Identification of Amino Acid Residues in APOBEC3G Required for Regulation by Human Immunodeficiency Virus Type 1 Vif and Virion Encapsidation

The human immunodeficiency virus type-1 (HIV-1) accessory proteinVif serves to neutralize the human antiviral proteins apolipoproteinB mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G[A3G]) and A3F. As such, the therapeutic blockade of Vif functionrepresents a logical objective for rational drug design. Tofacilitate such endeavors, we have employed molecular geneticsto define features of A3G that are required for its interactionwith Vif. Using alanine-scanning mutations and multiple differentsubstitutions at key residues, we confirm the central role playedby the aspartic acid at position 128 and identify proline 129and aspartic acid 130 as important contributory residues. Theoverall negative charge of this 3-amino-acid motif appears criticalfor recognition by Vif, as single lysine substitutions are particularlydeleterious and a double alanine substitution at positions 128and 130 is far more inhibitory than single-residue mutationsat either position. Our analyses also reveal that the immediatelyadjacent 4 amino acids, residues 124 to 127, are important forthe packaging of A3G into HIV-1 particles. Most important aretyrosine 124 and tryptophan 127, and mutations at these positionscan ablate virion incorporation, as well as the capacity toinhibit virus infection. Thus, while pharmacologic agents thattarget the acidic motif at residues 128 to 130 have the potentialto rescue A3G expression by occluding recognition by Vif, carewill have to be taken not to perturb the contributions of theneighboring 124-to-127 region to packaging if such agents areto have therapeutic benefit by promoting A3G incorporation intoprogeny virions.

INTRODUCTION

Top

Introduction

Proteins of the vertebrate apolipoprotein B mRNA-editing enzyme,catalytic polypeptide-like 3 (APOBEC3) family protect cellsagainst invasion by a broad range of viruses and mobile geneticelements, such as retroviruses, hepadnaviruses, endogenous retroviruses,and retrotransposons (3, 5, 8, 16, 30, 42, 46, 47, 52, 56).These proteins belong to a larger family of proteins that containone or two copies of the signature histidine/cysteine-X-glutamicacid-X_23-28-proline-cysteine-X₂-cysteine motif that is characteristicof cytidine and adenosine deaminases found in species rangingfrom bacteria to vertebrates (7, 20, 22, 47). Expression ofAPOBEC3 proteins can lead to their encapsidation into progenyvirions through recruitment to substrate viral or transposoncapsid structures, and this appears to involve interactionswith both Gag (or Gag-like) proteins and RNA (6, 12, 14, 28,43, 54, 63).

Following reverse transcription of the retroelement genomicRNA into single-stranded DNA, the APOBEC proteins can deaminatecytidines to uridines, causing deleterious mutations that resultin the loss of genetic integrity and protein function, a processthat is commonly referred to as hypermutation (19, 30, 64).More recently, it has emerged that hypermutation is not theonly means by which APOBEC3 proteins can inhibit infectivityor transposition. Specifically, it has been reported that inhibitionof retrotransposons and hepatitis B virus occurs in the absenceof detectable hypermutation (5, 36, 42, 52, 56) and that mutantversions of the human APOBEC3G (A3G) and A3F proteins with inactivedeaminase catalytic domains can still suppress human immunodeficiencyvirus type 1 (HIV-1) infection (2, 21, 38). In the latter case,the inhibition of infection correlates well with reduced accumulationsof HIV-1 reverse transcripts in challenged cells (2, 21). TheA3G and A3F proteins both contain duplications of the cytidinedeaminase (CDA) catalytic motif, but they are not equivalent,as only the C-terminal CDA motif can mediate cytidine deamination(21, 38): the molecular basis for this domain difference remainsto be defined.

Lentiviruses, such as HIV-1, encode a virion infectivity factor(Vif) protein that prevents the antiviral effects of APOBEC3family members, in particular, A3G and A3F (3, 47, 58, 65).The mode of action by which HIV-1 Vif counters A3G-mediatedantiviral function has been elucidated in some detail. HIV-1Vif interacts with human A3G and thereby recruits a cullin 5-elonginB/C E3 ubiquitin ligase so that A3G is polyubiquitinated andtargeted to the proteasome for degradation (9, 32-34, 48, 61,62). The elimination of A3G at the time of virus productionconsequently prevents its encapsidation, although Vif has alsobeen reported to occlude A3G encapsidation directly (48) andto reduce A3G translation (53). The interactions of Vif withcomponents of this E3 ligase complex are mediated by a histidine-cysteine-cysteine-histidinezinc binding motif that provides a binding site for cullin 5(35, 59) and a BC box that binds to elongin C (62).

Our molecular understanding of the Vif-A3G interaction has beenadvanced by the observation that a single-amino-acid differencebetween the A3G proteins of humans and African green monkeys(AGMs) at position 128 modulates the species-specific interactionwith the Vif proteins of HIV-1 or AGM-derived simian immunodeficiencyvirus (SIV_AGM) (4, 31, 44, 60). Most notably, replacement ofthe aspartic acid residue that naturally occurs at this position(D128) in human A3G with the lysine found at the correspondingposition in AGM A3G abrogates the interaction with HIV-1 Vifbut confers sensitivity to the SIV_AGM Vif protein. The A3G-Vifinteraction is of considerable interest, as it provides a compellingtarget for novel therapeutic strategies for treating HIV-1 infections.Specifically, successful disruption of the Vif-A3G interactionis predicted to rescue A3G expression, induce virion packaging,and therefore stimulate natural antiviral activity. Similarly,pharmacologic suppression of proteasome-mediated A3G degradationhas already been shown to enhance A3G expression and therebyinhibit HIV-1 infectivity (61). The rational design of inhibitorsof the Vif-A3G interaction will be facilitated by detailed moleculardescriptions of the determinants present in both Vif and A3G.Accordingly, and with the current absence of structural information,we have undertaken a molecular genetic approach to address thisquestion.

Extensive site-directed mutagenesis of human A3G has enabledus to map a 3-amino-acid motif comprising aspartic acid-proline-asparticacid at positions 128 to 130 as being crucial for the interactionwith HIV-1 Vif. Further substitutions in this region with aminoacids carrying side chains with different properties revealedthat the negative charges at positions 128 and 130 play a centralrole in the HIV-1 Vif interaction but that there is redundancybetween the two positions. In addition, a 4-amino-acid regionof A3G immediately N-terminal to residue 128 is essential forvirion packaging and hence for antiviral efficacy. In developingtherapeutic moieties that interfere with the Vif-A3G interaction,consideration will have to be given to retaining efficient A3Gpackaging.

MATERIALS AND METHODS

Top

Materials and Methods

Plasmids and cloning. The wild-type (parental) human A3G cDNA was inserted into pCMV4-HA(1) as PCR-amplified HindIII-XbaI fragments to create (i) pA3G,in which the natural stop codon of the A3G gene preceded thetriple hemagglutinin (HA) tag, or (ii) pA3G-HA, in which theHA tag was fused in frame to the C terminus of hA3G. An overlappingPCR-based strategy was used to generate derivatives that encodedmutated A3G proteins. Briefly, for each mutation, N- and C-terminalfragments of A3G containing the desired point mutation in an $~$ 20-nucleotide region that was common to both fragments weregenerated by PCR. These were used as the template for a secondPCR in which only the outer primers were present. The resultingmutated cDNA was then inserted into pCMV4-HA to yield vectorsexpressing untagged or tagged proteins, depending upon the 3'primer that was used. Bacterial A3G expression vectors wereproduced by subcloning A3G cDNAs into pTrc99A as Asp718-SmaIfragments (2). The wild-type and vif-deficient ( ${Delta}$ vif) HIV-1 proviruses,pIIIB and pIIIB/ ${Delta}$ vif; the HIV-1 Vif expression vector, pcVIF;and its corresponding control, pc ${Delta}$ VIF, have all been describedpreviously (48, 50).

Single-cycle infectivity assays. Stocks of wild-type or ${Delta}$ vif HIV-1 were prepared by cotransfectionof 35-mm-diameter monolayers of 293T cells with 0.5 µgof pA3G expression vector and 2.0 µg of pIIIB or pIIIB/ ${Delta}$ vifusing polyethylenimine (13). After 24 h, the supernatants wereharvested and virus levels were quantified by p24^g^ag enzyme-linkedimmunosorbent assay (Perkin-Elmer). The producer cells werelysed in sodium dodecyl sulfate (SDS) containing loading dyefor the analysis of protein expression. Viral stocks were storedat –80°C, and volumes corresponding to 5 ng p24^g^agwere used to infect 10⁵ TZM-bl indicator cells (11, 40, 57).The induced expression of ß-galactosidase in whole-celllysates was measured 24 h after the initiation of infectionusing the Galacto-Star system (Applied Biosystems).

Analysis of protein expression by immunoblotting. Whole-cell lysates prepared from virus-producing cells wereresolved by SDS-polyacrylamide gel electrophoresis and analyzedby immunoblotting using primary antibodies specific for A3G(rabbit polyclonal [38] or Hsp90 [Invitrogen], horseradish peroxidase-conjugatedsecondary antibodies) and enhanced chemiluminescence (Pierce).In experiments examining the effects of Vif on A3G accumulation,35-mm cultures of 293T cells were transfected with 0.2 µgpA3G (or a mutant derivative) and 0.2 µg pcVIF or pc ${Delta}$ VIF.After 24 h, the cells were lysed in loading buffer and analyzedby immunoblotting using primary antibodies specific for A3G,Hsp90, or Vif (319 mouse monoclonal antibody) (50).

Coimmunoprecipitation assays. 293T cells were transfected with 2 µg pA3G-HA (or a mutantderivative) and 2 µg pcVIF or pc ${Delta}$ VIF in 35-mm cultures.After 24 h, the cells were washed and lysed in 1 ml lysis buffer(1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mMEDTA, and complete protease inhibitor cocktail [Roche]). Thelysates were cleared by centrifugation in a bench-top centrifugeat 21,000 x g for 10 min, and 850 µl of each was incubatedwith the 12CA5 antibody and protein A-agarose (Invitrogen) for2 h at 4°C. The agarose beads were washed three times withlysis buffer and resuspended in 50 µl loading dye; 10µl of the immunoprecipitated samples, as well as 10 µlof the cleared lysate, was resolved by SDS-polyacrylamide gelelectrophoresis (11% gel) and analyzed by immunoblotting usingprimary antibodies specific for HA, Vif, or Hsp90.

Packaging assays. Virus stocks containing 20 ng p24^g^ag were spun in a bench-topcentrifuge at 21,000 x g for 2 h at 4°C through a 20% (wt/vol)sucrose cushion (500 µl) in a 2-ml Eppendorf tube. Theviral pellets were resuspended in loading dye and analyzed byimmunoblotting using antibodies specific for A3G or p24^g^ag (mousemonoclonal 24-2 [49]). Whole-cell lysates from the correspondingproducer cells were assessed for A3G expression.

E. coli mutation assay. The KL16 strain of Escherichia coli was transformed with pTrc99A-based,IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducibleA3G expression vectors or the empty vector. Individual colonieswere picked and grown to saturation in LB medium containing100 µg/ml ampicillin and 1 mM IPTG. Appropriate dilutionswere spread onto agar plates containing either 100 µg/mlampicillin or 100 µg/ml rifampin and incubated overnightat 37°C. Mutation frequencies were recorded as the numberof rifampin-resistant colonies per 10⁹ viable cells (which wereenumerated using the ampicillin-containing plates) (38, 39).

RESULTS

Top

Results

Modeling the A3G structure. We previously exploited the availability of the crystal structuresof some nucleotide cytidine deaminases to produce a model forAPOBEC proteins by the combined use of structure predictionand sequence alignment (22). This analysis demonstrated thatmost structural features of cytidine deaminases can readilybe assigned counterparts in APOBEC proteins. In particular,a mixed sheet composed of five ß-strands and two ${alpha}$ -helicesthat contain the catalytic site residues was readily predicted.One key difference that emerged was the presence of the additional ${alpha}$ -helix 3 between ß-strands 4 and 5 in APOBEC proteins,which is absent from the nucleotide cytidine deaminases. Basedon the crystal structures of the human adenosine-to-inosineediting enzyme ADAR2 (29), the tRNA adenosine deaminases fromAquifex aeolicus (15, 25) and Staphylococcus aureus (27), Saccharomycescerevisiae cytosine deaminase (23, 24), and APOBEC2 (an orphanAPOBEC family member whose crystal structure was reported whilethis work was being reviewed) (41), it is now believed that ${alpha}$ -helix 3 of APOBEC protein CDA cores crosses the ß-sheet,resulting in a parallel positioning of ß-strands 4and 5. As a consequence, this helix is located toward the sameface of the ß-sheet as helices ${alpha}$ 1 and ${alpha}$ 2 (Fig. 1A).Because this conformation may limit the accessibility of ${alpha}$ 3 andthe immediately adjacent residue D128 to interacting factors,such as Vif, we set out to analyze in detail the importanceof the region between predicted ß-strands 4 and 5of the amino-terminal CDA domain of A3G using site-directedmutagenesis. In particular, we wished to probe the contributionsof the residues surrounding D128 for involvement in regulationby HIV-1 Vif and to investigate the role of ${alpha}$ 3 in A3G function(Fig. 1B).

View larger version (27K):
[in this window]
[in a new window]

FIG. 1. (A) Structure model of the N-terminal CDA domain of A3G. Zinc-coordinating residues are indicated by circles, ${alpha}$ -helices are in yellow, and ß-strands are in pink. The position of residue D128 is indicated in the predicted loop between ß4 and ${alpha}$ 3. (B) The amino acid sequence of residues 114 to 153 of A3G. The positions of predicted ${alpha}$ -helix (yellow) and ß-strand (pink) regions are indicated, as are residues 119, 128, and 146. The motifs defined here as being important for HIV-1 packaging and Vif responsiveness are indicated.

Alanine-scanning mutagenesis of A3G. We initially used site-directed mutagenesis to construct a seriesof A3G expression vectors encoding proteins with single alaninesubstitutions covering residues 119 to 146. This region containstwo naturally occurring alanine residues, A121 and A134 (Fig.1B), and they were not included in our analysis. Wild-type orvif-deficient ( ${Delta}$ vif) HIV-1 stocks were produced using 293T cellsin the presence of mutant or wild-type A3G proteins and usedin single-cycle challenges of TZM-bl indicator cells to assayA3G-mediated antiviral activity. All mutants with alterationsbetween residues 131 and 146 displayed antiviral propertiescomparable to those of the wild-type protein in that virus infectionwas efficiently suppressed in the absence of Vif but was restoredin the presence of Vif to levels close to that of vector-onlycontrols (data not shown). From this, we concluded that theregion spanning residues 131 to 146 is not involved in the interactionwith HIV-1 Vif and that the predicted helix ${alpha}$ 3 most likely playsa predominantly structural role. We did not examine this regionfurther and instead focused our attention on residues 119 to130. Mutations in this more N-terminal region gave rise to proteinswith altered phenotypes that can be organized into two categories(Fig. 1B). First, alanine substitutions at positions 128 to130 yielded proteins that showed increased capacities to inhibitwild-type HIV-1 infection, indicating resistance to the actionof Vif. Second, various proteins with mutations between residues119 and 127 display decreased abilities to inhibit HIV-1/ ${Delta}$ vif,caused by reduced expression or packaging into virus particles.The further analyses of each class of mutated protein are discussedin turn below.

Analysis of A3G mutations affecting regulation by Vif. The aspartic acid at position 128 plays a pivotal role in theresponsiveness of A3G to HIV-1 Vif (4, 31, 44, 60). In additionto D128A, mutants P129A and D130A also emerged with Vif-resistantphenotypes (Fig. 2A). Though the disruption of the capacityof Vif to regulate A3G was not nearly as marked for the D128Amutant as it was for P129A, a similarly modest loss of Vif responsivenesswas also seen for D130A. Since these three residues may constitutethe core of a site required for the Vif interaction, we constructedadditional mutant proteins in which the chemical natures ofthe three side chains were changed. The aspartic acids at positions128 or 130 were altered to lysine, asparagine, glutamic acid,or serine to assess the consequences of replacing a negativelycharged side chain with a side chain that was positively charged,basic, alternatively negatively charged, or polar. In addition,the proline at position 129 was changed to glycine to test whetherthe inherent flexibility of this amino acid might be able tocompensate for the bend that would be imposed by the naturallyoccurring proline. Single-cycle infectivity assays were performedwith this set of A3G mutants (as well as with the initial alaninesubstitutions) using wild-type or ${Delta}$ vif virus stocks, and proteinexpression was examined in whole-cell lysates by immunoblotting(Fig. 2A).

View larger version (34K):
[in this window]
[in a new window]

FIG. 2. Mutational analysis of residues 128, 129, and 130 of A3G. (A) Analysis of the side chain requirements for Vif responsiveness at residues 128, 129, and 130. WT, wild type. (B) Simultaneous mutation of residues 128 and 130 to alanine increases the Vif resistance of A3G. Single-cycle infectivities of virus stocks produced in the presence of mutant or wild-type A3G (or the pCMV4 parental vector) were measured as relative luciferase units and are presented as percent infectivity relative to wild-type virus produced in the absence of A3G. The infectivities of wild-type HIV-1 stocks are shown as black bars and ${Delta}$ vif stocks as white bars. The data are the means of three infections performed with independently produced virus samples, and the error bars represent standard deviations. Below, immunoblots show the expression of A3G in the corresponding producer 293T cells for one set of transfections. The Hsp90 immunoblot is shown as a loading control.

The phenotypic trends for the mutations at residues 128 and130 showed strong similarity, although subtle differences werealso evident. In both cases, the strongest inhibition of wild-typeHIV-1 infection (and hence resistance to Vif) was observed uponthe introduction of lysine (mutants D128K and D130K), thoughthe effect was greater at position 128. Among the other substitutionsat this residue, the D128N mutation exhibited the most substantialloss of inhibition by Vif, whereas D128E and D128S behaved muchlike D128A. In comparison, at position 130, the introductionof asparagine, glutamic acid, or serine had negligible effectson susceptibility to Vif. The P129G mutant showed a phenotypevery similar to that of P129A in that it inhibited wild-typeHIV-1 infection very effectively, indicative of the loss ofregulation by Vif. As anticipated, reductions in Vif responsivenessfor this collection of mutants were accompanied by increasedlevels of protein accumulation in cell lysates (Fig. 2A, bottom).Together, these results suggest that the interaction betweenA3G and Vif is largely determined by electrostatic interactionsinvolving residues 128 and 130, but with the dependency foraspartic acid at position 128 being more stringent.

The observation that reversing the negative charges on two closelypositioned residues had marked effects on regulation by Vifbut that the mere loss of one aspartic acid often had a relativelyminor effect (e.g., the serine substitutions) implied that theremay be redundancy between D128 and D130. To address this idea,a double-alanine-substitution mutant, D128A-D130A, was constructedand analyzed as described above (Fig. 2B). This protein wasa considerably more potent inhibitor of wild-type HIV-1 infectionthan either single mutant, with infectivity being reduced bymore than 90%. Consistent with this, examination of producerlysates revealed that Vif had little effect on its expression.Thus, while the removal of the aspartic acid residues does notcompletely abrogate responsiveness to Vif, these amino acidsclearly play central and somewhat overlapping roles in the interactionof A3G with Vif.

The proline at position 129 also appears to be a key featurefor recognition by Vif. As this could be due to either the structuralor hydrophobic properties of the proline, we also analyzed anA3G mutant with valine at position 129, because the hydrophobicityof valine is similar to that of proline but it does not causea bend in a peptide backbone. Though the infectivity of wild-typeHIV-1 produced in the presence of the P129V mutant was mildlyimproved compared to that with P129A, the P129V protein waspoorly expressed (data not shown). This suggests that the prolineat position 129 is a key structural feature of A3G that contributesto correct protein folding, as well as being important for helpingto establish an interaction with HIV-1 Vif.

Having examined the effects of Vif on the expression of A3Gmutant proteins only in the context of virus-producing cells,we next ventured to verify the Vif-resistant properties of asubset of mutants by expressing them in the presence or absenceof Vif in 293T cells independently of other viral proteins (Fig.3A). This analysis confirmed the stability of the D128K, P129A,P129G, and D130K mutants in the presence of Vif. In contrast,the D128A and D130A mutants generally showed some Vif-induceddegradation in this setting, confirming that these mutants haveonly moderately Vif-resistant phenotypes.

View larger version (46K):
[in this window]
[in a new window]

FIG. 3. Vif-resistant A3G proteins do not interact with HIV-1 Vif. (A) Analysis of the stability of wild-type (WT) or mutant A3G proteins in the absence or presence of HIV-1 Vif. Proteins were detected by immunoblotting of cell lysates from transfected 293T cells using specific primary antibodies, and Hsp90 was measured as a loading control. (B) Coimmunoprecipitation of HIV-1 Vif with HA-tagged versions of wild-type or mutant A3G. The pCMV4 sample served as the negative control. Whole-cell lysates (top) were analyzed by immunoblotting using antibodies specific for A3G, HIV-1 Vif, or Hsp90 (loading control). The immunoprecipitates (IP) (bottom) were analyzed with antibodies specific for A3G or HIV-1 Vif. The band common to all coprecipitated Vif samples represents antibody light chains.

To this point, we have referred to the A3G-Vif interaction inonly a genetic sense. To address whether the noted trends extendedto biochemical interactions, the abilities of mutant A3G proteinsto interact with Vif were assessed by coimmunoprecipitation(Fig. 3B). Cell lysates were prepared from coexpressing 293Tcells and either examined directly by immunoblotting (upperthree panels) or immunoprecipitated using an HA-specific antibodythat recognized the C-terminally HA-tagged A3G proteins; thepresence of A3G (fourth panel) or Vif (bottom panel) in theprecipitates was then monitored by immunoblotting. We were ableto coimmunoprecipitate Vif with wild-type A3G-HA and the D128Aand D130A mutants, but not with the D128K, P129A, P129G, andD130K mutants. This confirmed that the partially Vif-resistantD128A and D130A proteins maintained an interaction with Vif(albeit at slightly reduced levels compared to wild-type A3G),but the profoundly Vif-resistant mutants did not. Thus, thecapacity of altered A3G proteins to escape Vif-induced downregulationcorrelates well with their respective inabilities to form complexeswith Vif.

Analysis of A3G mutants with reduced antiviral activities. We next investigated the mutant proteins with changes betweenpositions 119 and 127 that showed generalized reductions inantiviral activity (Fig. 4A). Most of the alanine-scanning mutantsshowed normal expression in producer cell lysates and stillaccumulated to lower levels in the presence of Vif. MutantsF119A and R122A, however, were expressed at lower levels, likelyaccounting for their respective losses of antiviral activity.In contrast, Y124A, Y125A, F126A, and W127A each showed levelsof expression comparable to that of wild-type A3G, suggestingthat their losses of antiviral activity, which were more pronouncedfor Y124A and W127A, may be caused by the disturbance of a functionalmoiety of A3G. The F119A, R122A, Y124A, and W127A mutants weretherefore investigated to see whether editing activity (Fig.4B) or virion packaging (Fig. 4C) had been compromised.

View larger version (38K):
[in this window]
[in a new window]

FIG. 4. Alanine-scanning mutagensis of residues 119 to 127 of A3G. (A) Single-cycle infectivities of virus stocks produced in the presence of A3G mutant proteins. The experiments were performed, and are reported, as for Fig. 2. (B) Editing activities of wild-type (WT) and mutant A3G proteins as measured in a bacterial reporter assay. (C) Packaging of wild-type and mutant A3G into HIV-1 particles. Virions purified from supernatants of 293T cells cotransfected with ${Delta}$ vif and A3G expression vectors were analyzed by immunoblotting, together with corresponding producer cell lysates.

To begin to address whether the loss of antiviral function wasdue to impaired protein activity, we used an E. coli-based assaythat measures the DNA-editing capability of APOBEC proteins.In this assay, APOBEC-driven mutation of the bacterial rpoBgene is registered by the emergence of rifampin-resistant colonies,which can be readily counted (38, 39). Mutants R122A, Y124A,and W127A all showed editing activities similar to that of wild-typeA3G, but editing by the F119A mutant was reduced to backgroundlevels (Fig. 4B). Immunoblotting analysis showed that all proteinswere expressed at similar levels (data not shown). The wild-typeediting activities of R122A, Y124A, and W127A were perhaps notsurprising, as previous studies had demonstrated that the editingactivity of A3G is associated with the C-terminal CDA domainand not the N-terminal CDA domain in which these mutations arelocated. However, the inactivity of the F119A mutant proteinwas unexpected, suggesting that this mutation may have causedthe loss of editing through misfolding of the protein in bacteria.In contrast, the R122A, Y124A, and W127A proteins presumablystill adopted active conformations.

As A3G needs to be packaged into HIV-1 virions to exert itsantiviral affect, we next produced ${Delta}$ vif virus particles in thepresence of wild-type A3G or F119A, R122A, Y124A, or W127A andanalyzed their encapsidation into virions by immunoblottingthem (Fig. 4C). Consistent with their lower expression levelsin cell lysates, only small amounts of the F119A and R122A proteinswere detected in virus particles. In contrast, the Y124A andW127 mutant proteins were efficiently expressed but showed severedefects in packaging efficiency compared with wild-type A3G.Thus, the loss of antiviral activity of Y124A and W127A correlateswell with the reduced packaging of these proteins.

To investigate and compare further the expression and packagingphenotypes of mutant proteins with alterations at positions119 or 127, we constructed another set of mutants in which thesenatural aromatic residues were replaced with amino acids carryinghydrophobic, polar, or alternative aromatic side chains (leucine,serine, or tyrosine, respectively). Single-cycle infectivityand protein expression assays were performed as described above(Fig. 5A). At position 119, all of these substitutions restoredexpression levels, Vif responsiveness, and inhibition of HIV-1/ ${Delta}$ vifinfectivity to levels close to those of wild-type A3G. In fact,the behavior of the F119Y protein that contains the alternativearomatic residue was indistinguishable from that of the parentalprotein in this experiment. On the other hand, none of thesesubstitutions at position 127 restored the antiviral propertiesof the protein. Indeed, W127L and W127S both showed an evenmore pronounced loss of antiviral activity, as evidenced bytheir complete inability to inhibit infection by HIV-1/ ${Delta}$ vif.Investigation of the packaging properties of these proteinsrevealed that all of the substitutions at position 119 restoredencapsidation to close to wild-type levels but that the samesubstitutions at position 127 did not (Fig. 5B). At best, W127Yexhibited a marginal increase in packaging efficiency comparedto W127A, but the presence of W127L or W127S in virions wasundetectable. Thus, we conclude that the presence of tryptophanat position 127 is required for the correct packaging of A3Ginto HIV-1 particles.

View larger version (37K):
[in this window]
[in a new window]

FIG. 5. Mutational analysis of A3G residues 119 and 127. (A) Analysis of the side chain requirements for antiviral function at residues 119 and 127. Experiments were performed as for Fig. 2. (B) Roles of residues 119 and 127 in virion incorporation. Packaging was determined as for Fig. 4C. (C) Residues spanning positions 124 to 127 contribute to virion incorporation.

Finally, we sought to establish whether the reductions in antiviralactivity associated with other mutations preceding residue 127could also be explained by packaging defects. Accordingly, weanalyzed the virion encapsidation of mutant proteins with aminoacid substitutions at positions 123 to 128 (Fig. 5C). Consistentwith their strong antiviral phenotypes, the L123A and D128Aproteins were efficiently packaged. As in Fig. 4C, Y124A andW127A were packaged at very low levels, whereas the Y125A andF126A proteins showed intermediate levels of incorporation.In sum, the severity of the packaging defects for this set ofmutant proteins correlated closely with the extent to whichtheir abilities to inhibit HIV-1/ ${Delta}$ vif had been impaired (Fig.5A). Thus, our analysis identified the aromatic 4-amino-acidY124-Y125-F126-W127 motif as being important for the incorporationof A3G into HIV-1 particles.

DISCUSSION

Top

Discussion

The primary intent of this structure-function study was to betterunderstand molecular determinants of the interaction betweenA3G and the antagonistic HIV-1 Vif protein. A detailed knowledgeof the interaction between these proteins is of particular interestfor the rational design of future therapeutic approaches thataim to harness natural protection against HIV-1 infection bypreventing the Vif-mediated degradation of A3G. We chose tomutate residues 119 to 146 of A3G because the region containsresidue 128, which has previously been shown to be pivotal infacilitating the formation of A3G-Vif complexes (4, 31, 44,60).

In addition to confirming the role of residue 128 in the interactionwith Vif, we identified two additional amino acids, P129 andD130, whose alteration created proteins that displayed Vif-resistantphenotypes (Fig. 1B). More specifically, we found that insensitivityto Vif was conferred by mutating the aspartic acid residue atposition 128 or 130 to the positively charged residue lysine.However, we did observe that residue 128 is more sensitive tomutation than 130, which can accommodate several amino acidchanges and still behave like wild-type A3G, implying that D128may play a more prominent role in the Vif interaction (Fig.2A). Nevertheless, simultaneous mutation of residues D128 andD130 to alanine yielded a protein that was markedly more resistantto Vif than either singly mutated protein (Fig. 2B), suggestinga substantial degree of redundancy between these positions.In sum, these data indicate that the Vif-A3G interaction isdependent on electrostatic interactions, which is fully consistentwith recent data demonstrating that mutation of positively chargedresidues in HIV-1 Vif can compensate for the D128K mutationin A3G (45). In addition to these electrostatic determinants,there also appears to be a rather specific structural requirementfor the interaction, as evidenced by the strong Vif-resistantphenotypes of mutant A3G proteins carrying either alanine orglycine in place of proline at position 129 (Fig. 2A). Thoughit is most likely that Vif contacts A3G directly in this region,we emphasize that direct binding awaits formal demonstration.For this reason, we use the term "interaction" to encompassboth direct and indirect contacts.

While our current study has provided novel insight into theinteraction between HIV-1 Vif and A3G, the 3-amino-acid motifD128-P129-D130 is unlikely to represent a complete descriptionof motifs within APOBEC proteins that are capable of interactingwith HIV-1 Vif. More specifically, (i) one study reported thatresidues 54 to 124 of A3G are sufficient for coimmunoprecipitationwith Vif, which would suggest the presence of an additionalpoint (or points) of contact (9); (ii) the Vif proteins of HIV-2and SIV_mac are able to mediate the degradation of both wild-typeA3G and the D128K mutant, indicating that these Vif proteinshave different requirements for functional interactions withA3G (17, 60); and (iii) the interaction between HIV-1 Vif andhuman A3F is different from the interaction with A3G, as illustratedby the demonstration that amino acid differences in HIV-1 Vifcan differentially affect the ability to neutralize A3F versusA3G (51, 55). Indeed, the human A3F protein contains an ERDmotif at the position equivalent to the DPD motif of A3G (definedhere), yet mutation of the glutamic acid in A3F does not affectsensitivity to Vif (10, 26); moreover, the naturally Vif-resistanthuman A3B protein also has this ERD motif at the equivalentposition, again implying that an interaction with Vif requiresmore than just acidic amino acids at this position. Lastly,like A3G, A3F contains two CDA domains, and we noted that theC-terminal domain contains a DTD motif within the predictedß4- ${alpha}$ 3 loop. However, mutation of the first asparticacid in this motif to lysine (D310K) did not affect downregulationby Vif (data not shown). Taken together, these assorted findingsindicate that the DPD motif at residues 128 to 130 in humanA3G represents a Vif interaction domain that is unique to thisprotein. A full understanding of the molecular determinantsof this interaction will clearly depend on future structuralanalyses of Vif-A3G complexes. In this regard, it is interestingthat the recently resolved structure of APOBEC2 (41) revealsthat the corresponding glutamic acid-proline-glutamic acid motif(residues 159 to 161) also straddles the N terminus of helix ${alpha}$ 3 (Fig. 1A) and that the acidic side chains protrude outwardfrom the helix, rendering them accessible for interactions withprotein ligands.

In addition to defining this Vif interaction motif, our analysesrevealed the existence of an adjacent motif that plays a centralrole in the packaging of A3G into virus particles (Fig. 1B).Alanine substitution mutations in this 4-amino-acid clusterspanning residues 124 to 127 generated proteins displaying reducedlevels of antiviral activity against HIV-1/ ${Delta}$ vif (Fig. 4A) thatcorrelated with losses in virion incorporation (Fig. 4C and5C). Specifically, mutants Y124A and W127A were almost entirelyexcluded from virions and had minimal effects on infectivity,whereas the Y125A and F126A proteins showed reduced levels ofpackaging and intermediate infectivity phenotypes. Further mutationanalysis of residue 127 indicated that the presence of tryptophanat this position is particularly important, as all substitutionstested resulted in substantial diminutions of antiviral activity(Fig. 5A). A principal role for residues 124 and 127 in A3Gfunction is also reflected in the strong conservation of theseresidues within A3G proteins from different species, whereasmore variation was observed at the less important positions125 and 126 (not shown).

It has been reported by several groups that incorporation ofA3G into virus particles depends on a combination of its interactionwith the NC domain of Gag and nonspecific RNA binding (6, 12,14, 28, 43, 54, 63). Although the losses of antiviral activityfor mutants in the 124-to-127 region correlated with defectsin packaging, we found, using a previously described pull-downassay (12, 63) and lysates from transfected 293T cells, thatproteins with mutations at positions 124, 125, and 126 interactedwith HIV-1 Gag in an RNA-dependent manner but that the W127Amutant protein did not (data not shown). Thus, packaging capabilitiescannot simply be ascribed to Gag-A3G-RNA interactions as monitoredby this assay system. The reasons for these discrepancies arenot yet evident and will require further investigation; however,one potential contributing factor to A3G packaging may be therequirement for spatial proximity between A3G and the cellularsites of viral particle assembly, and it is conceivable thatthis motif may participate in determining correct subcellularlocalization. A further possibility is that this motif may bindto an as-yet-unidentified molecular partner that may specifyvirus incorporation. In sum, further work is required to definethe role of residues 124 to 127 in HIV-1 packaging and to determinehow this region combines with other conserved elements of theN-terminal CDA of A3G that also contribute to encapsidation(18, 37, 38).

In this study, we demonstrated that crucial determinants forA3G interaction with Vif and its incorporation into HIV-1 particlesare localized to a 7-amino-acid element spanning residues 124to 130. Consistent with the notion that this region interactswith viral and cellular factors, structure predictions for A3Gand comparisons with APOBEC2 place it in a loop region thatconnects ß4 to ${alpha}$ 3 (Fig. 1). On the basis of our alaninescan, the residues that confer these two attributes appear tobe distinct from each other. The W127L protein provides theonly data that may dispute this: it is no longer packaged (Fig.5B) yet has lost susceptibility to Vif-induced degradation (Fig.5A). Since other mutations at position 127 remain Vif sensitive,it seems most likely that the W127L mutant is misfolded andconsequently broadly nonfunctional (though it remains formallypossible that this mutation genuinely influences both attributes).As discussed above, pharmacologic inhibition of the Vif-A3Ginteraction represents a potential therapeutic approach forsuppressing HIV-1 replication. Should such an inhibitory agentinteract with A3G at the DPD motif, it will be important topreserve the biological activities of the neighboring YYFW motif:failure to do so could lead to reduced packaging and antiviralefficacy. Further work defining the precise functions and structuresof these motifs will inform rational drug design in this area.

ACKNOWLEDGMENTS

The following reagent was obtained through the NIH AIDS Researchand Reference Reagent Program, Division of AIDS, NIAID, NIH:TZM-bl from John Kappes, Xiaoyun Wu, and Tranzyme Inc.

This work was supported in part by grant number 106637-38-RFHFfrom the American Foundation for AIDS Research (amfAR). Workin the Malim laboratory is supported by the United Kingdom MedicalResearch Council, and M.H.M. is an Elizabeth Glaser Scientistsupported by the Elizabeth Glaser Pediatric AIDS Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Infectious Diseases, King's College London School of Medicine, 2nd Floor, New Guy's House, Guy's Hospital, London Bridge, London SE1 9RT, United Kingdom. Phone: 44 0 20 7188 0149. Fax: 44 0 20 7188 0147. E-mail: michael.malim{at}kcl.ac.uk

Published ahead of print on 31 January 2007.

REFERENCES

Top