Identification of a receptor for an extinct virus
Steven J. Solla, Stuart J. D. Neilb, and Paul D. Bieniasza,1
aThe Howard Hughes Medical Institute, The Aaron Diamond AIDS Research Center, and Laboratory of Retrovirology, The Rockefeller University, New York,
NY 10016; andbDepartment of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, United Kingdom
Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved September 28, 2010 (received for review
August 23, 2010)
The resurrection of endogenous retroviruses from inactive mo-
lecular fossils has allowed the investigation of interactions be-
tween extinct pathogens and their hosts that occurred millions
of years ago. Two such paleoviruses, chimpanzee endogenous
retrovirus-1 and -2 (CERV1 and CERV2), are relatives of modern
MLVs and are found in the genomes of a variety of Old World
primates, but are absent from the human genome. No extant
CERV1 and -2 proviruses are known to encode functional pro-
teins. To investigate the host range restriction of these viruses,
we attempted to reconstruct functional envelopes by generating
consensus genes and proteins. CERV1 and -2 enveloped MLV par-
ticles infected cell lines from a range of mammalian species. Us-
ing CERV2 Env-pseudotyped MLV reporters, we identified copper
transport protein 1 (CTR1) as a receptor that was presumably
Expression of human CTR1 was sufficient to confer CERV2 permis-
siveness on otherwise resistant hamster cells, and CTR1 knock-
down or CuCl2treatment specifically inhibited CERV2 infection of
human cells. Mutations in highly conserved CTR1 residues that have
rendered hamster cells resistant to CERV2 include a unique deletion
tions in hamster CTR1 are accompanied by apparently compensat-
coordinating residues, and this may represent an evolutionary
barrier to the acquisition of CERV2 resistance in primates.
endogenous retrovirus|copper transport
of proviruses if integration into the host germ line occurs (1). This
route of transmission results in an organism-wide presence of
provirus in the genomes of progeny. Such endogenization events
have occurred numerous times during primate evolution and
many proviruses have become fixed in host populations (2–5).
Endogenous retroviruses thus represent a record of ancient in-
fections and these “paleoviruses” can provide information about
the evolution of host–virus interactions (6, 7).
Endogenous γ-retroviruses are abundant in primate genomes
and among them, chimpanzee endogenous retroviruses-1 and -2
(CERV1 and CERV2) and their relatives are the groups most
closely related to the modern prototype γ-retrovirus, MLV (3, 4).
CERV1 and -2 are assumed to be extinct, although without ex-
haustive sampling, it is nearly impossible to definitively demon-
strate that intact exogenous relatives are not currently replicating
in some modern primate species. They are of particular interest
because of their peculiar absence from the human genome, al-
though homologs exist in the genomes of chimpanzee, bonobo,
gorilla, and Old World monkeys. Thus, both viruses apparently
replicated after the divergence of the human and chimpanzee
lineages approximately 6 million years ago, with zoonoses ulti-
specific property protected human ancestors from CERV1 and -2
infection during the time that they were becoming endogenized in
nonhuman primates, some 1 to 6 million years ago.
Previously, we investigated whether host antiretroviral factors
that restrict modern retroviruses were able to target CERV1 and -2
he ability of retroviruses to integrate into the genomes of
target cells allows for the possibility of Mendelian inheritance
during exogenous replication (6). TRIM5α is a restriction factor
that blocks the replication of a variety of retroviruses, including
certain MLV strains (8–11). However, we found no evidence
that TRIM5α was involved in a protection of human ancestors
from CERV1 or -2 infection. Conversely, inspection of CERV1
and -2 sequences revealed that many proviruses displayed G-
to-A hypermutation within GG or GA dinucleotides (6, 7),
characteristic of APOBEC3-mediated mutation (12–14). APO-
BEC3 proteins were therefore capable of acting on these vi-
ruses and may have been involved in limiting their host range.
In addition to restriction factors, cell-surface receptor usage is
often a primary determinant of viral host range. Indeed, MLV
tropism is partly determined by sequences in the viral envelope,
which can direct the use of a variety of MLV cell-surface re-
ceptors, including cationic amino acid transporter-1, inorganic
phosphate transporters, or xenotropic/polytropic receptor (15–
22). To examine the host range and receptor usage of CERV1
and -2 during the time that they replicated, we reconstituted func-
tional envelope genes and proteins. MLV particles carrying a recon-
stituted CERV2 envelope protein displayed a broad species tro-
pism in cell culture and were used to identify copper transport
protein 1 (CTR1) as a receptor that was likely used by CERV2
during ancient infections. The only mammalian species tested that
was nonpermissive to CERV2 was hamster, and its resistance to
infection was explained by mutations in the CTR1 extracellular
domain. This work demonstrates that reconstruction of ancient
endogenous viral envelope genes from molecular fossils can allow
the discovery of host proteins that have been used as receptors by
presumptively extinct viruses in prehistoric times.
Reconstruction of Functional CERV1, CERV2, and RhERV2-A Envelope
Genes and Proteins. To investigate the tropism of the aforemen-
tioned extinct retroviruses, we attempted to reconstruct functional
envelope genes and proteins. Specifically, consensus envelope
sequences were derived by aligning all 50 CERV1 and 8 CERV2
env genes, which were identified by BLAST searches of the chim-
panzee genome. Consensus env genes were also generated from
CERV2 homologs present in the rhesus macaque genome, which
fall into two distinct groups: RhERV2-A, consisting of 27 env
genes, and RhERV2-B, consisting of 34 env genes. All env nucle-
otide sequences were unique, except for one pair of identical
sequences in each of the RhERV2 groups. Phylogenies of CERV1
and CERV2/RhERV2 env genes and their respective consensus
DNA sequences are shown in Fig. 1A. The CERV2/RhERV2
phylogentic tree was constructed using TM domain DNA se-
Author contributions: S.J.S. and P.D.B. designed research; S.J.S. performed research;
S.J.D.N. contributed new reagents/analytic tools; S.J.S. and P.D.B. analyzed data; and
S.J.S. and P.D.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The hamster CTR1 sequence reported in this paper has been deposited in
the GenBank database (accession no. HQ290320).
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 9, 2010
| vol. 107
| no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1012344107
significant homologyto the CERV2 and RhERV2-A SU domains.
For each virus, nucleotides encoded by the majority of the env
genes at each position in the alignment were included in the
consensus sequence. Where no majority existed, which occurred
in some situations where more than two nucleotide variants oc-
curred at a given position, the nucleotide encoded by the largest
fraction of individuals was selected. In-frame insertions or dele-
the 34 sequences carried abundant G to A mutations within GG
and GA dinucleotides, which were likely the result of APOBEC3-
induced hypermutation. At these sites, G was assigned to the
consensus sequence regardless of whether or not the majority
sequences. The consensus CERV1, CERV2, RhERV2-A, and
557, 651, 626, and 465 amino acids, respectively. Each sequence
contained features typical of γ-retroviral envelope proteins (Fig.
S1), although both the consensus RhERV2-B env gene and all
portion of the SU protein. These consensus env genes were syn-
thesized using panels of overlapping oligonucleotides and PCR-
based synthesis and inserted into a mammalian expression vector
(SI Materials and Methods).
Each consensus Env expression vector was cotransfected with
an MLV Gag-Pol expression plasmid and an MLV vector carrying
a GFP reporter (MLV-GFP) to generate putatively pseudotyped
MLV particles. Three envelope proteins (CERV1, CERV2, and
RhERV2-A) were functional, in that they supported pseudotype
infection of human 293T cells (Fig. 1B), but the fourth (RhERV2-
B,whichencoded a large deletion in SU) didnot. Further analyses
revealed that CERV1 Env supported infection of several primate
cell lines but did not support infection of cells from New World
displayed a broad species tropism in that it supported infection of
human,chimpanzee,rhesusmacaque,African green monkey, owl
monkey, dog, rat, and mouse cell lines (Fig. 1C). Notably, the
ability of CERV1- and CERV2-enveloped particles to infect hu-
man cells argues against the notion that a lack of functional re-
ceptors is responsible for the absence of these viruses from the
Identification of a Receptor for CERV2 and RhERV2-A. MLVparticles
bearing the resurrected CERV2 envelope could efficiently infect
humancells, yieldingan infectious titer that was four to seven times
Fig. S2). Therefore, we used CERV2-enveloped MLV particles to
screen a HeLa cDNA library for genes encoding receptors that
were used by CERV2. Moreover, Chinese hamster ovary (CHO)-
derived CHO-745 cells were resistantto CERV2infectionand pro-
vided a suitable target cell with which to perform such a screen.
Twenty independent pools of a HeLa cDNA-IRES-Zeo retroviral
library (105clones per pool) were separately introduced into CHO-
These cells were challenged, in duplicate, with CERV2-enveloped
virions carrying a neomycin-resistance gene. All but one of these
40 infections yielded G418-resistant colonies (from 1 to 24 colonies
per 2 × 105challenged cells). The G418-resistant colonies were
replated in 20 dishes that corresponded to the original 20 pools of
the cDNA library and were challenged in a second round of selec-
count) were obtained in 3 of the 20 G418-resistant pools. These
three G418 and hygromycin-resistant pools displayed a >100-fold
increase in permissivity, relative to unmanipulated CHO-745 cells,
to a CERV2-pseudotyped MLV vector that carried a DsRED re-
porter gene (Fig. 2A). Importantly, resistance to virions carrying
amphotropic MLV envelope (A-MLV) was maintained in these
selected populations of cells, suggesting that their increased per-
missivity was envelope-dependent and -specific.
PCR primers directed to the retroviral library vector were used
to obtain a 1.7-kb amplicon from all three CERV2-permissive cell
lines that was not amplified when DNA from unmanipulated
CHO-745 cells was used. Sequence analysis revealed that, in all
inserted into a retroviral vector and independently transduced
into CHO-745 cells. This manipulation rendered CHO-745 cells
as permissive to CERV2 infection as the CHO-745 cells that were
derived by library transduction and selection (Fig. 2A). Notably,
expression of huCTR1 also rendered CHO cells permissive to an
MLV pseudotype carrying the RhERV2-A consensus envelope
(Fig. 2B). In contrast, huCTR1 did not render CHO cells per-
missive to CERV1-, MPMV-, GALV-, or A-MLV–pseudotyped
virions. Thus, huCTR1 specifically induced permissivity to
CERV2- and RhERV2-A-pseudotyped vector infection.
Inhibition of CERV2 Infection by CTR1 Depletion or Copper, but Not by
Endosome Acidification. To determine whether CTR1 is necessary
for CERV2 infection, two different human cells lines were
% cells infected
nucleotide sequences. CERV2/RhERV2 TM-encoding nucleotide sequences
and complete CERV1 env nucleotide sequences were analyzed. Only those
CERV1 (n = 45), CERV2 (n = 7), RhERV2A (n = 24), and RhERV2B (n = 17)
proviruses that had complete env genes were included in the analyses.
ClustalX software was used to derive neighbor-joining trees, which were
formatted using FigTree software. The consensus sequences are indicated
by asterisks. (Scale bars, 0.01 substitutions per nucleotide position.) (B) MLV
particles (50 μL) carrying a GFP reporter and pseudotyped with the indicated
envelope proteins were used to infect 104293T cells. Cells were analyzed by
FACS 2 d after infection. (C) CERV2 enveloped MLV-GFP particles were used
to infect cell lines from a variety of species: human (293T, HeLa), chimpanzee
(PtSF), rhesus macaque (FrhK-4), African green monkey (COS-7), owl monkey
(OMK), dog (D-17), cat (CRFK), pig [PK(15)], rat (Rat2), mouse (MDTF), and
hamster (CHO-745), as in B.
(A) Phylogenetic analyses of CERV2, RhERV2, and CERV1 envelope
Soll et al.PNAS
| November 9, 2010
| vol. 107
| no. 45
transfected with an siRNA pool targeting huCTR1 or a control
luciferase-specific siRNA. The activity of the huCTR1 siRNA
pool was confirmed by knockdown of tagged huCTR1 ectopically
expressed in 293T cells (Fig. 3A). The huCTR1 siRNA trans-
fection reduced CERV2 infection of 293T or HT1080 cells by
approximately threefold compared with cells transfected with
control siRNA, but susceptibility to A-MLV–enveloped MLV
was unaffected (Fig. 3B). To further investigate the requirement
for huCTR1 in CERV2 infection, we tested whether its normal
ligand, copper, could inhibit its function as a viral receptor.
Copper II chloride treatment did in fact inhibit CERV2 infection
of HeLa cells by up to 10-fold and only had minor effects on
infection by vesicular stomatitis virus (VSV)-G and MLV en-
veloped virions (Fig. 3C). These data strongly suggest that CTR1
is the major receptor used by CERV2, but do not formally ex-
clude the possibility that another receptor could also be used at
The protein huCTR1 is naturally endocytosed in response to
copper treatment (24), and it was possible that endocytosis might
be required for CERV2 infection. Therefore, we asked whether
endosome acidification was required for CERV2 entry. Pre-
treatment of cells and infection in the presence of the endosome
acidification inhibitor, ammonium chloride, demonstrated that
infection by CERV2 enveloped MLV was endosomal pH-
independent in both CHO-huCTR1 and human TE671 cells
(Fig. S3). This finding suggests that, unlike VSV-G–mediated
entry, CERV2 infection does not require the low pH environ-
ment of endocytic compartments.
Human CTR1 Induces Attachment of CERV2 Enveloped Particles to
Cells. To determine whether CTR1 facilitates CERV2 Env-
mediated attachment to target cells, GFP-labeled virus-like par-
ticles (VLPs) were generated by expressing MLV Gag-GFP in
the presence or absence of the CERV2 envelope. These fluores-
cent VLPs were applied to CHO cells, or a derivative expressing
huCTR1 (CHO-huCTR1) and bound VLPs were enumerated by
microscopic examination. CERV2-enveloped VLPs bound to
unmanipulated CHO cells only at low levels, below which par-
ticles lacking a viral envelope glycoprotein bound (Fig. 4). Con-
versely, approximately four times as many CERV2-enveloped
VLPs bound to CHO-huCTR1 cells (P < 0.0001), and treatment
of CHO-huCTR1 cells with CuCl2inhibited this binding by ap-
proximately twofold (P = 0.011) (Fig. 4B). Attachment of VLPs
that lacked Env protein to CHO cells, presumably mediated by
interactions between plasma membrane components, was not
affected by huCTR1 (P = 0.6854). Overall, these data demon-
strate that huCTR1 confers on CHO cells the ability to bind
CERV2 enveloped VLPs, and therefore suggest that CTR1
served to mediate both attachment and infection of CERV2.
Determinants of CTR1 Receptor Function.Next, we sought to explain
why CHO cells were nonpermissive, and human cells were per-
missive to CERV2 infection. Presumably, this could be explained
either by sequence differences between primate and hamster
CTR1 (haCTR1), or by low or absent expression of haCTR1 in
CHO cells. CTR1 has three transmembrane helices, a 64 amino
acid (in the case of huCTR-1) variable extracellular N-terminal
domain, and a small invariant three amino acid extracellular loop
between helices 2 and 3 (25–27) (Fig. 5A). Notably, stable ex-
pression of haCTR1 in CHO cells did not confer CERV2 per-
missivity, indicating that low CTR1 expression could not explain
the nonpermissivity of CHO cells (Fig. 5B). However, CHO cells
that were engineered to stably express a chimeric haCTR1 in
which the N-terminal extracellular domain was replaced with its
huCTR1 counterpart were as permissive to CERV2 infection as
reciprocal chimera were nonpermissive (Fig. 5B). A C-terminal
proteins and a Western blot analysis demonstrated that all four
CTR1 proteins were equivalently expressed (Fig. 5B). Thus, the
N-terminal extracellular domain of CTR1 is a key determinant of
CERV2 receptor function.
Alignment of CTR1 amino acid sequences from primates
revealed a high degree of conservation (Fig. S4). CTR1 proteins
but the rhesus macaque CTR1 has two amino acid differences.
Both changes occur within the 40-residue N-terminal portion of
% cells infected
CERV2 A-MLVVSV-G no env
CERV2 RhERV2-A CERV1MPMV GALV VSV-G no env A-MLV
fection in hamster cells. (A) Following two rounds of selection using CERV2
pseudotyped MLV, cells from three separate pools of cDNA library trans-
duced CHO-745 cells (#9, #11, and #18) were tested for sensitivity to infection
by MLV particles pseudotyped with the indicated envelope and carrying
a DsRED reporter construct. Unmanipulated CHO-745 cells, as well as cells
stably transduced with a huCTR1 expressing retroviral vector, are included
for comparison. (B) Unmanipulated CHO cells, CHO stably transduced with
huCTR1, and FRHK cells were infected as in A with MLV particles pseudo-
typed with the indicated Env proteins.
HuCTR-1 expression confers sensitivity to CERV2 Env-mediated in-
[CuCl2] ( M)
siLuc HT -1080
siCTR1 HT -1080
0.01 0.11 10
infection of human cells. (A) 293T cells were cotransfected with plasmids
expressing huCTR1 with an N-terminal HA tag and siRNAs targeting CTR1 or
luciferase, as indicated, and subjected to Western blot analysis. Blots were
probed with anti-HA (Upper) or anti-tubulin (Lower). Glycosylated forms of
CTR1 can be observed above the nonglycosylated protein. (B) 293T or
HT1080 cells were transfected twice with siRNAs targeting CTR1 or luciferase
before infection with MLV particles carrying a GFP reporter and CERV2 or
A-MLV Env proteins. (C) HeLa cells were incubated for 2 h in CuCl2before
overnight infection, at the same CuCl2concentration, with the indicated
CTR1 knockdown or copper treatment inhibits CERV2 pseudotype
| www.pnas.org/cgi/doi/10.1073/pnas.1012344107 Soll et al.
for copper uptake (28). In fact, inspection of CTR1 sequences
from a wide range of mammals, frog, and zebrafish revealed con-
the remainder of the protein is highly conserved in these diverse
species(Fig. S4). Notably, a mutant huCTR1with a 40 aminoacid
N-terminal truncation was able to confer CERV2 permissivity to
a similar degree as the intact huCTR-1 (Fig. 5C). Thus, the most
divergent region of CTR1 is completely dispensable for CERV2
receptor function. Rather, CERV2 appears to target the con-
served membrane proximal portion of the extracellular domain.
Consistent with this notion, haCTR1 mutants that were altered
to mimic huCTR1 in the conserved membrane proximal portion
of the extracellular domain were functional CERV2 receptors
(Fig. 5 A and C). Specifically, combined introduction of R(53)N
and Y(59)S mutations (numbered according to huCTR1) into
haCTR1 resulted in partial receptor function. Notably, haCTR1
is unique among the CTR1 proteins examined in that it lacks
three methionines from the conserved MMMMXM motif (Fig.
S4). The addition of three methionines to reconstitute this motif
in an otherwise unaltered haCTR1 did not result in a functional
receptor, however this change did significantly improve the re-
ceptor function of the R(53)N/Y(59)S haCTR1 mutant (Fig. 5 A
and C). The reciprocal mutations in huCTR1 inhibited CERV2
receptor function. Specifically, a three-methionine deletion in the
huCTR1 MMMMXM motif decreased receptor function and
the N53R/S59Y mutant huCTR1 was inactive. AnN-terminalHA
tag was included in all CTR1 proteins tested and expression in
transduced CHO cells was confirmed by Western blot and FACS
analysis (Fig. 5C and Table S1). Modest variation in expression
levels did not correlate with CERV2 receptor function. Thus,
mutations in the conserved extracellular portion of haCTR1 rel-
ative to primate CTR1 confer resistance to CERV2.
Finally, to determine if the use of CTR1 as a viral receptor is
directly related to its cellular function, mutants that have been
previously shown to be deficient in copper transport were tested
for viral receptor function. Two huCTR1 mutations (M81I and
Y156A) were previously shown to inhibit transport function (29),
and a third (C189S) has been shown to decrease huCTR1 trimer
ability of huCTR1 to confer CERV2 permissivity to CHO cells
(Fig.5C).Therefore, theability ofCTR1toserve asa receptorfor
CERV2 can be uncoupled from its copper transport function.
Determining the tropism of extinct ancient viruses requires the
reconstitution of functional viral proteins from molecular fossils.
By resurrecting a functional CERV2 envelope we were able to
determine the identity of a receptor for a presumptively extinct
virus. Specifically, we determined that CERV2, and its relative
RhERV2-A, used the copper transporter CTR1 as its receptor
expression was sufficient to allow CERV2- and RhERV-2A–
pseudotyped MLV particle binding and infection in otherwise
resistant hamster cells; siRNAs directed against CTR1, or copper
treatment, inhibited CERV2 infection of human cells. Copper
treatment caused a 10-fold decrease in HeLa cell infection and
magnitude is likely the result of differing dynamic ranges in the
binding and infection assays, but it remains possible that copper
can inhibit entry without entirely preventing virion binding.
The exploitation of transport proteins for cell entry is a com-
mon feature of γ-retrovirus biology. MLV, gibbon ape leukemia
virus, feline leukemia virus (FeLV), porcine endogenous retro-
virus, and the baboon endogenous retrovirus/RD114 feline en-
dogenous retrovirus/simian type D retrovirus interference group
all use transporters as receptors (15–18, 30–41). Interestingly,
FeLV-A recurrently evolves into FeLV-B or -C, which display
shifted tropisms because of their use of different transport pro-
teins. Aside from their expression on the surface of a wide variety
of cells, no common property of transport proteins has been
shown to promote their ability to serve as receptors for viruses.
Indeed, in the case of CTR1, we demonstrated that viral receptor
activity could be uncoupled from its normal transport function, as
has previously been shown for the ecotropic MLV receptor (42).
The ability of CERV2 pseudotypes to infect human cells and
to use huCTR1 as a receptor indicates that species-specific en-
velope:receptor incompatibility does not explain the absence of
endogenous copies of CERV2 from the human genome. Addi-
tionally, huCTR1 is broadly expressed (43), which is to be ex-
pected given the use of copper ions by crucial cellular enzymes
(e.g., superoxide dismutase and cytochrome c oxidase). In par-
ticular, the expression of CTR1 in human testes, ovaries, and fetal
tissues implies that a lack of endogenization is not explained by
the absence of receptor expression in the germ line. Although we
cannot exclude the possibility that huCTR1 reverted from a
replication, this evolutionary course is unlikely, given the perfect
and chimpanzee CTR1.
Other plausible explanations for the absence of CERV1 and -2
from human DNA include the possibilities that human ancestors
were ecologically isolated from viral reservoirs during the time of
itsexogenous replication, or thatendogenous proviruses were lost
during passage through genetic bottlenecks. It is also conceivable
that behavioral changes in nascent humanity limited acquisition
CERV2 + Cu
Bound VLPs per cell
CERV2 + Cu
Images of CERV2 particles bound to CHO cells or a derivative expressing
huCTR1, as indicated. A single image representing a projection of a z-series
of fluorescent images that capture the entire thickness of the cell monolayer
is shown (Left), and a merge of fluorescence and phase-contrast images is
also shown (Right). (B) Quantitation of the number of MLV Gag-GFP particles
with (circles) or without (triangles) CERV2 Env protein that bound to CHO or
CHO-huCTR1 cells, as indicated. Particles were incubated with cells in the
absence (open symbols) or presence (filled symbols) of CuCl2. For quantita-
tion, 15 individual cells under each condition were chosen at random and
the number of GFP particles per cell was counted. Each symbol represents an
individual cell and the mean number of particles per cell is indicated by
a horizontal line. P values were calculated using the unpaired, two-tailed
t test (Graphpad Prism).
HuCTR1 promotes binding of CERV2 particles to CHO cells. (A)
Soll et al. PNAS
| November 9, 2010
| vol. 107
| no. 45
ordissemination oftheseviruses.Thepossibility also remainsthat
human ancestors were protected from these γ-retroviruses by
cytidine deaminases (6), other unknown restriction factors, or
effectively suppressive adaptive immune responses.
CERV2 and, furthermore, CTR1 conservation coupled with the
why this evolutionary course was unlikely to occur. Interestingly,
the vast majority of CTR1 sequence diversity among diverse ani-
mals is concentrated in its N-terminal extracellular domain, par-
ticularly the extreme N-terminal 40 amino acids that we found to
be dispensable for virus receptor function (Fig. 5C and Fig. S4).
That CERV2 targets the extracellular domain’s most conserved
portion likely explains why cells from diverse species are suscep-
tible to CERV2 pseudotype infection (Fig. 1C). The extracellular
copper-binding residues that are the most crucial to CTR1’s
transport function are found in the highly conserved MMMMXM
motif (28), which is positioned at the N-terminal boundary of the
extracellular domain that is critical for CERV2 receptor function.
Although this motif is conserved from human to zebrafish,
an extended N terminus that encodes several additional putative
copper-coordinating residues (Fig. S4). We found that the three-
methionine deletion, along with point mutations at two other
conserved residues, are responsible for the resistance of hamster
cells to CERV2 infection. Possibly, these changes could represent
the result of selection in the hamster lineage by a pathogenic
in response to changes in copper availability or requirement, and
lossofCERV2receptor functionis a fortuitous by-product. Inany
case, the divergence observed in the haCTR1 N-terminal domain
motif, which represents an evolutionaryhurdle thata primatehost
may have needed to overcome(and apparently did not) to acquire
CERV2 resistance through receptor evolution, without forfeiting
optimal CTR1 function. Thus, the work described here demon-
strates that paleovirology has the potential to both identify novel
host factors that were usurped by ancient viruses, as well as indi-
cate the limitations that hosts may have faced in avoiding such
Materials and Methods
Generation of Consensus Envelope Genes. Envelope sequences were collected
from Ensembl using TBLASTN and were used to derive majority consensus
sequences. Overlapping oligonucleotides used to synthesize consensus genes
were designed using Genedesign from The Johns Hopkins University (http://
www.genedesign.org). For envelope gene construction, two sequential PCR
reactions were carried out and the resulting products were cloned into
pCAGGS (see SI Materials and Methods for a detailed description).
Receptor Screen. Twenty pools of 2 × 105pgsA-745 cells were transduced
with a retroviral HeLa cDNA library constructed in the LM8iresZeo MLV
vector. The library-transduced cells underwent two rounds of challenge and
selection with CERV2 Env-pseudotyped MLV virions. Genomic DNA was
extracted from surviving cells and used as PCR template with primers
designed to anneal to DNA flanking the LMN8iresZeo cloning site (see SI
Materials and Methods for a detailed description).
VLP Binding Assay. GFP-labeled virus particles were generated by cotrans-
fection of 293T cells with MLV Gag-GFP and CERV2 envelope-expression
plasmids. CHO cells, or a derivative expressing huCTR1, were incubated in
medium either with or without CuCl2followed by VLP-containing super-
natent supplemented with or without CuCl2. The cells were washed, fixed,
--------------- M D H S H H M G M S Y M D S N
M G M N H M G M N H M G M N H M D H M D H M D H -
M G M N H M G M N H M G M N H M D H
- M D N N
S Y M D
S T M Q P S H H H P T T S A S H S H G G G D S S M M M M P M
S T M P P - H H H P - T T A S H S H G G G D -
S T MP S H H H P T T A S H S H G G G D S S M M M M P M
---- M P M
T F Y F G F K N V E L L F S G L V I N T A G E
T F Y F G F K R V E L L F Y G L V I N T P G E
T F Y F G F K V E L L FG L V I N TG E
indicated. An amino acid alignment of human and hamster CTR1 N-terminal extracellular domains is shown to the right. The MMMMXM copper coordination
motif and two other residues that contribute to CERV2 receptor function are indicated by boxes. (B) CERV2 pseudotype infection of CHO cell lines expressing
C-terminally HA tagged huCTR1 (hu), haCTR1 (ha), or chimeras in which the N-terminal extracellular domains were exchanged (hu-ha and ha-hu). Expression
of CTR1 was detected on Western blots probed with an αHA antibody (Lower). (C) CHO cells stably expressing N-terminally HA-tagged CTR1 proteins were
infected with CERV2 pseudotypes (chart), and were analyzed by Western blotting with anti-HA antibodies (Lower). Mutations of huCTR1 (huWT) or haCTR1
(haWT) were introduced, as indicated, including a deletion of 40 amino acids at the N terminus of huCTR1 (40del), deletion of methionine residues 41 to 43 in
huCTR1 (3Mdel), or addition of three methionine residues at the orthologous position in haCTR1 (ham 3M+).
Determinants of CTR1 viral receptor function. (A) An Illustration of CTR1 topology with residues that are crucial to normal CTR1 transporter function
| www.pnas.org/cgi/doi/10.1073/pnas.1012344107Soll et al.
DAPI stained, and visualized by microscopy (see SI Materials and Methods for
a detailed description).
RNA Interference. A Dharmacon siGENOME smart pool was used to knock-
down huCTR-1 expression. Small interfering RNA transfections were done
twice, and cells were infected the day after the second transfection. To
validate siRNA activity, siRNAs were cotransfected with a plasmid expressing
HA-CTR1 whose expression was assessed by Western blot analysis (see SI
Materials and Methods for a detailed description).
ACKNOWLEDGMENTS. We thank Theodora Hatziioannou for the cDNA
library construction protocol, and Chetankumar Tailor (The Hospital for
Sick Children, Toronto, Canada) and various members of the P.D.B. labo-
ratory for helpful discussions and reagents. This work was supported by
National Institutes of Health Grant R01AI64003 (to P.D.B.)
1. Weiss RA, Payne LN (1971) The heritable nature of the factor in chicken cells which
acts as a helper virus for Rous sarcoma virus. Virology 45:508–515.
2. Martin MA, Bryan T, Rasheed S, Khan AS (1981) Identification and cloning of
endogenous retroviral sequences present in human DNA. Proc Natl Acad Sci USA 78:
3. Polavarapu N, Bowen NJ, McDonald JF (2006) Identification, characterization and
comparative genomics of chimpanzee endogenous retroviruses. Genome Biol 7:R51.
4. Jern P, Sperber GO, Blomberg J (2006) Divergent patterns of recent retroviral
integrations in the human and chimpanzee genomes: Probable transmissions be-
tween other primates and chimpanzees. J Virol 80:1367–1375.
5. Anderssen S, Sjøttem E, Svineng G, Johansen T (1997) Comparative analyses of LTRs of
the ERV-H family of primate-specific retrovirus-like elements isolated from marmoset,
African green monkey, and man. Virology 234:14–30.
6. Perez-Caballero D, Soll SJ, Bieniasz PD (2008) Evidence for restriction of ancient
primate gammaretroviruses by APOBEC3 but not TRIM5alpha proteins. PLoS Pathog
7. Lee YN, Bieniasz PD (2007) Reconstitution of an infectious human endogenous
retrovirus. PLoS Pathog 3:e10.
8. Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD (2004) Retrovirus
resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc Natl
Acad Sci USA 101:10774–10779.
9. Keckesova Z, Ylinen LM, Towers GJ (2004) The human and African green monkey
TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc
Natl Acad Sci USA 101:10780–10785.
10. Stremlau M, et al. (2004) The cytoplasmic body component TRIM5alpha restricts HIV-1
infection in Old World monkeys. Nature 427:848–853.
11. Yap MW, Nisole S, Lynch C, Stoye JP (2004) Trim5alpha protein restricts both HIV-1
and murine leukemia virus. Proc Natl Acad Sci USA 101:10786–10791.
12. Bishop KN, et al. (2004) Cytidine deamination of retroviral DNA by diverse APOBEC
proteins. Curr Biol 14:1392–1396.
13. Harris RS, et al. (2003) DNA deamination mediates innate immunity to retroviral
infection. Cell 113:803–809.
14. Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that
inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–650.
15. Albritton LM, Tseng L, Scadden D, Cunningham JM (1989) A putative murine
ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein
and confers susceptibility to virus infection. Cell 57:659–666.
16. Miller DG, Edwards RH, Miller AD (1994) Cloning of the cellular receptor for
amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia
virus. Proc Natl Acad Sci USA 91:78–82.
17. van Zeijl M, et al. (1994) A human amphotropic retrovirus receptor is a second
member of the gibbon ape leukemia virus receptor family. Proc Natl Acad Sci USA 91:
18. Stoye JP, Coffin JM (1987) The four classes of endogenous murine leukemia virus:
Structural relationships and potential for recombination. J Virol 61:2659–2669.
19. Yang YL, et al. (1999) Receptors for polytropic and xenotropic mouse leukaemia
viruses encoded by a single gene at Rmc1. Nat Genet 21:216–219.
20. Tailor CS, Nouri A, Lee CG, Kozak C, Kabat D (1999) Cloning and characterization of
a cell surface receptor for xenotropic and polytropic murine leukemia viruses. Proc
Natl Acad Sci USA 96:927–932.
21. Elder JH, et al. (1977) Biochemical evidence that MCF murine leukemia viruses are
envelope (env) gene recombinants. Proc Natl Acad Sci USA 74:4676–4680.
22. Fischinger PJ, Nomura S, Bolognesi DP (1975) A novel murine oncornavirus with dual
eco- and xenotropic properties. Proc Natl Acad Sci USA 72:5150–5155.
23. Zhou B, Gitschier J (1997) hCTR1: A human gene for copper uptake identified by
complementation in yeast. Proc Natl Acad Sci USA 94:7481–7486.
24. Petris MJ, Smith K, Lee J, Thiele DJ (2003) Copper-stimulated endocytosis and
degradation of the human copper transporter, hCtr1. J Biol Chem 278:9639–9646.
25. Klomp AE, et al. (2003) The N-terminus of the human copper transporter 1 (hCTR1) is
localized extracellularly, and interacts with itself. Biochem J 370:881–889.
26. De Feo CJ, Aller SG, Siluvai GS, Blackburn NJ, Unger VM (2009) Three-dimensional
structure of the human copper transporter hCTR1. Proc Natl Acad Sci USA 106:
27. Eisses JF, Kaplan JH (2002) Molecular characterization of hCTR1, the human copper
uptake protein. J Biol Chem 277:29162–29171.
28. Puig S, Lee J, Lau M, Thiele DJ (2002) Biochemical and genetic analyses of yeast and
human high affinity copper transporters suggest a conserved mechanism for copper
uptake. J Biol Chem 277:26021–26030.
29. Eisses JF, Kaplan JH (2005) The mechanism of copper uptake mediated by human
CTR1: A mutational analysis. J Biol Chem 280:37159–37168.
30. O’Hara B, et al. (1990) Characterization of a human gene conferring sensitivity to
infection by gibbon ape leukemia virus. Cell Growth Differ 1:119–127.
31. Kavanaugh MP, et al. (1994) Cell-surface receptors for gibbon ape leukemia virus
and amphotropic murine retrovirus are inducible sodium-dependent phosphate
symporters. Proc Natl Acad Sci USA 91:7071–7075.
32. Mendoza R, Anderson MM, Overbaugh J (2006) A putative thiamine transport protein
is a receptor for feline leukemia virus subgroup A. J Virol 80:3378–3385.
33. Ericsson TA, et al. (2003) Identification of receptors for pig endogenous retrovirus.
Proc Natl Acad Sci USA 100:6759–6764.
34. Quigley JG, et al. (2000) Cloning of the cellular receptor for feline leukemia virus
subgroup C (FeLV-C), a retrovirus that induces red cell aplasia. Blood 95:1093–1099.
35. Tailor CS, Willett BJ, Kabat D (1999) A putative cell surface receptor for anemia-
inducing feline leukemia virus subgroup C is a member of a transporter superfamily.
J Virol 73:6500–6505.
36. Takeuchi Y, et al. (1992) Feline leukemia virus subgroup B uses the same cell surface
receptor as gibbon ape leukemia virus. J Virol 66:1219–1222.
37. Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA (1997) Two sets of human-
tropic pig retrovirus. Nature 389:681–682.
38. Shalev Z, et al. (2009) Identification of a feline leukemia virus variant that can use
THTR1, FLVCR1, and FLVCR2 for infection. J Virol 83:6706–6716.
39. Marin M, Tailor CS, Nouri A, Kabat D (2000) Sodium-dependent neutral amino acid
transporter type 1 is an auxiliary receptor for baboon endogenous retrovirus. J Virol
40. Tailor CS, Nouri A, Zhao Y, Takeuchi Y, Kabat D (1999) A sodium-dependent neutral-
amino-acid transporter mediates infections of feline and baboon endogenous
retroviruses and simian type D retroviruses. J Virol 73:4470–4474.
41. Rasko JE, Battini JL, Gottschalk RJ, Mazo I, Miller AD (1999) The RD114/simian type D
retrovirus receptor is a neutral amino acid transporter. Proc Natl Acad Sci USA 96:
42. Wang H, Kavanaugh MP, Kabat D (1994) A critical site in the cell surface receptor for
ecotropic murine retroviruses required for amino acid transport but not for viral
reception. Virology 202:1058–1060.
43. Su AI, et al. (2004) A gene atlas of the mouse and human protein-encoding
transcriptomes. Proc Natl Acad Sci USA 101:6062–6067.
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| November 9, 2010
| vol. 107
| no. 45