90% of the WT Vif's effectiveness in rescuing HIV-1ΔVif infectivity against A3G (Fig. (Fig.2B),2B), and they reduced intracellular A3G expression as efficiently as did WT Vif (Fig. (Fig.2C).2C). VifL24S was only modestly defective (Fig. (Fig.2B),2B), whereas VifL24S-myc (Fig. (Fig.4A,4A, lane 5) and VifV25S-myc (lane 6) showed greater impairment in their abilities to suppress the antiviral activity of A3F (Fig. (Fig.4A).4A). Although VifL24S could rescue more than 50% of HIV-1ΔVif infectivity against A3G compared to the WT Vif, it lost the ability to enhance HIV-1ΔVif infectivity against A3F (Fig. (Fig.4A).4A). VifL24S was also defective in reducing the intracellular expression of A3F (Fig. (Fig.4B,4B, lane 5) compared to WT Vif (Fig. (Fig.4B,4B, lane 2). Similarly, VifV25S was almost as effective as the WT against A3G (Fig. (Fig.4A)4A) but showed a greater defect in terms of its A3F-related defense (Fig. (Fig.4A).4A). VifV25S reduced the intracellular expression of A3F (Fig. (Fig.4B,4B, lane 6) less efficiently than did WT Vif (Fig. (Fig.4B,4B, lane 2). Both VifL24S-myc and VifV25S-myc were also impaired in terms of their abilities to reduce the intracellular expression of A3C and A3DE (data not shown), as well as in their activities against both A3C and A3DE (data not shown). Thus, the LV portion of the SLV/Ix4Yx9Y motif seems to be the most important portion of the sequence in terms of the inactivation of A3F, A3C, and A3DE.

FIG. 4.
Effectiveness of VifL24S and VifV25S against A3F versus A3G. (A) VifL24S and VifV25S are required for intracellular A3F reduction. 293T cells were cotransfected with NL4-3ΔVif and A3F-V5 expression vector plus an expression vector for WT Vif or ...

The K22, K26, and Y30 residues in HIV-1 Vif influence the Vif-A3G interaction.

Mutation of K22, K26, or Y30 abolished the HIV-1 Vif activity against A3G. However, these Vif mutants maintained their abilities to inactivate A3F, A3C, and A3DE, indicating they could still assemble with the Cul5-elongin B/C E3 ubiquitin ligase complex and target certain APOBEC3 molecules for polyubiquitination and degradation. Therefore, it is possible that these residues are involved in Vif-A3G binding. To test this hypothesis, we characterized the interaction of WT and mutant Vif molecules with A3G by coimmunoprecipitation analysis. In order to block the Vif-induced, proteasome-mediated degradation of A3G, the cells were treated with 10 μM MG132 for 16 h prior to being harvested. For coimmunoprecipitation assays, Vif-A3G was used at a ratio of 1:1 to limit the degradation of A3G. This condition was different from the virus infectivity assay and was one potential limitation of the study. The Vif-myc proteins were immunoprecipitated from cell lysates, and coprecipitation of A3G-HA was assessed by immunoblotting.

A3G-HA was efficiently coimmunoprecipitated with WT Vif-myc (Fig. (Fig.5A,5A, lane 2), and this interaction was specific, since A3G-HA was not detected in the absence of Vif (Fig. (Fig.5A,5A, lane 1). Even though high levels of A3G-HA were detected in cells expressing VifK22E-myc (Fig. (Fig.5A,5A, lane 3), VifK26A-myc (lane 4), VifY40A-myc (lane 5), or VifRH41/42AA-myc (lane 6), significantly less A3G-HA was coimmunoprecipitated with these Vif mutants than with WT Vif (Fig. (Fig.5A).5A). VifY30A-myc also showed an impaired ability (90% reduction) to interact with A3G-HA compared to WT Vif. The impaired recognition of A3G by these Vif mutants was correlated with their decreased abilities to suppress the antiviral activity of A3G (Fig. (Fig.2B)2B) and to reduce its intracellular expression (Fig. (Fig.2C)2C) compared to the WT Vif. Thus, these results identified K22, K26, and Y30, in addition to YRHHY (40-44), as possible A3G-binding residues in HIV-1 Vif.

FIG. 5.
Impaired interaction of VifK22E, VifK26A, VifY40A, and VifRH41/42AA with A3G. (A) 293T cells were cotransfected with an expression vector for A3G-HA plus a control vector (VR1012), an expression for the WT Vif-myc, or one of the indicated Vif mutants. ...

It has been reported that deletion of amino acids 23 to 43 in HIV-1 Vif does not affect mutant Vif binding to A3G, as assessed by coimmunoprecipitation experiments (14). These results raise the possibility that HIV-1 Vif may interact with A3G through more than one interface. We have previously demonstrated that the V55xIPLx4-5LxΦx2YWxL72 motif is important for the interaction of Vif with A3G. Most of the critical residues in this region are hydrophobic amino acids. If hydrophobic interactions play a dominant role in mediating Vif-A3G binding, then mutations of charged residues, such as E22, K26, and RH41/42, would be expected to have a lesser effect on Vif-A3G coprecipitation in the presence of low concentrations of nonionic detergent. Indeed, when cell lysates were prepared using Triton X-100 at a concentration of only 0.1% instead of 1%, significant coprecipitation of A3G-HA with VifK22E-myc (Fig. (Fig.5B,5B, lane 3), VifK26A-myc (lane 4), VifY40A-myc (lane 5), and VifRH41/42AA-myc (lane 6) was observed compared to WT Vif-myc (lane 2). This result is in sharp contrast to the results obtained when the cell lysates were prepared using 1% Triton X-100 (Fig. (Fig.5A).5A). These observations are consistent with the concept that when hydrophobic interactions between Vif and A3G are neutralized by nonionic detergent, the importance of the charged interactions between the two molecules becomes more apparent.

DISCUSSION

In the current study, we have identified previously unrecognized residues in HIV-1 Vif that are critical for A3G suppression. In particular, we have determined that residues K26 and Y30 are important for the interaction between Vif and A3G. Although K22 of HIV-1 Vif has previously been suggested to play a role in A3G suppression (57), our study has demonstrated for the first time that the VifK22E mutant also has a reduced ability to interact with A3G (Fig. (Fig.5).5). Mutations of HIV-1 Vif K22, K26, and Y30 resulted in reduced A3G-Vif interaction, suggesting that these residues may be involved in Vif binding to A3G. However, it is also possible that mutations of these residues affected Vif folding and therefore A3G binding. The SLV/IKx3Yx9Y motif in HIV1 and HIV-2/SIVsmm/SIVmac Vif molecules is more divergent in SIVagm Vif (GIVKYx10Y). However, we have observed that the invariable K residue is also critical for SIVagm Vif-mediated African green monkey A3G degradation (data not shown). Interestingly, the HIV-1 VifK22E, VifK26A, and VifY30A mutants all maintained their abilities to suppress several other human cytidine deaminases (A3F, A3C, and A3DE) (Fig. (Fig.3),3), indicating that changes at these positions did not affect global folding of the Vif molecule. Combining our identified residues with the previously identified A3G-interacting amino acids 40 to 44 (YRHHY) in HIV-1 Vif (39, 49), we can now point to the K22x3K26x3Y30x9YRHHY44 motif of HIV-1 Vif as a unique A3G interaction and suppression domain.

Although SLV is part of the original SLV/Ix4Yx9Y motif (44), individual replacement of each amino acid had no or only minor effect on the mutants' activities against A3G (Fig. (Fig.2).2). VifL24S could still significantly reduce the level of A3G intracellular expression compared to the BC box mutant VifL145A (Fig. (Fig.2C),2C), and it maintained >50% of the A3G suppression produced by WT Vif (Fig. (Fig.2B).2B). In contrast, VifL24S was almost completely ineffective against A3F (Fig. (Fig.4).4). VifV25S was also less effective against A3F than against A3G (Fig. (Fig.4).4). We could not rule out the possibility that L24 and V25 are involved in the folding of the Vif molecule, since mutations of these amino acids, and particularly L24, affected the neutralization of multiple APOBEC3 subtypes. However, mutation of L24 affected the defense against A3F to a greater extent than that against A3G, raising the possibility that L24, and to a lesser extent V25, may be more involved in A3F, A3C, and A3DE neutralization and that they are part of an extension of the previously identified A3F interaction motif spanning W11QVDRMR17. Structural information concerning Vif and the various APOBEC3 proteins will be required if we are to thoroughly understand the significance of the L24 and V25 residues for Vif activity. It is somewhat surprising that we did not observe any functional significance of the relatively conserved S23 residue. VifS23A maintained its function against A3G, A3F, A3C, and A3DE; it is not clear whether this residue of HIV-1 Vif has a function independent of APOBEC3 suppression.

It is well established that HIV-1 Vif can functionally interact with various human cytidine deaminases (34). However, HIV-1 Vif inactivates human A3G and A3F through distinct motifs (19, 39, 46, 49, 53, 57, 62, 69, 77). We have recently demonstrated that A3F, A3C, and A3DE are suppressed by HIV-1 Vif through similar mechanisms (77). Mutations within amino acids 40 to 44 of HIV-1 Vif that disrupt A3G binding and inactivation have no effect on A3F, A3C, or A3DE binding and suppression (77). On the other hand, mutations within amino acids 11 to 17 and 74 to 79 of HIV-1 Vif disrupt the binding and suppression of A3F, A3C, and A3DE but have little effect on A3G (77). This pattern continued in our current study. Mutants VifK26A and VifY30A were defective in neutralizing A3G but maintained their abilities to inactivate A3F, A3C, and A3DE. However, VifL24S lost the ability to suppress A3F, A3C, and A3DE but showed only a modest reduction in its defensive activity against A3G. Future studies are needed to determine whether there are any subtle differences in the recognition of A3C, A3DE, and A3F by HIV-1 Vif.

The K22x3K26x3Y30x9YRHHY44 motif of HIV-1 Vif is rich in positively charged amino acids. Furthermore, the region between K26 and Y40, although not conserved among HIV/SIV Vif molecules, always has a high content of positively charged amino acids, albeit not at fixed positions (Fig. (Fig.1).1). Interestingly, one region of A3G that has been suggested to be important for its interaction with Vif is rich in negatively charged amino acids, including D128, D130, and E134 (6, 20, 32, 33, 50, 52, 68). It is plausible that electrostatic interactions between Vif and A3G play an important role in Vif-mediated A3G binding, ubiquitination, and/or degradation.

We have previously identified a motif (V55xIPLx4-5LxΦx2YWxL72) in HIV-1 Vif as being important for the interaction between HIV-1 Vif and A3G (19). This motif is rich in hydrophobic amino acids, and changes from hydrophobic to hydrophilic amino acids disrupt the Vif-A3G interaction (19). Thus, both hydrophobic and charged interactions are important for HIV-1 Vif-mediated suppression of A3G. Interestingly, in the current study, substitution of charged amino acids affected the Vif-A3G interaction more obviously in the presence of a 1% concentration of the nonionic detergent Triton X-100 than in the presence of a 0.1% concentration (Fig. (Fig.5).5). We also observed that VifK22E, VifK26A, and VifRH41/42AA exhibited more profound defects in A3G binding when the hydrophobic interactions between Vif and A3G were disrupted by Triton X-100 (Fig. (Fig.5).5). These data suggest that hydrophobic interactions between Vif and A3G are more dominant than the charged interactions between these two molecules. These results also provide an explanation for previous apparent discrepancies regarding the participation of residues between 23 and 44 of HIV-1 Vif in the Vif-A3G association: Some studies had reported a defect in A3G binding by Vif mutants within the YRHHY region in the presence of high concentrations of nonionic detergents (39, 49). On the other hand, Goila-Gaur et al. observed an interaction between A3G and an internal deletion (23 to 43) Vif mutant (14) when 0.1% Triton X-100 was used; in this case, it is likely that hydrophobic interactions between Vif and A3G could still occur.

Why is a weaker charged interaction still important for A3G suppression? It is possible that the high-affinity hydrophobic interactions mediate the initial association between Vif and A3G that is necessary for the recruitment of A3G into the Vif-Cul5 E3 ubiquitin ligase complex. Subsequently, charged amino acids mediate a more precise and specific interaction between Vif and A3G. This secondary interaction may place the A3G molecule in the most favorable position for ubiquitination (Fig. (Fig.6A).6A). Alternatively, the charged interaction may help to retain A3G within the E3 complexes (reducing the disassociation of A3G) for progressive ubiquitination (polyubiquitination) when different E2-conjugating enzymes are used (Fig. (Fig.6B).6B). A third possibility is that the charged interaction triggers a conformational change in A3G for subsequent ubiquitination and/or recognition and degradation by the proteasome. Amino acids between 23 and 42 in HIV-1 Vif have been found to influence the folding of A3G (14).

FIG. 6.
Model depicting the roles of the positively charged amino acids in HIV-1 Vif activity. A patch of positively charged and hydrophilic residues (K22x3K26x3Y30x9YRHHY44) and a cluster of hydrophobic residues (V55xIPLx4-5LxΦx2YWxL72) in HIV-1 Vif ...

The precise roles of different domains of HIV-1 Vif in APOBEC3 suppression require further study. The insights to be gained from exploring these possible interfaces between Vif and APOBEC3 proteins have the potential to contribute to the design of novel HIV inhibitors.

Acknowledgments

We thank M. Malim, N. Landau, and Y. H. Zheng for critical reagents and D. McClellan for editorial assistance. The following reagents were obtained through the AIDS Research Reagents Program, Division of AIDS, NIAID, NIH: MAGI-CCR5 cells, the Vif polyclonal antibody, and the p24 MAb.

This work was supported by a grant from the NIH (AI062644) to X.-F.Y.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 June 2009.

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