RNA-Dependent Oligomerization of APOBEC3G Is
Required for Restriction of HIV-1
Hendrik Huthoff1, Flavia Autore2, Sarah Gallois-Montbrun1, Franca Fraternali2, Michael H. Malim1*
1Department of Infectious Diseases, King’s College London, London, United Kingdom, 2Randall Division of Cell and Molecular Biophysics, King’s College London, London,
The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able
to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine
deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the
crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential
to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical
crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain
mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific
residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template
structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of
arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and
oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins
associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively
charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function.
Citation: Huthoff H, Autore F, Gallois-Montbrun S, Fraternali F, Malim MH (2009) RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of
HIV-1. PLoS Pathog 5(3): e1000330. doi:10.1371/journal.ppat.1000330
Editor: Thomas J. Hope, Northwestern University, United States of America
Received June 4, 2008; Accepted February 5, 2009; Published March 6, 2009
Copyright: ? 2009 Huthoff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grant 106637-38-RFHF from the American Foundation for AIDS Research (amfAR), the U. K. Medical Research
Council, the Wellcome Trust [083498/Z/07/Z], and Tibotec Pharmaceuticals Ltd. HH is a Wellcome Trust fellow, FA is supported by a short-term fellowship of the
European Molecular Biology Organization, SG-M is a Fellow of the European Molecular Biology Organization, and MHM is an Elizabeth Glaser Scientist. None of the
sponsors had a role in the study design, execution, or manuscript preparation.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The human protein APOBEC3G (A3G) belongs to a family of
cellular polynucleotide cytidine deaminases and is a potent
inhibitor of HIV-1 in the absence of the viral protein Vif .
Vif-deficient HIV-1 (HIV-1/Dvif) is subject to A3G mediated
cytidine to uridine deamination of single-stranded DNA that is
generated during reverse transcription, a process also referred to as
DNA editing or hypermutation [2–4]. In addition, A3G further
suppresses infection by inhibiting reverse transcription in a poorly
understood manner that seems to be independent of the
deamination activity of the protein [5–9]. A3G is incorporated
into progeny virions during particle assembly at the plasma
membrane by associating with the NC domain of the viral Gag
protein in an RNA dependent manner [10–15]. The viral Vif
protein prevents the antiviral properties of A3G by targeting it for
proteasomal degradation . Specifically, Vif interacts with A3G
and recruits the cullin5-elonginB/C-Rbx ubiquitin ligase complex,
resulting in the polyubiquitylation and degradation of A3G .
This reduction of intracellular levels in A3G results in a substantial
decrease in the packaging of A3G into virus particles and,
therefore, suppresses its antiviral properties.
The recent reports of the crystal structure of APOBEC2 (A2) 
and the NMR and crystal structures of the C-terminal cytidine
deaminase (CDA) domain of A3G [19,20] offer opportunities to
investigate the structure-functionorganization ofAPOBEC proteins
with greater incisiveness. Although the physiological function of A2
is as yet unknown, its structure shows all the hallmarks of a cytidine
deaminase, being a five-stranded mixed b-sheet which presents on
one face two a-helices containing the H/C-X-E-X23–28-P-C-X2-C
catalytic centre that coordinates a zinc ion. Surprisingly, A2
associates into tetramers in a manner unprecedented among
cytidine deaminases. Whereas tetrameric free-nucleotide cytidine
deaminases of bacteria [21,22], yeast  and vertebrates 
adopt a globular structure in which each subunit interacts with the
other three, A2 forms a linear tetramer in which monomers interact
with either one or two of the other subunits . An A2 monomer
contains a single CDA domain and two of these form a dimer by
joining the b-sheets present in each monomer in a side-by-side
fashion such that one wide b-sheet is formed. The tetramer is
assembled from two such dimers through head-to head interactions
at one edge of the extended b-sheets.
In contrast to A2, A3G contains two CDA domains in a single
polypeptide chain, which are termed the N- and C-terminal CDA
domains (N-CDA and C-CDA, respectively). Indeed, the CDA
fold observed in the A2 crystal structure closely matches the
structure of the truncated A3G C-CDA domain as observed by
NMR and crystallography [19,20]. Differences arise mainly at the
peripheral loops, which are generally longer in A3G than in A2.
The A3G C-CDA fragment is exclusively monomeric, both in
PLoS Pathogens | www.plospathogens.org1 March 2009 | Volume 5 | Issue 3 | e1000330
solution and in crystalline form [19,20]. However, there is
mounting evidence that A3G not only oligomerizes [12,25–30],
but can also assemble into large RNP complexes that accumulate
in cellular microdomains that are associated with RNA regulation,
such as P-bodies and stress granules [30–34]. We therefore asked
whether the tetrameric structure of A2 may hold clues not only
into how A3G oligomerizes, but also into its participation in other
interactions. Here, we show that hydrophobic residues in A3G
that are equivalent to those that mediate A2 oligomerization are
required for RNA-dependent oligomerization, packaging into
HIV-1 virions and the inhibition of HIV-1 infection. In addition,
we present a homology model of an A3G dimer that reveals a
positively charged pocket at the predicted dimer interface.
Mutation of basic residues within this pocket also affects
oligomerization, RNA interactions, virion packaging and virus
Differential contributions of A3G N- and C-CDA domains
to packaging, DNA editing, and oligomerization
The tetramer interface of A2 is formed of extensive hydropho-
bic, polar and electrostatic interactions, many of which are
clustered in a loop termed L1 . In particular, residues F155,
(Figure 1A), whereas R153, E158 and E159 are involved in salt-
bridges as well as in hydrogen bonding. Upon alignment of the A2
amino acid sequence with the N- or C-terminal CDA sequences of
A3G, we identified a highly similar loop sequence in both CDA
domains of A3G in which both charged and hydrophobic residues
are conserved (Figure 1B). Arginines equivalent to R153 in A2 are
present at positions 122 and 313 in A3G, whereas the tyrosines at
positions 124 and 315 in A3G are at the equivalent position of
F155 in A2. Although F155 does not make any direct interactions
across the tetramer interface of the A2 crystal, it is involved in a
cluster of hydrophobic packing interactions that sandwich M156
between Y61 and F155 at the tetramer interface. A tryptophan
equivalent to W157 in A2 is present only in the N-CDA of A3G at
position 127. W157 of A2 makes extensive hydrophobic
interactions across the tetramer interface, notably with Y214
and W157 of the adjacent subunit.
We have previously reported a mutational analysis of residues
122–146 of A3G to define the site of interaction with Vif, which
was mapped at positions 128–130 . That analysis also revealed
that substitutions at positions Y124 and W127 yield A3G proteins
that are inefficiently packaged into virus particles and therefore
lose their antiviral properties. Given the involvement of the
conserved counterparts of these residues in A2 oligomerization, we
sought to establish whether this region would have an analogous
activity in A3G. To investigate this possibility, and to compare the
contribution that the N- or C-CDAs of A3G may make to
oligomerization, we introduced identical mutations (alanine,
leucine and phenylalanine) at residues Y124 and Y315; these
residues were chosen because they are present at equivalent
positions in both the N- and C-CDA of A3G, and because the
mutant proteins are expressed well. In contrast, the introduction of
substitutions at the conserved arginine at position 122 resulted in
poor expression , and mutants of R122 were therefore not
examined further. The construction of mutations at position W127
has been described previously .
Figure 1. The tetramer interface of A2 and sequence alignment
with A3G. (A) Detail of the A2 tetramer interface, highlighting residues
that mediate oligomerization interactions. One subunit is shown in blue
(left) and one in orange (right). Residues F155 and W157 are shown in
blue, other residues from the left-hand subunit that contribute
interactions are shown in yellow, and residues from the right-hand
subunit are in green; all are indicated by labels. (B) Sequence alignment
of A2 with the A3G N- and C-CDA domains corresponding to the L1
loop of A2. Arrows indicate the position of b-strands, and a barrel
indicates the position of an a-helix in the A2 crystal structure. Below the
alignment, residues in A3G are indicated that correspond to F155 and
W157 in A2.
APOBEC3G is a human protein that inhibits the replication
of HIV-1 in CD4+ T cells. It gains entry to the virus particles
that are released from infected cells and subsequently
interferes with viral genome replication, which in the case
of HIV-1 is reverse transcription. APOBEC3G is a cytidine
deaminase, and it catalyses the deamination of cytidines to
uridines in viral single-stranded DNA replication interme-
diates, resulting in the generation of defective progeny
viruses. In addition, APOBEC3G can inhibit reverse
transcription by a poorly characterized deamination-
independent mechanism. HIV-1 has evolved the viral Vif
protein to counteract the antiviral properties of APO-
BEC3G. Vif associates with APOBEC3G and targets it for
proteasomal degradation, such that intracellular levels of
APOBEC3G are reduced and packaging into virions is
averted. Based on the structure of a human homolog of
APOBEC3G, APOBEC2, we performed a mutational analysis
of amino acids that have the potential to mediate the
assembly of APOBEC3G into multi-component complexes.
We report that these amino acids affect the association of
APOBEC3G with itself and cellular RNA, and that the same
attributes are also required for packaging into virions and
antiviral function. Thus, the processes of APOBEC3G self-
association, RNA binding, and virion packaging are
functionally linked and essential for virus inhibition.
Oligomerization of APOBEC3G
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We first tested these mutant A3G proteins for their ability to
inhibit HIV-1/Dvif infection (Figure 2A). The substitution of Y124
or Y315 to alanine or leucine caused marked losses of antiviral
function, whereas substitutions to the chemically more similar
phenylalanine resulted in less marked disruption. Determination of
the A3G content of virus particles revealed that all mutations at
position Y124 result in poor packaging, whereas packaging was
maintained with mutations at position Y315 (Figure 2B). Interest-
ingly, the Y124F mutation yielded low but clearly detectable levels
of A3G in virions in comparison to mutants Y124A and Y124L,
which likely explains why this protein showed a less severe loss of
antiviral activity. We next determined the extent to which wild
type or mutant A3G can act as a mutagen in a bacterial DNA
editing assay (Figure 2C). In this analysis, we included two mutants
of W127 (W127A and W127Y), which have previously been
shown to have substantial packaging defects . Editing activity
was maintained following substitutions at positions Y124 and
W127, but mutations at position Y315 caused a loss of editing.
Together, these results indicate that the loss of antiviral activity
imparted by mutations at residues Y124 and W127 corresponds to
reduced packaging, whereas DNA editing activity is unaffected.
Conversely, mutations at residue Y315 ablate DNA editing but not
packaging into virions, which is consistent with the critical
involvement of Y315 in substrate DNA binding at the catalytically
active C-CDA domain [19,20].
To begin to address the ability of A3G to oligomerize, we
performed a yeast two-hybrid experiment (Figure 2D). Mutations
were introduced into the prey-construct and assayed with a wild
type A3G bait. Again, we observed a marked difference between
the effects of substitutions in the N- and C-CDA domains of A3G.
Mutations at Y124 and W127 resulted in a lack of reporter gene
activity, whereas mutations at Y315 displayed wild type levels of
Figure 2. Characterization of A3G proteins with mutations at Y124, W127, and Y315. (A) Single-cycle infectivity of HIV-1/Dvif viruses
produced in the presence of wild type or mutant A3G, indicated on the x-axis, as measured in relative luciferase units and presented as percent
infectivity relative to the wild type A3G (RLU, y-axis). The empty vector control is indicated by -. The data are the average of three independent
experiments, and errors bars represent the standard deviation of the three independent transfections. (B) Expression of wild type and mutant A3G
proteins in 293T producer cells and packaging into HIV-1/Dvif virions as determined by immunoblotting. The empty vector control is indicated by -.
(C) Relative editing activity of wild type and mutant A3G proteins as determined in a bacterial mutator assay relative to activity of wild type A3G from
12 independent experiments. The empty vector control is indicated by -. Immunoblots beneath the graph show the expression of A3G in equal
volumes of the bacterial cultures. (D) Interaction of wild type and mutant A3G proteins (prey) with wild type A3G (bait) in a yeast two-hybrid assay as
determined by b-galactosidase activity in OD540units. A series of controls include: C1, empty bait and prey vectors; C2, the positive control with
Tsg101-bait and Vps28-prey; C3, wild type A3G-bait and empty prey vector; C4, empty bait vector and wild type A3G-prey. The data are the average
of three independent experiments. Beneath the graph, immunoblots using anti-HA antibody show the expression in equal volumes of the yeast
cultures of A3G prey, which uniquely carry a triple HA –tag.
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activity and, hence, interaction. This result suggests that residues
Y124 and W127 of the N-CDA domain play critical roles in A3G
oligomerization, whereas Y315 of the C-CDA does not.
RNA-dependent oligomerization of A3G is mediated by
the N-terminal CDA domain
To investigate further the oligomerization of A3G, we next
performed a series of immunoprecipitation and chemical cross-
linking experiments. Wild type or mutant A3G was co-expressed
with HA-tagged wild type A3G (A3G-HA) and then co-
immunoprecipitated from cell lysates using a monoclonal anti-
HA antibody. Aliquots were or were not treated with RNAse A
and analyzed by immunoblotting (Figure 3A). Consistent with the
results of the yeast two-hybrid experiments, wild type A3G and the
Y315A mutant were efficiently co-precipitated with A3G-HA,
whereas co-precipitation of the Y124A mutant was strongly
reduced and a mere trace amount of W127A was detected in the
immunoprecipitate. In all cases, co-precipitation with A3G-HA
was substantially inhibited by the treatment with RNAse A,
indicating that RNA is required for stable A3G oligomerization.
We then performed a chemical crosslinking experiment in which
cell lysates from 293T cells expressing wild type A3G were treated
with BM(PEO)3, a compound with two reactive maleimide moieties
separated by an 18 A˚linker that form irreversible covalent bonds
with the sulfohydrils of cysteines . After crosslinking and
resolutionby SDS-PAGE, A3G was detected as a band migrating at
,80 kD as well as at ,40 kD where the untreated, and presumably
monomeric, A3G migrates (Figure 3B, lanes 1 and 2). Treatment
with RNase A prior to crosslinking resulted in complete
disappearance of the band at ,80 kD; this was maintained,
however, when the RNase treatment was performed after the
crosslinking reaction (Figure 3B, lanes 3 and 4, respectively). To
verify that the crosslinked A3G species migrating at ,80 kD
represents an A3G oligomer, we also perfomed an experiment in
which myc-tagged A3G (A3G-myc) was co-expressed with A3G-
HA, subjected to crosslinking and then immunoprecipitated with
the anti-HA antibody (Figure 3C). Samples were split into two
aliquots which were (or were not) subjected to RNase A treatment
after crosslinking. Indeed, A3G-myc was detected in the immuno-
precipitate as a species migrating at ,80 kD after treatment with
BM(PEO)3and this was unaffected by treatment with RNase A. In
the control sample without the crosslinker, detection of monomeric
A3G-myc in the immunoprecipitate was abolished by the treatment
with RNase A. This result indicates that the species migrating at
,80 kD is indeed formed by intermolecular crosslinking between
A3G-HA and A3G-myc.
To assess in greater detail the oligomerization characteristics of
some of our mutant A3G proteins, we next performed a
Figure 3. Oligomerization of wild type and mutant A3G proteins. (A) Co-immunoprecipitation of wild type and mutant A3G with HA-tagged
wild type A3G (A3G-HA). Immunoblots on the left show whole cell expression of A3G, A3G-HA, and the cellular control protein Hsp90. On the right,
blots show A3G and A3G-HA in the immunoprecipitate, with or without RNase A treatment. (B) Immunoblot showing A3G after chemical crosslinking
with BM(PEO)3in the lysate of transfected 293T cells. Lane 1, untreated control; lane 2, BM(PEO)3treated; lane 3, BM(PEO)3treated after incubation
with RNase A; lane 4, BM(PEO)3treated before incubation with RNase A. Relative molecular mass markers (in kD) are indicated on the right. (C)
Immunoblot showing the immunoprecipitation of myc-tagged A3G (A3G-myc) with A3G-HA, with or without BM(PEO)3and subsequent RNase A
treatment. Samples were immunoprecipitated with anti-HA antibody, and immoblots were probed with the anti-myc antibody. An asterisk indicates
the position of a band generated by crossreactivity to the heavy chain of the 3F10 antibody used for immunoprecipitation. (D) Immunoblot showing
the effect of BM(PEO)3treatment on wild type and mutant A3G in the lysates of transfected 293T cells. The control sample transfected with the empty
vector is indicated by -.
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crosslinking experiment with untagged wild type A3G or A3G
harbouring the Y124A, Y315A or W127A mutations. The
,80 kD crosslinked species appeared at a low level with the
Y124A mutant and was barely detectable with the W127A
mutant, whereas it was efficiently generated with the Y315A
mutant (Figure 3D). Crosslinking and co-immunopreciptitaton of
A3G with the Y124F or W127Y mutations resulted in increased
levels oligomerization in comparison to the respective alanine
mutations (Figure S1), which correlates with the less disruptive
effects on virus packaging of these substitutions relative to the
alanine mutations (Figure 1B and ). We note that we were
unable to detect these subtle differences with the yeast-two hybrid
system (Figure 1D).
To assess the possibility that the ,80 kD species may be due to
two A3G monomers being bound in close proximity on the same
RNA molecule, we performed crosslinking experiments in which
A3G was co-expressed with the RNA helicase Mov10. Mov10 is
known to associate with A3G in ribonucleoprotein complexes in
an RNA-dependent manner, though it is unknown whether these
proteins interact directly . We were unable to detect any
intermolecular crosslinks between A3G and Mov10 (results not
shown), further suggesting that the ,80 kD crosslinked species
forms as a consequence of A3G intermolecular contacts. Together,
these results indicate that residues Y124 and W127 play central
roles in the N-CDA mediated oligomerization of A3G, and that
this interaction is also dependent on the presence of RNA.
Modelling of an A3G dimer
In a complementary approach for addressing the mode of A3G
oligomerization, we constructed homology models of A3G dimers
using the A2 crystal structure as a template (Figure 4A). In these
models, the N- and C-CDA domains of one A3G polypeptide
together form the extended b-sheet that is the equivalent of an A2
dimer. Models with either the N-CDA or C-CDA at the oligomer
interface, which corresponds to the tetramer interface of A2, were
then assembled and subjected to energy minimization. In conver-
gence with the results of the experiments described above, models
with N-CDA at the oligomer interface (Figure 4C) proved
energetically more favourable than models with the C-CDA at the
dimer interface by ,2000 to ,3000 kJ/mol, depending on
interactions with the solvent (Table S1). N-terminal dimerization
than W127 (Table S2). We next assessed the effect of the Y124A and
W127A mutations by determining the interactions that are lost upon
introduction of these mutations into the A3G structure model with
the N-terminal CDA at the dimer interface (Table S3). This analysis
intersubunit interactions, while the W127A mutation affects mostly
intersubunit interactions in this structure model.
Importantly, inspection of the charge distribution over the
surface the model revealed a conspicuous clustering of positive
charges that are located in a rather large pocket at the predicted
dimer interface (Figure 4D): notably, residues Y124 and W127 are
also located within this pocket (Figure 5A). In contrast, this
positively charged surface is absent from the A2 structure
(Figure 4B). To assess the accuracy of this modelling effort, the
C-CDA domain from our model was superimposed with the NMR
 and X-ray  structures for this domain (Figure S2). The
overall agreement was good with both structures, as evidenced by
a root mean square deviation (RMSD) of less than 5 A˚, but our
model displayed slightly more similarity to the X-ray structure
(RMSD NMR: 4.920 A˚and RMSD X-ray: 3.650 A˚).
Mutational analysis of basic residues at the oligomer
The presence of clustered charged and aromatic residues at the
A3G oligomer interface is suggestive of a binding site for RNA. In
Figure 4. Structure of A2 and homology model of A3G. Ribbon representation of the A2 crystal structure (A) and the A3G homology model
with the N-terminal CDA domain at the dimer interface (C). Monomer subunits of A2 are shown in turquoise, blue, orange, and yellow. Monomer
subunits of A3G are shown in magenta and green. Zinc ions are shown as red spheres. To the right is a space-filling representation of the A2 crystal
structure (B) and the A3G model (D) highlighting charge distribution. Red indicates negative charge and blue positive charge. The potential is ranged
from 210kT (red) to the maximal positive value +10kT (blue).
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particular, the basic residues R14, R24, R29, R30, K63, K99, R102,
R122, R136, K141 and R142 all lie within the aforementioned
pocket and we sought to test this feature of our model. All these
arginines and lysines were mutated to alanine and their antiviral
properties assessed in single-cycle infectivity assays; the R24A and
R30A proteins had the most profound loss of antiviral function,
whereas mutations at other positions had no or modest effects, as
exemplified by R136A (Figure 6A, and data not shown).
As the basic residues may act cooperatively to bind RNA, we
also produced a set of doubly mutated proteins in which the R24A
or R30A mutation was combined with alanine substitutions at
R63, R99, R102, R136, K141 or R142. Only the R24A+R136A
and R30A+R136A composite mutants showed further reductions
in virus inhibition, resulting in phenotypes similar in severity to the
W127A mutation (Figure 6A, and data not shown). Analysis of the
levels of A3G present in virus particles revealed that mutants
R24A, R30A, R24A+R136A and R30A+R136A were each
packaged less efficiently than wild type A3G, but that the
R136A mutant was still packaged well (Figure 6B). None of the
R24A, R30A and R136A mutants showed any loss of editing
activity in bacteria, either as single or double mutants, indicating
that these proteins were not misfolded (Figure 6C).
We next assessed the oligomerization properties of these mutant
A3G proteins by co-immunoprecipitation and chemical cross-
linking. In both assay systems, we consistently observed that the
R24A and R30A mutants oligomerized less efficiently than the
wild type protein, and this was accentuated further by the
additional R136A substitution (Figure 6D and 6E). Together,
these results demonstrate that the removal of basic residues from
the predicted oligomer interface creates proteins with very similar
phenotypes to the Y124A and W127A mutants. Specifically, these
mutated proteins display limited antiviral activity, packaging, and
oligomerization, and this is consistent with the close spatial
proximity of these residues in our structure model (Figure 5).
Impaired A3G oligomerization correlates with reduced
To determine whether oligomerization-defective mutants of
A3G are reciprocally deficient for associating with cellular RNA,
we performed semi-quantitative reverse transcription coupled
PCR on immunoprecipitates of wild type or mutant HA-tagged
A3G to detect the presence of the Y1, Y4 and 7SL RNAs; these
RNA molecules have each previously been shown to be present in
A3G-associated RNPs [13,32,37]. Indeed, these RNAs were
readily detected in association with wild type A3G and the
oligomer-forming Y315A mutant (Figure 7A). In contrast, much
less Y1, Y4 and 7SL RNA was detected in the immunoprecipitates
of W127A, R24A, R24A+R136A and R30A+136A A3G, as well
as in the A3F and luciferase negative controls. Modest exceptions
were the Y124A and R30A mutants, for which low levels of Y4 as
well as 7SL RNA, respectively, were detected. These differences
were not due to different amounts of protein in the immunopre-
cipitate, as demonstrated by immunoblotting (Figure 7B).
Thus, mutations of hydrophobic and basic residues at the
predicted A3G oligomer interface caused a loss in association with
cellular RNA. These mutations do not, however, affect the ability
of A3G to assemble into high molecular weight ribonucleoprotein
complexes in 293T cells, as evidenced by velocity sedimentation of
A3G-containing cell lysates through sucrose gradients (Figure S3).
Moreover, all A3G-containing complexes maintained sensitivity to
RNase treatment, suggesting that the mutations have not imparted
pleiotropic defects in nucleic acid interactions or the capability to
assemble into large RNP complexes.
We have performed a mutational study of residues in the N- and
C-CDA domains of A3G whose counterparts in A2 are involved in
key interactions that support oligomerization of A2. Our results
Figure 5. The dimer interface of the A3G homology model. (A) Detail of the predicted A3G dimer interface highlighting residues Y124 and
W127 (A) and R24, R30, and R136 (B) from each subunit.
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show that mutations in the N-CDA, but not the C-CDA, are
associated with reductions in A3G RNA-dependent oligomeriza-
tion and packaging into virions. Upon modelling of an A3G dimer
onto the template A2 crystal structure, we identified a positively
charged pocket at the oligomer interface formed between two N-
CDAs that bore the hallmarks of a nucleic acid binding site
Figure 7. Association of wild type and mutant A3G with cellular RNAs. (A) Reverse transcription coupled PCR of RNA recovered from cell
extracts and immunoprecipitates (indicated, respectively, by Cell and IP) of 293T cells transfected with wild type or mutant A3G-HA to detect selected
cellular RNAs (Y1, Y4, and 7SL as indicated on the left). Negative controls were performed with HA-tagged A3F and luciferase (Luc). The control PCR
reactions using immunoprecipitate with Taq polymerase instead of the RT enzyme were all negative and are not shown. (B) Immunoblot with anti-HA
antibody showing the wild type and mutant A3G proteins present in the immunoprecipitate on which RT PCR was performed. Also included are A3F-
HA and luciferase-HA (Luc) negative controls.
Figure 6. Characterization of A3G proteins with mutations at R24, R30, and R136. (A) Single-cycle infectivity of HIV-1/Dvif viruses produced
in the presence of wild type or mutant A3G. See the legend to Figure 2A. (B) Expression of wild type and mutant A3G proteins in 293T producer cells
and packaging into HIV-1/Dvif virions as determined by immunoblotting. (C) Relative editing activity of wild type and mutant A3G proteins as
determined in a bacterial mutator assay relative to activity of wild type. See the legend to Figure 2C. (D) Co-immunoprecipitation of wild type and
mutant A3G with HA-tagged wild type A3G (A3G-HA). Immunoblots on the top show whole cell expression of A3G, A3G-HA, and the cellular control
protein Hsp90, as indicated by Cell at the left of the blot. At the bottom, blots show A3G and A3G-HA in the immunoprecipitate, as indicated by IP to
the left of the blot. (E) Immunoblot showing the effect of BM(PEO)3treatment on wild type and mutant A3G in the lysates of transfected 293T cells.
The control reaction with the W127A was performed and blotted in parallel but was run on a separate gel.
Oligomerization of APOBEC3G
PLoS Pathogens | www.plospathogens.org7 March 2009 | Volume 5 | Issue 3 | e1000330
(Figure 5). Indeed, mutation of basic residues within this pocket
also resulted in losses of antiviral function, packaging into virus
particles, oligomerization and association with cellular RNA
(Figures 6 and 7). Consistent with previous work showing that
only the C-CDA of A3G is responsible for DNA editing [5,38–40],
this attribute was unaffected by disruption of this basic pocket.
Thus, our findings demonstrate further segregation of functions
between the N- and C-CDA domains of A3G.
Throughout our chemical crosslinking experiments, we consis-
tently observed the generation of oligomeric A3G migrating at
,80 kD, which is twice the relative molecular mass of untreated
A3G, which migrates at ,40 kD (Figures 3 and 6). This result
suggests that A3G oligomerizes as a discrete dimer, an assertion
that is further supported by the fact that we did not detect slower
migrating species at ,120 (trimer) or ,160 kD (tetramer). We
note, however, that we have not formally demonstrated dimer-
ization of A3G, as attempts to perform analytical ultracentrifuga-
tion were unsuccessful owing to the poor solubility of purified full-
length A3G at high concentrations (results not shown). Nonethe-
less, dimerization of A3G via the N-terminal CDA domain
remains the simplest model to explain our current results.
Although this conclusion is at odds with a recent study proposing
oligomerization of A3G via the C-CDA domain , that study is
also contradicted by the observations that the C-CDA of A3G
appears as a monomer by both ultracentrifugation  and
Importantly, we have furthermore demonstrated that the
oligomerizaton of A3G is dependent on the presence of RNA, as
evidenced by the disruption of oligomers upon treatment with
RNase (Figure 3). These observations are explained by our
combined modelling and structure-function analyses, which
predict that the oligomer interface between the A3G N-terminal
CDA domains produces a positively charged pocket that requires
occupation by RNA to allow effective oligomerization. Thus, we
propose that the formation of A3G oligomers requires hydro-
phopic and basic residues that mediate protein-protein interac-
tions between the A3G subunits and/or protein-RNA interactions,
in a manner similar to that proposed for PKR and RIG-I [42–44].
We acknowledge, however, that the precise contribution of these
residues to RNA-dependent oligomerization of A3G must await
advances in the biochemical characterization of this protein.
An additional piece of evidence supporting the interdependence
between the oligomerization of A3G and the association with
RNA comes from the analysis of Y1, Y4 and 7SL RNA in A3G
RNPs (Figure 7). In general terms, we observed that oligomeri-
zation-impaired mutants of A3G exhibited much reduced co-
immunoprecipitation of these RNA molecules. Indeed, correla-
tions between oligomer formation and RNA interaction were
excellent in that the R30A and Y124A mutants displayed partial
A3G-A3G interactions as well as intermediate levels of RNA
interactions (Figures 3, 5, and 7). An additional instructive
observation was made upon velocity sedimentation of cell lysates
with oligomerization-impaired A3G, which demonstrated that
assembly into RNase-sensitive high molecular weight RNP
complexes was not noticeably affected by these mutations (Figure
S3). This demonstrates that A3G’s assembly into at least two
intermolecular complexes is RNA-dependent: the oligomerization
of A3G and its recruitment into RNP complexes. Importantly, our
mutational analysis shows that oligomerization can be disrupted
selectively without grossly preventing RNP association. This
suggests either that (1) there is a certain degree of specificity to
the identity of RNAs that are required for A3G oligomerization,
but not to the RNAs that promote RNP association, or that (2)
recruitment of A3G to RNase-sensitive RNP complexes is driven
predominantly by protein-protein interactions. Specifically, RNA-
dependent A3G RNP formation through protein-protein interac-
tions could be mediated by proteins that bind A3G directly and
additionally bind RNA.
Throughout these studies, we have highlighted a tight
correlation between the packaging into HIV-1 virions and the
RNA-dependent oligomerization of wild type and mutant A3G
proteins. Here, we have presented a structure model of an A3G
dimer that readily accommodates these attributes. Indeed, the
packaging of A3G into virus particles has been reported to require
binding to RNA and this has been interpreted as reflective of an
RNA-dependent interaction between the HIV-1 Gag protein and
A3G [10–14,45]. Although the identity of RNA required for
[10,13,14,37,45], specificity with regards to the RNA molecules
that mediate oligomerization of A3G may impart some of the
selectivity for the establishment of an A3G-Gag interaction and
The structure of A3G is also of considerable interest with regard
to the binding of the HIV-1 Vif protein and efforts to manipulate
this interaction therapeutically. Previous analyses have shown that
Vif interacts with a three amino-acid core motif in A3G at residues
128–130 [35,46–49], which is directly adjacent to residues Y124
and W127. This would position the residues of A3G that interact
with Vif in close proximity to the oligomer interface. In our
previous study, we found that mutant proteins with substitutions at
position Y124 or W127 remain responsive to regulation by the Vif
protein , suggesting that oligomerization is not a prerequisite
for binding of Vif. Indeed, the interaction of Vif with A3G in co-
immunoprecipitation experiments is resistant to treatment with
RNase [30,34]. Similarly, mutations at residues 128–130 in A3G
affect the interaction with Vif but not packaging into virus particles
 or generation of the dimeric species by chemical crosslinking
(results not shown). Thus, while the residues that mediate Vif-
binding and RNA-dependent oligomerization are in close
proximity, they appear to be functionally distinct.
We have presented evidence for the RNA-dependent oligomer-
ization of A3G via its N-CDA domain. A structure model of an
A3G dimer based on the A2 crystal structure readily rationalizes
the RNA-dependency of oligomerization as it revealed a clustering
of positive charge near the predicted dimer interface. Further-
more, the model proved consistent with the contribution of basic
residues at the interface to RNA-dependent oligomerization and
packaging of A3G into virus particles. We thus propose that this
model can serve as a guide for the further dissection of the
structure-function relationships of domains and motifs within
A3G. Ultimately, this may help endeavours aimed at therapeutic
intervention with the interaction between the HIV-1 Vif protein
and A3G. In particular, such efforts should strive to preserve the
antiviral functions of A3G by interrupting the interaction with Vif,
while maintaining the interactions that mediate association with
RNA, oligomerization and virion packaging.
Materials and Methods
Plasmids and cloning
Wild type and mutant A3G expression plasmids for infectivity
studies, immunoprecitation, crosslinking and the bacterial editing
assay were generated as described previously . A3G expression
plasmids for the yeast two-hybrid experiments were generated by
cloning of the EcoRI fragment from the pCMV4-A3G plasmids
into the EcoRI site of the pGBKT7 (bait) and pHB18 plasmids
(prey) . Proper orientation and sequence of the insert was
confirmed by restriction digest or sequencing.
Oligomerization of APOBEC3G
PLoS Pathogens | www.plospathogens.org8 March 2009 | Volume 5 | Issue 3 | e1000330
Single-cycle infectivity assays
Stocks of HIV-1/Dvif  were prepared by cotransfection of
35-mm diameter monolayers of 293T cells with 0.5 mg of pA3G
expression vector and 1.0 mg of pIIIB/Dvif using polyethylenimine
(PEI). After 24 hr, the supernatants were harvested and volumes
corresponding to 5 ng p24Gagused to infect 105TZM-bl indicator
cells. The producer cells were lysed in SDS-containing loading dye
for the analysis of protein expression. The induced expression of b-
galactosidase in whole cell lysates was measured 24 hr after the
initiation of infection using the Galacto-Star system (Applied
Analysis of protein expression by immunoblotting
Whole cell lysates prepared from virus producing cells,
immunoprecipitates and purified HIV-1 virions were resolved by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 11% gel)
and analysed by immunoblotting using primary antibodies specific
for A3G , myc (ab9106; Abcam), HA (12CA5), Hsp90 (sc7947:
Santa Cruz) and p24Gag. Blots were resolved using either
horseradish peroxidase-conjugated secondary antibodies and
enhanced chemiluminescence (Pierce) or fluorescent secondary
antibodies using the LI-COR infrared imaging technology (LI-
COR UK LTD).
Virus stocks containing 20 ng p24Gagwere spun in a benchtop
centrifuge at 210006g for 2 h at 4uC through a 20% w/v sucrose
cushion (500 ml) in a 2 ml eppendorf tube. Viral pellets were
resuspended in loading dye and analyzed by immunoblotting.
Whole cell lysates from the corresponding producer cells were
assessed for A3G and Hsp90 expression in parallel.
E. coli mutation assay
The KL16 strain of E. coli was transformed with pTrc99A-
based, IPTG-inducible A3G expression vectors or the empty
vector . Individual colonies were picked and grown to
saturation in LB medium containing 100 mg/ml ampicillin and
1 mM IPTG. Appropriate dilutions were spread onto agar plates
containing either 100 mg/ml ampicillin or 100 mg/ml rifampicin
and incubated overnight at 37uC. Mutation frequencies were
recorded as the number of rifampicin-resistant colonies per 109
viable cells, which were enumerated using the ampicillin-
containing plates. Colony counts were recorded in this manner
on 12 rifampicin- and 12 ampicilin-containing plates for each
construct, in sets of 4 of each at one time. To average the repeat
experiments, the average colony count for wild type A3G was set
at 100 and all other scores were normalized to this value.
Yeast two-hybrid assay
Yeast Y190 cells were transformed with 1 mg of each of the
pGBKT7 (bait) and pHB18 (prey) plasmids . The Wild type
A3G cDNA was inserted into the bait construct, and Wild type
A3G and mutant derivative inserts thereof were cloned into the
prey construct. Transformants were selected on medium lacking
tryptophan and leucine for 3 days at 30uC. Pools of .20
transformed yeast colonies were scraped into b-Gal assay buffer
(60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM
MgSO4, 50 mM 2-mercaptoethanol, 0.01% SDS, pH 7.0) and
normalized according to optical density in a final volume of
500 ml. Cells were lysed by addition of 25 ml of chloroform and
vortexing. The b-Gal substrate chlorophenol red-b-D-galactopyr-
anoside was added to a final concentration of 4 mM and samples
were incubated at room temperature for 30 min. After centrifu-
gation to remove cellular debris, absorbance was determined at
540 nm. Repeat experiments were normalized to the OD540of
samples with Tsg101 (bait) and Vps28 (prey) which was set at 4.0.
293T cells were transfected with 1 mg pA3G-HA and 1 mg
pA3G (wild type or mutant) in 35-mm cultures. After 24 h, the
cells were lysed in 600 ml lysis buffer (0.5% Triton X-100,
287 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, Na2HPO4,
pH 7.2 and complete protease inhibitor cocktail from Roche). The
lysates were cleared by centrifugation in a benchtop centrifuge at
210006g for 10 min and 500 ml of each incubated with the 3F10
HA-specific antibody raised in rat (Roche) and protein G-agarose
(Invitrogen) for 2 h at 4uC. 50 ml of the cleared lysate was kept to
analyse protein expression levels. After binding to the beads the
samples were washed twice with lysis buffer and split into two
aliquots of 250 ml. To one aliquot of the samples 25 U of bovine
pancreatic RNase A (Sigma) was added, and all samples were
tumbled at room temperature for 30 min. The agarose beads were
then washed three times with lysis buffer, and resuspended in
50 ml loading dye. 10 ml of the immunoprecipitated samples as
well as 10 ml of the cleared lysate were resolved by SDS-PAGE
(11% gel) and analyzed by immunoblotting using primary
antibodies specific for HA, A3G or Hsp90.
293T cells were transfected with 2 mg pA3G (wild type or
mutant) in 35-mm cultures. After 24 h, the cells lysed in 600 ml
lysis buffer (0.5% Triton X-100, 287 mM NaCl, 2.68 mM KCl,
1.47 mM KH2PO4, Na2HPO4, pH 7.2 and complete protease
inhibitor cocktail from Roche). The lysates were cleared by
centrifugation in a benchtop centrifuge at 210006g for 10 min.
Samples were then split into aliquots of 100 ml to which 10 U of
RNase A was, or was not, added either prior to or after addition of
1.25 ml of 20 mM BM(PEO)3(Thermo Scientific) in DMSO. After
incubation at 20uC for 1 h, 1 ml of 1 M DTT was added to
quench the reaction. After the addition of 25 ml loading dye,
samples were analysed by SDS-PAGE and immunoblotting. In the
experiment describing crosslinking of A3G-myc to A3G-HA,
293T cells were transfected with 2 mg of each plasmid. After 24 h,
cells were lysed in 600 ml lysis buffer and cleared by centrifugation.
Samples were split into aliquots of 250 ml which were treated, or
not, with 2.5 ml of 20 mM BM(PEO)3in DMSO. After addition of
2 ml 1 M DTT samples were incubated with the 3F10 anti-HA
antibody (Roche) and protein A-agarose beads. Subsequent
immunoprecipitation was performed in the manner described
above and the gel resolved samples analysed using a myc-specific
Reverse transcription coupled PCR
293T cells in a 10 cm dish were transfected with 12 mg of wild
type or mutant A3G-HA expression vector and lysed after 24 h in
lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5
and complete protease inhibitor cocktail from Roche). The cell
lysates were precleared overnight using an irrelevant monoclonal
antibody and A3G ribonucleoprotein complexes were subjected to
immunoprecipitation with the 3F10 rat anti-HA antibody using
protein G-coupled agarose beads. Following immunoprecipitation,
associated RNAs were recovered with the miRNAeasy mini kit
(Qiagen). RNA was detected by semi-quantitative RT-PCR using
the SuperScript III One-Step RT-PCR system with platinum Taq
DNA polymerase (Invitrogen) (cDNA synthesis at 55uC for
30 min, denaturation at 95uC for 2 min, 15 amplification cycles
of 95uC for 15 sec, 56uC for 30 sec and 68uC for 1 min, and a
Oligomerization of APOBEC3G
PLoS Pathogens | www.plospathogens.org9 March 2009 | Volume 5 | Issue 3 | e1000330
final extension step at 68uC for 5 min) using specific primers for Y
and 7SL RNAs . Products were resolved by electrophoresis on
a 1.5% agarose gel and stained with ethidium bromide.
Homology modelling of an A3G dimer
The structure of the dimer model of A3G was obtained by
homology modelling using as a template the crystal structure of
APOBEC2 (A2) (2NYT pdb entry). To generate the 3D-model,
the alignment between A3G and APOBEC2 was submitted to the
comparative structural modeling program MODELLER 8v2 .
100 best solutions for the MODELLER objective function have
been considered. Models were produced with either the N- or C-
terminal CDA domain at the dimer interface.
293T cells were transfected with 2 mg pA3G (wild type or
mutant) in 35-mm cultures. After 24 h, the cells were lysed in
250 ml lysis buffer (0.626% NP40, 100 mM NaCl, 50 mM KAc,
10 mM EDTA, 10 mM Tris pH 7.4 and complete protease
inhibitor cocktail from Roche). The lysates were cleared by
centrifugation in a benchtop centrifuge at 1626 g for 10 min
followed by 180006 g for 30 sec. Samples were then split into
aliquots of 100 ml to which 10 U of RNase A (Sigma) was, or was
not, added. Samples were then loaded on top of a 10–15–20–30–
50% sucrose step gradient in lysis buffer and centrifuged for
45 min at 1630006 g at 4uC. After centrifugation, samples of
78 ml were sequentially removed from the top of the gradient,
added to 30 ml of loading dye and analysed by immunoblotting.
tion (B) of A3G proteins with the Y124A, Y124F, W127A, or
W127Y mutations. Refer to the legend for Figure 3 for details.
Chemical crosslinking (A) and co-immunoprecipita-
Found at: doi:10.1371/journal.ppat.1000330.s001 (1.21 MB TIF)
homology model of A3G (magenta) with the ten NMR models
(blue, RMSD 4.920 A˚) (A) and the crystal structure (yellow,
RMSD 3.650 A˚) (B)
Found at: doi:10.1371/journal.ppat.1000330.s002 (9.41 MB TIF)
Superposition of the C-CDA domain from the
through a sucrose gradient. The direction of the gradient is
indicated at the top of the figure and treatment with RNase by
a+at the right of the figure. Samples were examined by
immunoblot using the A3G-specific antibody.
Found at: doi:10.1371/journal.ppat.1000330.s003 (2.03 MB TIF)
Velocity sedimentation of wild type or mutant A3G
terminal (C-C) models for A3G oligomerization
Found at: doi:10.1371/journal.ppat.1000330.s004 (0.01 MB PDF)
Energy decomposition of N-terminal (N-N) and C-
dimer formation as calculated with POPS
Found at: doi:10.1371/journal.ppat.1000330.s005 (0.05 MB PDF)
Solvent-accessible surface area (SASA) buried upon
W127A mutations into the structure model of the A3G dimer
Found at: doi:10.1371/journal.ppat.1000330.s006 (0.01 MB PDF)
Interactions lost upon introduction of the Y124A and
Conceived and designed the experiments: Hh MHM. Performed the
experiments: Hh FA SGM. Analyzed the data: Hh FA. Contributed
reagents/materials/analysis tools: Hh SGM FF MHM. Wrote the paper:
Hh FA SGM FF MHM. Performed molecular modeling: FA FF.
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PLoS Pathogens | www.plospathogens.org11 March 2009 | Volume 5 | Issue 3 | e1000330