Evolutionarily conserved and non-conserved
retrovirus restriction activities of artiodactyl
Stefa ´n R. Jo ´nsson1,2,3,4, Guylaine Hache ´1,2,3, Mark D. Stenglein1,2,3,
Scott C. Fahrenkrug3,5, Valgerdur Andre ´sdo ´ttir4and Reuben S. Harris1,2,3,*
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,2Institute for
Molecular Virology,3Arnold and Mabel Beckman Center for Transposon Research, Minneapolis,
MN 55455, USA,4University of Iceland, Institute for Experimental Pathology, Keldur v/Vesturlandsveg,
112 Reykjavı ´k, Iceland and5Department of Animal Sciences, University of Minnesota,
St Paul, MN 55108, USA
Received August 4, 2006; Revised September 16, 2006; Accepted September 18, 2006
The APOBEC3 proteins are unique to mammals.
Many inhibit retrovirus infection through a cDNA
cytosine deamination mechanism. HIV-1 neutralizes
this host defense through Vif, which triggers
APOBEC3 ubiquitination and degradation. Here, we
report an APOBEC3F-like, double deaminase domain
protein from three artiodactyls: cattle, pigs and
sheep. Like their human counterparts, APOBEC3F
and APOBEC3G, the artiodactyl APOBEC3F proteins
are DNA cytosine deaminases that locate predomi-
nantly to the cytosol and can inhibit the replication
of HIV-1 and MLV. Retrovirus restriction is attri-
butable to deaminase-dependent and -independent
mechanisms, as deaminase-defective mutants retain
significant anti-retroviral activity. However, unlike
human APOBEC3F and APOBEC3G, the artiodactyl
APOBEC3F proteins have an active N-terminal DNA
cytosine deaminase domain, which elicits a broader
dinucleotide deamination preference, and they are
resistant to HIV-1 Vif. These data indicate that DNA
cytosine deamination; sub-cellular localization and
retrovirus restriction activities are conserved in
mammals, whereas active site location, local muta-
tional preferences and Vif susceptibility are not.
Together, these studies indicate that some proper-
ties of the mammal-specific, APOBEC3-dependent
retroelement restriction system are necessary and
conserved, but others are simultaneously modular
and highly adaptable.
Expression of many of the human APOBEC3 (A3) proteins
has been shown to inhibit the infective potential and mobility
of a broad and growing number of retroviruses and retrotrans-
posons [reviewed by (1–4)]. Humans encode seven A3
proteins, Homo sapiens (Hs) HsA3A, HsA3B, HsA3C,
HsA3DE, HsA3F, HsA3G and HsA3H, in tandem on
chromosome 22 (2,5–7). HsA3A has recently been shown
to inhibit the mobility of both long terminal repeat (LTR)
and non-LTR retrotransposons (8–10). HsA3B can also
inhibit L1 and Alu retrotransposition, as well as the replica-
tion of SIV and to a lesser extent HIV-1 (8–13). HsA3C
potently inhibits SIV, but it has shown little activity against
other substrates (11,13). HsA3DE was recently shown to pos-
sess weak antiviral activity (14). HsA3G was the first member
of this family to be associated with HIV restriction (15).
HsA3F and HsA3G are both capable of potently inhibiting
a variety of exogenous and endogenous retroelements
(9,10,12,13,15–25). Finally, although HsA3H elicited DNA
cytosine deaminase activity, it was unable to restrict SIV or
HIV-1 replication (26). Several simian (e.g. chimpanzee),
one carnivore (cat) and one rodent (mouse) APOBEC3
protein have also been shown to possess retroelement restric-
tion activities [e.g. (20,25,27–30)]. APOBEC3 proteins from
other mammals have yet to be examined.
Three themes appear to be emerging from these studies.
First, the A3 proteins deaminate cytosines to uracils (C!U)
within single-strand DNA (ssDNA). This property enables the
A3 proteins to target the cDNA replication intermediates of
all of the aforementioned retroviruses and retrotransposons.
Second, retroelement restriction is mediated by at least two
distinct mechanisms—by retroviral cDNA cytosine deamina-
tion (the hallmark activity of this family of proteins) and by a
*To whom correspondence should be addressed. Tel: +1 612 624 0457; Fax: +1 612 625 2163; Email: email@example.com
? 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
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Published online 11 October 2006Nucleic Acids Research, 2006, Vol. 34, No. 195683–5694
understood [e.g. (12,31–34)]. Finally, many of the retroele-
ments (especially the retroviruses) that have been examined
in detail can evade A3-dependent restriction. For instance,
the virion infectivity factor (Vif) of HIV-1 and SIV recruits
a cellular ubiquitin ligation complex to purge cells of A3G,
the Bet protein of several different foamy viruses appears
to directly bind and neutralize A3G and other retroviruses
have simply evolved to exclude A3 proteins from nascent
virions (e.g. MLV and HTLV) (13,28–30,33,35–39).
Strong evidence indicates that the conflict between host
A3 proteins and invasive retroelements is ancient. Phylo-
genome sequences indicate that the A3 proteins are at least
as old as the mammalian lineage, because rodents encode
one and primates seven A3 proteins (2,6). Other vertebrates,
such as birds and fish do not have A3 proteins per se, but
they do encode activation-induced deaminase (AID), an A3
orthologue that uses DNA cytosine deamination to trigger
immunoglobulin gene hypermutation and isotype switch
recombination [recently reviewed by (40–42)]. Comparative
studies of A3 proteins from humans and non-human primates,
New World monkeys, such as the tamarin and the woolly
monkey, have demonstrated that the mammalian A3 proteins
have been under a strong positive selection for at least 33 mil-
lion years (26,43,44). The strong and likely ongoing positive
selection and the unparalleled A3 gene expansion from one in
rodents to seven in primates combine to suggest that the A3
proteins form a highly flexible and adaptable innate host
defense system, which may very well be capable of readily
adapting to new and potentially invasive retroelements.
The evolutionary gap between rodents and primates is
?90–100 million years [e.g. (45)]. This large genetic distance
enables some comparative studies, but it limits others. There-
fore, to close some of this distance, to enable more extensive
comparative studies, and to examine potentially novel
A3-virus conflicts, we have cloned and characterized A3
proteins from representative artiodactyls: cattle (Bos taurus;
Bt), sheep (Ovis aries; Oa) and pigs (Sus scrofa; Ss). These
studies focused on artiodactyl A3 proteins that were predicted
to be similar in size (ca. 400 amino acids) and domain
domains) to the mouse Mus musculus (Mm) A3 protein and
the well-characterized HsA3F and HsA3G proteins. The
results of our studies emphasize the importance of DNA cyto-
sine deamination in retrovirus restriction, but they also high-
light the existence of a conserved deaminase-independent
restriction mechanism. Moreover, non-conserved properties,
such as Vif susceptibility, active site location and local
mutational preferences combine to suggest mechanistic
flexibilities that the APOBEC3 proteins might employ
while adapting to new and potentially threatening genetic
mechanism that is notwell
MATERIALS AND METHODS
Artiodactyl A3F cDNA sequences and expression
from pTrc99A (GE Healthcare). The SsA3F expression
cDNA [GB# BI346898 (46)] using 50-NNNNGAGCTCAGG-
TACCACCATGGATCCTCAGCGCCTGAGAC and 50-NN
ing the PCR product with KpnI and SalI, and ligating it
into a similarly digested pTrc99A. The BtA3F expression
construct was made in the same manner by PCR amplifying
a cDNA [GB# BE684372 (47)] using oligonucleotides 50-
CGAGGC and 50-NNNNGTCGACCTAAATTGGGGCCGT-
TAGGAT. OaA3F was obtained by first amplifying a frag-
ment by degenerative PCR using oligonucleotides 50-TWYR-
TVTCCTGGAGCCCCTG and 50-CCRKWWGTWGTAGA-
GGCGR, which were made to conserved regions of
APOBEC3 proteins using template cDNA from sheep macro-
phages. The amplified fragment was sequenced and used to
make OaA3F-specific oligonucleotides for 30RACE to obtain
the remainder of the OaA3F coding sequence, 50-AACCAG-
GTCTATGCTGGGACT and 50-CTGGGGATGTACCAGA-
ATGTG were used with an oligo dT primer 50-AAGCAGTG-
GTAACAACGCAGAGTACT30VN. The OaA3F expression
plasmid was made by amplification from cDNA of sheep
macrophages using oligonucleotides 50-NNNNGAGCTCAG-
GTACCACCATGCCCTGGATCAGCGACCAC and 50-NN-
same manner as the BtA3F and SsA3F constructs. E!Q
zinc-binding domain mutants were constructed using standard
site-directed mutagenesis protocols (Stratagene). The follow-
ing oligonucleotides were used: SsA3F (E87!Q) 50-CCGA-
GGGCGGGTCGG; SsA3F (E267!Q) 50-ACAAGAAAAA-
GCGACATGCACAAATTCGTTTTATTGACAAG and 50-
CTTGT; BtA3F (E80!Q) 50-GTGGGACTCGCTGCCACA-
CCCAACTCC;GCTTCCTGTCTTGG and 50-CCAAGACA-
BtA3F (E260!Q) 50-ACAAGAAGCAGCGGCATGCACA-
CTTCCTGTCTTGG and 50-CCAAGACAGGAAGCGGC-
GTTGGCTGTGGCAGTGAGTCCCAG; OaA3F (E247!Q)
TGACAAG and 50-CTTGTCAATAAAGCGAATTTGTGC-
ATGCCGCTGCTTCTTGT. Eukaryotic expression plasmids
were derived from pcDNA3.1(+) (Invitrogen) by subcloning
the KpnI/SalI-flanked A3 cDNAs from the E.coli expression
Additional DNA constructs
HsA3F, HsA3G and HsAID expression plasmids were
described previously (31,48). MmA3 was made in the same
manner as the artiodactyl constructs using oligonucleotides
CTGGGA and 50-NNNNGTCGACATCAAGACATCGGG-
GGTCCAAGCTG. E!Q zinc-binding domain mutants
were constructed using standard site-directed mutagenesis
protocols (Stratagene). The following oligonucleotides were
5684Nucleic Acids Research, 2006, Vol. 34, No. 19
used: HsA3G (E67!Q) 50-CTTAAGTACCACCCACAGA-
GGGTGGTACTTAAG; HsA3G(E259!Q) 50-CCTTGAA-
GGCCGCCATGCACAGCTGTGCTTCCTGG and 50-CCA-
CAG; HsA3F(E251!Q) 50-CCCATTGTCATGCACAAAG-
GTGCTTCCTC and 50-GAGGAAGCACCTTTGTGCATG-
ACAATGGG; MmA3(E73!Q) 50-CCAGTATAAAAAGC-
AGATTTGAGCGTGGATGTTGTCCTTG and 50-CAAG-
CATGCTGTTTGCCTTTC and 50-GAAAGGCAAACAGC-
expression plasmids were derived from pcDNA3.1(+) (Invit-
rogen) and pEGFP-N3 (Clontech) by inserting KpnI and SalI
or SacI and SalI-flanked A3 cDNAs from the E.coli expres-
sion plasmids. The coding sequences of HsAID, HsA3F,
HsA3G and MmA3 used in this study are identical those rep-
resented by GenBank accession nos NP_065712, NP_660341,
NP_068594 and AAH03314, respectively. The coding
sequence of HsA3B has two amino acid substitutions,
W228L and D316N (12), which distinguish it from GenBank
Amino acid alignments and phylogenetic
Protein alignments and phylogenetic analyses of the
conserved, zinc-binding deaminase cores were done using
the Clustal W software (49). A rooted phylogenetic tree
was constructed using the neighbor-joining method. The
GenBank accession numbers of the sequences used
in these comparisons were
NP_112436 (MmA1), NP_037039 (RnA1), NP_006780
NP_033824 (MmA2), XP_217334 (RnA2), XP_528775
(PtAID), NP_065712 (HsAID), NP_033775 (MmAID),
NP_663745 (HsA3A), NP_004891 (HsA3B), NP_055323
(HsA3C), Q96AK3 (HsA3DE), NP_660341 (HsA3F),
NP_068594 (HsA3G), NP_861438 (HsA3H), AAT44392
(PtA3G), ABD72578 (PtA3H), AAH03314 (MmA3) and
The intrinsic DNA cytosine deaminase activity of the A3
proteins was assayed by quantifying the accumulation
of RifRmutants in ung-deficient E.coli [e.g. (31,48)]. Briefly,
dilutions of A3- or vector control-expressing single colo-
nies were grown to saturation in a rich bacterial growth
medium containing 100 mg/ml ampicillin and 1 mM
isopropyl-b-D-thiogalactopyranoside (IPTG). Aliquots were
spread to plates containing rifampicin (100 mg/ml; Sigma)
to select for RifRmutants. Dilutions were spread to plates
containing ampicillin to determine the number of viable
cells. Mutation frequencies were calculated as the number
of RifRmutants per 107viable cells. For each experi-
mental condition, 8 or 12 independent transformants were
Cells were visualized on a Zeiss Axiovert 200 microscope at
400· total magnification. A total of 7500 HeLa cells were
seeded on LabTek chambered coverglasses (Nunc). After
24 h of incubation, these cells were transfected with 200 ng
of the pEGFP-N3-based DNA constructs. After an additional
24 h of incubation, images of the live cells were collected.
HsA3B, HsA3F and HsA3G–eGFP fusion constructs were
reported previously (12).
Green fluorescent protein (GFP)-based retrovirus
293T cells were grown in DMEM supplemented with 10%
FBS, penicillin and streptomycin. Viruses were produced
by lipid-mediated transfection (Fugene; Roche) of 50%
confluent 293T cells with the following plasmids. HIV-
GFP: 0.22 mg of CS-CG (50), 0.14 mg of pRK5/Pack1(Gag-
Pol), 0.07 mg of pRK5/Rev, 0.07 mg of pMDG (VSV-G
Env) and 0.5 mg of an A3 expression plasmid or an empty
vector control, as described previously (31,51). In some
experiments, an HIV-1 Vif or a DVif control plasmid
was included (0.5 mg). MLV-GFP was produced similarly,
except the proviral plasmid was pM3P-GFP and a MLV
gag/pol construct was used (18,51). After 48 h of incubation,
the virus-containing supernatants were clarified by low speed
centrifugation, filtered (0.45 mm), and quantified using a
reverse transcriptase activity-based enzyme-linked immuno-
sorbent assay (ELISA) (Cavidi Tech). Reverse transcriptase-
normalized supernatants were applied to fresh 293T cells, and
infection was allowed to proceed for 72 h. Infectivity (GFP
fluorescence) was measured by flow cytometry (FACSCal-
ibur, BD Biosciences). For experiments requiring the recov-
ery of retroviral DNA for hypermutation analyses, the viral
supernatants were treated with 50 U/ml DNase (Sigma)
prior to 293T cell infection.
Retroviral DNA sequence analyses
Genomic DNA was incubated with DpnI to remove possible
contaminating input CS-CG plasmid DNA. GFP was ampli-
fied using high-fidelity Phusion polymerase (Finnzymes)
and CS–CG-specific primers, 50-CGTGTACGGTGGGAG-
GTCTA and 50-TTGGTAGCTGCTGTGTTGCT. PCR prod-
ucts were cloned and sequenced as described previously
(51). Mutational analyses were performed using Sequencher
software (Gene Codes Corp.).
GenBank accession numbers
The OaA3F cDNA sequence has been assigned GenBank
DQ974645. The GenBank
(BE684372) and SsA3F (BI346898) have been updated to
The double deaminase domain APOBEC3F proteins of
All known A3 proteins have either one or two conserved,
zinc-binding deaminase domains, consisting of amino acids
Nucleic Acids Research, 2006, Vol. 34, No. 195685
HXE-X23–28-PCX2–4C (X can be any amino acid) (2,5–7).
The histidine and the two cysteines coordinate zinc and the
glutamate participates directly in the C!U deamination reac-
tion. NCBI BLAST searches using the human and mouse A3
deaminase domains as query polypeptides revealed several
artiodactyl ESTs, which suggested the presence of at least
one A3 protein in cattle and pigs. Corresponding cDNA
clones were obtained, sequenced and shown to encode A3
proteins with two putative zinc-binding, cytosine deaminase
domains (Figure 1A and Supplementary Figures S1 and S2;
Materials and Methods). The orthologous sheep double
domain A3 cDNA sequence was obtained using a combina-
tion of degenerate PCR and RACE (Figure 1A and Supple-
mentary Figures S1 and S2). All three of these A3 proteins
were similar in size to the 373 amino acid HsA3F protein,
except the pig A3 protein, which was slightly longer due to
a unique C-terminal, serine-rich extension (Figure 1A and
Supplementary Figures S1 and S2).
Nomenclature standards dictate that protein names should
be assigned based on the closest human orthologue [e.g.
(52,53)]. However, amino acid comparisons showed that the
N- and the C-terminal artiodactyl deaminase domains were
most similar to different human A3 proteins (data not
shown). To resolve this ambiguity and to facilitate name
assignments, we named the artiodactyl double domain pro-
teins after ‘the human double domain A3 protein with the
highest degree of active site identity’ (see below for experi-
mental demonstrations). The active sites of these artiodactyl
A3 proteins were 56–62% identical to HsA3F (Figure 1B
and Supplementary Figure S1). With the exception of
HsA3E, which has equivalent identity (but no demonstrated
activity), all of the other human A3 proteins had less identity.
Thus, we have named the double domain deaminase proteins
from cow, sheep and pig after HsA3F and, hereafter, will
refer to them as BtA3F, OaA3F and SsA3F, respectively.
Amino acid alignments of the active deaminase domains
plus five residues on each side showed that the cow and
sheep A3F active sites are 78% identical (Figure 1B and
Supplementary Figures S1 and S2). Both the cow and the
sheep proteins shared a lower level of identity with the pig
protein (56%). These identity differences are consistent
with the estimated times of divergence from their last
common ancestor, as cows and sheep were estimated to
have diverged from each other 14–20 million years ago,
whereas pigs diverged from this lineage approximately
35–55 million years ago [e.g. (54)].
Phylogenetic analyses of the artiodactyl A3F proteins
As described above, all A3 proteins have either one or two
Figure 1A]. These domains cluster into three distinct phylo-
genetic groups: Z1a, Z1b or Z2 (7). The human double Z
domain proteins, HsA3F and HsA3G, have a Z1a/Z1a and a
Z1a/Z1b organization, respectively, whereas the MmA3 pro-
tein has a Z1a/Z2 organization. Interestingly, all three of the
artiodactyl A3F proteins have a Z1a/Z2 organization
The active sites of most of the human DNA cytosine
deaminase-competent A3 proteins can be classed as Z1
[e.g. HsA3F is Z1a and HsA3G is Z1b (31,32)]. Recently,
the only human protein with a Z2 designation, HsA3H, was
also shown to possess DNA cytosine deaminase activity
(26). It is not clear, which Z domain(s) of MmA3 is active
(addressed below). Thus, the N-terminal Z1a or the
C-terminal Z2 domain of the artiodactyl A3F proteins appeared
to have the potential to catalyze DNA cytosine deamination.
The artiodactyl A3F proteins catalyze DNA cytosine
To test whether the artiodactyl A3F proteins have the
capacity to deaminate cytosines within ssDNA, the intrinisic
mutator activity of these proteins was monitored using an
E.coli-based mutation assay. RifRis attributable to base sub-
stitution mutations in the E.coli RNA polymerase B (rpoB)
gene, and it occurs in approximately 1 of every 5 million
bacterial cells. We have found previously that expression
of several A3 family members, including HsAID, rat
APOBEC1, HsA3C, HsA3F and HsA3G, can accelerate the
accumulation of RifRmutations from a few- to several
100-fold (48,51,55). The mutator phenotype is accounted
for by a pronouned C/G!T/A transition bias within rpoB.
This assay therefore provides a robust measure of intrisic
DNA cytosine deaminase activity.
Expression of each of the artiodactyl A3 proteins increased
the RifRmutation frequency in E.coli from 3- to 7-fold, levels
that were higher than those attributable to HsA3F but slightly
lower than than those caused by HsA3G (Figure 2A). Curi-
ously, expression of MmA3 failed to cause an E.coli mutator
phenotype, despite the fact that it is clearly active and capable
of deaminating the cDNA of a variety of retroelements [e.g.
(20,27) and below]. It is not clear why MmA3 is inactive in
this system, yet active in others.
Artiodactyl A3F DNA cytosine deamination preferences
were examined by sequencing the rpoB gene of at least 100
independent RifRmutants (Figure 2B and C). In contrast to
HsA3F and HsA3G, which preferentially deaminate cytosines
at rpoB nucleotide positions 1721 and 1691, 50-TC and 50-CC,
respectively (48,51), the artiodactyl A3F proteins showed less
biased rpoB mutation spectra. OaA3F preferentially deami-
nated cytosine 1576, which is part of a 50-GC dinucleotide.
SsA3F also preferred cytosine 1576. However, SsA3F also
clearly deaminated cytosine 1586, which is part of a 50-AC
dinucleotide. Interestingly, these two cytosines, C1576 and
C1586, are also preferred by HsAID [Figure 2B and
(48,55)]. The fact that HsAID and SsA3F appear to share a
50-purine-C deamination preference suggests that their com-
mon ancestor [perhaps an ancient AID (40)] may have had
a similar target preference. However, several groups have
reported that even subtle amino acid substitutions in HsA3
proteins can dramatically alter local DNA cytosine deamina-
tion preferences, and therefore it is not surprising that most of
the present day A3 proteins show non-AID-like mutational
preferences (13,31,56). For instance, BtA3F did not appear
to have any prominent rpoB local mutation preference, as
increased levels of C/G!T/A mutation were apparent at sev-
eral sites. In conclusion, all three of the artiodactyl A3F pro-
teins are capable of deaminating DNA cytosines to uracils,
which triggers a corresponding shift in the pattern of
C/G!T/A transition mutations within the rpoB mutation
5686Nucleic Acids Research, 2006, Vol. 34, No. 19
Figure 1. A comparison of artiodactyl A3F proteins. (A) A schematic of HsA3F, BtA3F, OaA3F and SsA3F. The conserved, zinc-binding deaminase domains are
boxed (*) and those that are catalytically active are additionally shaded. The numbers on the right indicate the total polypeptide length. (B) HsA3F, BtA3F,
OaA3F and SsA3F active site amino acid alignments [shaded regions from (A)]. The conserved HXE and PCXXC motifs are boxed. Amino acid positions are
indicated on the right. (C) A neighbor-joining phylogenetic tree indicating the evolutionary relationship of several representative mammlian A3 family members.
Branch lengths are proportional to the number of amino acid differences. Comparisons were done using the conserved Z domain amino acids, plus five additional
residues on either side. The Z1a, Z1b and Z2 phylogenetic clusters are indicated. HsA3D and HsA3E represent the N- and C-terminal domains of HsA3DE. See
the text for additional details. Bt, B.taurus (cow); Cf, Canis familiaris (dog); Hs, H.sapiens (human); Oa, O.aries (sheep); Pt, Pan troglodytes (chimpanzee); Rn,
Rattus norvegicus (rat); Ss, S.scrofa (pig).
Nucleic Acids Research, 2006, Vol. 34, No. 19 5687
The artiodactyl A3F proteins are predominantly
As an initial step toward understanding the potential retroele-
ment targets of the artiodactyl A3F proteins, the sub-cellular
distribution of these proteins was determined. A3-GFP
constructs were transfected into HeLa cells and the sub-cellu-
lar localization of the A3 proteins was determined by live cell
fluorescence microscopy. In agreement with prior work,
HsA3B and a GFP-only control localized to the nucleus and
cell-wide, respectively (12). In contrast, the artiodactyl A3F
proteins and MmA3 appeared predominantly cytoplasmic
(Figure 3). Many cells contained brightly fluorescing, punct-
ate cytoplasmic aggregations, which may represent P bodies
[e.g. (57)]. The significance of the cytoplasmic punctae
remains to be determined. Nevertheless, the cytoplasmic
localization pattern is nearly identical to that of HsA3F and
HsA3G suggesting that the property of localizing to the
cytoplasm is conserved and that the artiodactyl A3F proteins
might function similarly to inhibit the replication of LTR-
dependent retroviruses, such as HIV-1 or MLV [Figure 3;
compare with (9,12,16,58)]. One should note, however, that
the present data do not exclude the possibility that one or
more of these A3 proteins might also possess the nucleo-
cytoplasmic shuttling capability of AID, which also appears
predominantly cytoplasmic but is clearly capable of entering
and exiting the nuclear compartment where it triggers
antibody gene diversification processes (59,60).
Retrovirus restriction by artiodactyl A3F proteins
A clear trend in the genetic conflict between A3 proteins and
retroelements is that an A3 from a given host is either neutral-
ized or avoided by retroelements that are specific to the host
species. For instance, HIV-1 Vif counteracts both HsA3F and
HsA3G, and SIVagmVif inhibits African green monkey A3G
[e.g. (51,61–64). In many instances, cross-species compar-
isons enable potential species-specific mechanisms of neutral-
ization to be avoided and the restrictive potential of A3
proteins to be studied. We therefore asked whether the artio-
dactyl A3F proteins could inhibit the infectivity of HIV-1 and
MLV-based retroviruses. In these systems, a GFP gene
embedded in proviral DNA provides a measure of both trans-
fection efficiency (which correlates directly with virus
production levels) and viral infectivity [e.g. (18,51)].
Expression of HsA3F and HsA3G caused 4- and 24-fold
reductions in the infectivity of HIV-GFP, in agreement with
previous studies [Figure 4A and (51,64)]. MmA3 was also
capable of strongly inhibiting HIV-GFP. In comparison,
expression of BtA3F, OaA3F or SsA3F caused 30-, 8- and
29-fold decreases in the infectivity of HIV-GFP, respectively
(Figure 4A). These potent anti-HIV activities demonstrated
that the artiodactyl A3F proteins possess at least one retro-
virus restriction activity. These results further imply that
the artiodactyl A3F proteins are able to specifically associate
with the HIV-1 Gag–genomic RNA complex and thereby
gain access to assembling virus particles (addressed further
Expression of MmA3 has little effect on the infectivity of
MLV, presumably because MLV excludes (or simply avoids)
this A3 protein [Figure 4B and (13,27,37,39)]. In contrast,
HsA3F and HsA3G inhibit the infectivity of MLV-based
Figure 2. DNA cytosine deaminase activity of the artiodactyl A3F proteins in
E.coli. (A) RifRmutation frequencies for 12 independent E.coli cultures
expressing a vector control (light blue), HsAID (purple), HsA3G (dark blue),
HsA3F (red), BtA3B (brown), OaA3B (black), SsA3F (pink) or MmA3
(gray). Each data point corresponds to the mutation frequency obtained from
a single culture, and the median mutation frequency for each condition is
shown (horizontal bar). (B) A histogram summarizing the C/G!T/A
transition mutations detected in rpoB. Only cytosines that had greater than
two mutations are shown. Apart from C1586, all of the cytosines are located in
the non-template strand of the rpoB gene. The number of independent RifR
mutants that were sequenced is indicated in parentheses in the legend. For
purposes of presentation the Y-axis extends below zero (a dotted line marks
the actual base-line), and the histogram bars follow the color scheme (A).
(C) Pie graphs depicting the frequency that each of the four dinucleotides was
targeted by the indicated A3 protein. The total number of independent
sequences analyzed is shown in the center of each graph, and the deaminated
cytosine is underlined in the legend. The dinucleotide wedges of each pie are
colored as indicated.
5688Nucleic Acids Research, 2006, Vol. 34, No. 19
retroviruses, but to a lesser extent than HIV lacking Vif
[Figure 4B and, e.g. (18,51,64)]. Therefore, to ask whether
the artiodactyl A3F proteins possess similar restriction poten-
tials, the infectivity of MLV-GFP produced in the presence of
these A3 proteins was monitored. Interestingly, similar to
HsA3F or HsA3G, expression of the artiodactyl A3F proteins
reduced the infectivity of MLV-GFP by 2- to 4-fold. Thus,
the HIV-GFP and MLV-GFP infectivity data combined to
suggest that the artiodactyl A3F proteins have a relatively
broad retrovirus restriction potential.
The N-terminal zinc-binding, deaminase domain
of the artiodactyl A3F proteins catalyzes C!U
deamination, and this activity is necessary for full
levels of retrovirus restriction
All of the double domain deaminases thus far characterized
have catalytically competent C-terminal Z domains and
apparently inert N-terminal Z domains (10,12,31,32,34).
Most experiments have focused on HsA3B, HsA3F and
HsA3G, DNA cytosine deaminases that have Z1a- or Z1b-
type, C-terminal active sites (Figure 1). However, HsA3H,
which has a single Z2-type deaminase motif, was also
shown to possess DNA cytosine deaminase activity (26).
Thus, it was possible that either the N- or the C-terminal Z
domain (or both) of the artiodactyl A3F proteins would be
To begin to work-out the mechanism of retrovirus
restriction by artiodactyl A3F proteins and to test whether the
N- or the C-terminal (or both) Z domain of these proteins
catalyzes DNA cytosine deamination, the conserved gluta-
mate (E) of each active site was changed to glutamine (Q)
and the resulting mutants were tested for HIV-GFP restriction
activity. As reported previously, the glutamate of both the
N- and the C-terminal Z domain of HsA3G contributed
to inhibiting HIV-1 infectivity, but the C-terminal catalytic
glutamate appeared to be more important [Figure 5A and
(34,65,66)]. In contrast, both the N- and the C-terminal
BtA3F Z domain E!Q mutants appeared to retain full levels
of anti-HIV activity. Interestingly, the N-terminal OaA3F and
SsA3F Z domain E!Q mutants were less able than the
corresponding C-terminal domain mutants to inhibit the
infectivity of HIV-GFP, a result particularly clear for
SsA3F (Figure 5A). These data were essentially the inverse
of the HsA3F and HsA3G E!Q mutant studies, and they
suggested that the N-terminal, Z domain of these proteins
catalyzes retroviral cDNA C!U deamination. MmA3 was
clearly distinct, as both the N- and the C-terminal Z domain
Although both the N- and the C-terminal Z domain E!Q
mutants of the human and the artiodactyl A3 proteins showed
significant levels of anti-retroviral activity, we surmised that
bona fide catalytic site mutants should be unable to catalyze
retroviral cDNA C!U deamination [although they may still
inhibit retroviral infectivity; e.g. (32)]. Minus strand uracils
template the incorporation of plus strand adenines, ultimately
manifesting as retroviral plus strand G!A hypermutations.
Therefore, to directly test which Z domain(s) catalyzes
DNA cytosine deamination and to gain additional insight
into the artiodactyl A3F retrovirus restriction mechanism,
the GFP gene from the aforementioned HIV-GFP infectivity
experiments was amplified by high-fidelity PCR, cloned and
sequenced. HIV-GFP produced in the presence of a control
vector showed a low base substitution mutation frequency,
0.00014 mutations per base, which is attributable to errors
in RT and PCR (Figure 5B). In contrast, viruses produced
in the presence of HsA3F, HsA3G, all three of the artiodactyl
A3F proteins or MmA3 showed between 30- and 80-fold
more base substitution mutations, which were almost exclu-
sively plus strand G!A transition mutations (Figure 5B
and Supplementary Figure S3). As described previously
by Malim and co-workers, HsA3G with a C-terminal domain
E!Q mutation failed to cause retroviral hypermutation,
although this variant still significantly inhibited HIV-
GFP infectivity [Figure 5 and Supplementary Figure S3
Figure 3. Sub-cellular distribution of the artiodactyl A3F proteins in comparison to the orthologous human and mouse A3 proteins. HeLa cells showing
localization of the indicated, GFP-tagged A3 proteins or a GFP-only control. The scale bar indicates 10 mm.
Nucleic Acids Research, 2006, Vol. 34, No. 195689
and (32)]. The HsA3F C-terminal Z domain mutant was still
able to modestly inhibit HIV-GFP infectivity, without obvi-
ous signs of retroviral hypermutation. Interestingly, E!Q
substitutions in the N-terminal (but not the C-terminal)
domain of all three of the artiodactyl A3F proteins abolished
the accumulation of retroviral hypermutations (Figure 5B and
Supplementary Figure S3). Thus, these data combined to
demonstrate that the N-terminal Z domain of the artiodactyl
A3F proteins is catalytic and that both deaminase-dependent
and independent activities are required for full levels of retro-
virus restriction. In support of this conclusion, the N-terminal
Z domain glutamate of BtA3F is required for mutator activity
in E.coli, whereas the C-terminal Z domain glutamate is
dispensable (Figure 5C).
Retroviral hypermutation properties of artiodactyl A3F
BtA3F, OaA3F and SsA3F suggested that these proteins
would trigger retroviral hypermutation patterns biased toward
Figure 4. Retrovirus restriction activity of the artiodactyl A3F proteins.
(A) Infectivity of HIV-GFP produced in the presence of a vector control or
the indicated A3 protein. Data were normalized to the infectivity of HIV-GFP
produced in the presence of a control vector, which was assigned a value of
one. The mean and the SEM of three independent experiments are shown.
HIV-1 Vif is not encoded by the proviral vector nor is it included in trans in
these experiments (contrast with those shown in Figure 6). (B) Infectivity of
MLV-GFP produced in the presence of the indicated constructs. Parameters
are identical to those in (A).
Figure 5. Relative contributions of the N- and C-terminal zinc-binding
domains to HIV-GFP restriction. (A) Infectivity of HIV-GFP produced in the
presence of a vector control or the indicated non-mutant (+) or mutant A3
protein containing an N-terminal E!Q substitution or a C-terminal E!Q
substitution. Parameters are identical to those in Figure 4A. HIV-1 Vif is not
included in these experiments (contrast with Figure 6). (B) Frequencies of
plus strand retroviral G!A hypermutation observed in HIV-GFP DNA.
(C) The N-terminal Z domain glutamate of the indicated artiodactyl A3F
protein is required for mutator activity in E.coli, whereas the C-terminal Z
domain glutamate is largely dispensable. RifRmutation frequencies for eight
independent E.coli cultures expressing a vector control (light blue), HsA3G
(dark blue), BtA3F (brown) or OaA3F (black). The addition sign (+), N and C
refer to non-mutant, N-terminal Z domain E!Q mutant or C-terminal Z
domain E!Q mutant proteins, respectively. Other parameters are identical to
those in Figure 2A.
5690Nucleic Acids Research, 2006, Vol. 34, No. 19
50-YC, 50- GC, and 50-RC, respectively (R ¼ A or G; Y ¼ C
or T; Figure 2C). To test this prediction, we examined the
types of base substitution mutations and the local retroviral
cDNA deamination preferences attributable to expression of
the artiodactyl A3F proteins (Figure 5B and Supplementary
Figures S3 and S4). In terms of the dinucleotide mutation
preferences, the base immediately 50of the targeted cytosine
is a crucial target site determinant. HsA3F and HsA3G over-
whelmingly preferred 50-TC (84%) and 50-CC (84%), respec-
tively, whereas MmA3 preferred 50-TC (61%) and 50-CC
(29%). Similar dinucleotide preferences were reported previ-
ously for these proteins [e.g. (13,18,51,64,67)]. Roughly
sequences revealed that the cow and the sheep A3F proteins
preferred a pyrimidine (Y) 50of the deaminated cytosine (93
and 79%, respectively; Supplementary Figure S4). Similarly,
the pig A3F protein preferred 50-GC (47%). This is notable
because this is the only example of an A3 protein preferring
50-purine-C [the immunoglobulin gene deaminase AID also
has this preference; e.g. (68)]. In addition, the artiodactyl
A3F proteins preferred a T at the ?2 position, which is
more similar to HsA3F than HsA3G (which prefers a C at
The artiodactyl A3F proteins are fully resistant to
HsA3F, HsA3G and chimpanzee A3G are neutralized by
HIV-1 Vif [e.g. (51,64)]. However, many other monkey
A3G proteins (e.g. African green monkey) and MmA3 are
completely resistant (27,61–63,69). The full sets of interac-
tions that govern the A3-Vif conflict have not been deter-
mined, and the artiodactyl A3F proteins are likely to prove
useful in this regard. Therefore, HIV-GFP infectivity was
monitored in the presence or absence of HIV-1 Vif and
human, artiodactyl or mouse A3 proteins. As described
previously, expression of HIV-1 Vif neutralized HsA3F
and HsA3G (although the former to a lesser extent) and
caused a proportional recovery of HIV-GFP infectivity
[Figure 6; e.g. (13,51,64)]. Expression of HIV-1 Vif failed
to enhance the infectivity of HIV-GFP produced in the pres-
ence of MmA3 or any of the artiodactyl A3F proteins. Thus,
the artiodactyl A3F proteins were fully resistant to HIV-1 Vif.
Murine A3 and many of the human A3 proteins are capable
of inhibiting the infectivity of a broad number of retroviruses
and endogenous retroelements. Here, we have cloned and
characterized a double domain A3 protein from three cloven
hoof ungulates, the artiodactyls cattle, sheep and pigs. Artio-
dactyls are positioned between rodents and primates in most
mammalian phylogentic trees. Thus, the artiodactyl A3F
proteins can be considered evolutionary intermediates
between the rodent and primate enzymes, and the studies
presented here contribute to bridging this vast, 90–100 mil-
lion year gap. The artiodactyl A3F data help delineate a num-
ber of conserved activities that define this group of proteins,
including DNA cytosine deaminase activity, cytoplasmic sub-
cellular localization and deaminase-dependent and indepen-
dent retrovirus restriction activities. Moreover, the artiodactyl
A3F proteins also help define non-conserved activities, the
varying local DNA cytosine deamination preferences and
the differential resistances to viral countermeasures, such as
One of the most intriguing non-conserved features of the
artiodactyl A3F proteins is the fact that a DNA cytosine
deaminase activity resides in the N-terminal Z domain, and
not in the C-terminal Z domain like several of the human
double domain A3 proteins. These data highlight the modular
nature of the A3 proteins, which can clearly function as single
domain proteins, as double domain proteins with a catalyti-
cally active C-terminal Z domain or as double domain
proteins with a catalytically active N-terminal Z domain
(e.g. HsA3H, HsA3G and SsA3F, respectively). We have
not excluded the possibility that under some conditions or
with other A3 proteins that both the N- and the C-terminal
deaminase domains may be catalytically active. The mouse
A3 protein offers an additional dimension of intrigue, as
both the N- and the C-terminal zinc-binding deaminase
domains are required for retrovirus restriction and for DNA
cytosine deaminase activity (Figure 5 and Supplementary
Figure 2).Prior studieshave
N-terminal, zinc-binding domain of HsA3G mediates interac-
tions with both HIV-1 Vif and Gag (70–76). The C-terminal
domain of HsA3G dictates DNA cytosine deaminase activity,
and both domains appear to contribute to dimerization
activity (31,32,65). Thus, the division of activities between
the N- and the C-terminal domains suggests that the A3 pro-
teins are rapidly evolving as modular domains centered upon
the conserved, zinc-binding motif. Such modularity may very
well enable the A3 proteins to associate with each other to
form highly adaptable and potent anti-retroelement defenses.
For instance, in a multi-A3 protein complex, only one poly-
peptide needs to associate with the retroelement target in
order to direct the restrictive potential of the other member(s)
of the complex. Indeed, the first hints of such a combinatorial
restriction potential were observed in retroviral hypermuta-
tion loads upon HsA3F and HsA3G co-expression (51).
Taken together with previous studies, it is clear that the
mammalian A3 proteins can use both deamination-dependent
Figure 6. Artiodactyl A3F proteins are resistant to HIV-1 Vif. Parameters are
identical to those used in Figure 4A, except for the inclusion of plasmids
encoding HIV-1IIIB Vif (+) or a HIV-1IIIB DVif (?) control, which has
translation stop codons at 33 and 34 amino acids (79).
Nucleic Acids Research, 2006, Vol. 34, No. 195691
and -independent mechanisms to block the transmission of
retroviruses and retrotransposons [e.g. (9,12,31–34,65,77)].
As discussed above, our studies indicate that the DNA
cytosine deaminase activity can reside in either the N- or
the C-terminal Z domain and that this domain appears to be
the dominant contributor to restriction of HIV-based retro-
virus substrates. Similar observations have been made previ-
ously for HsA3G (34,65,66). In contrast, other studies have
indicated that either Z domain can mediate full (32,77)
or partial (16) levels of HIV-1 restriction. These differential
results may be attributable to differences in A3 protein
expression levels. A resolution to this apparent paradox
may be achieved by working-out the mechanism(s) of DNA
The studies presented here represent the first essential steps
toward understanding the retroelement restriction activities of
a third major branch of the vertebrate tree (the other two
constituting rodents and primates). Ungulates, and specific-
ally the artiodactyls cattle, sheep and pigs, provide humans
with a number of benefits from food products to xenotrans-
plantation possibilities. The possible conflict(s) between
BtA3F and bovine immunodeficiency virus (BIV) and those
between OaA3F and the sheep lentivirus, maedi-visna virus
(MVV), will be of particular future interest. Ongoing studies
have indicated that Vif-deficient MVV accumulates retroviral
G!A hypermutations with an OaA3F-like dinucleotide
mutation spectrum, suggesting that at least OaA3F is active
in vivo [(78); S. Franzdo ´ttir and V. Andre ´sdo ´ttir, unpublished
data]. Artiodactyl and human comparative studies may also
contribute to understanding how HIV-1 uses Vif to neutralize
human APOBEC3 proteins.
Supplementary Data are available at NAR Online.
The authors thank R. LaRue for assistance with comparisons
and nomenclature, T. Smith for providing cDNA clones
and assistancewith artiodactyl
N. Martemyanova for providing expert technical assistance,
M. Titus for microscopy facilities, N. Somia for sharing
valuable reagents, and M. Murtaugh, P. Hackett, T. Smith and
Harris laboratory members for helpful discussions. This work
was supported by NIH grant AI064046. RSH is a Searle
Scholar and a University of Minnesota McKnight Land Grant
Professor. SRJ was the 2004–2005 Val Bjornson Icelandic
Exchange Scholarship recipient and is supported in part by a
grant from the Icelandic Research Fund. M.D.S. and G.H.
were supported in part by a 3M Science and Technology
Graduate Fellowship and an NSERC Graduate Studentship,
respectively. Assistance with flow cytometry was provided by
the University of Minnesota Comprehensive Cancer Center
Flow Cytometry Core Facility. University of Minnesota
Advanced Genetic Analysis Facility assisted with DNA
sequencing. Funding to pay the Open Access publication
charges for this article was provided by Spring Point Project.
Conflict of interest statement. None declared.
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