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: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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| vol. 107
| no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1012344107
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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