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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1093-1099
RED CELLS
Cloning of the cellular receptor for feline leukemia virus
subgroup C (FeLV-C), a retrovirus that induces red cell aplasia
John G. Quigley,
Cara C. Burns,
Maria M. Anderson,
Eric D. Lynch,
Kathleen M. Sabo,
Julie Overbaugh, and
Janis L. Abkowitz
From the Divisions of Hematology, Microbiology, and Medical
Genetics, Department of Medicine, University of Washington, Seattle;
and Division of Human Biology, Fred Hutchinson Cancer Research Center,
Seattle, WA.
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Abstract |
Feline leukemia virus-C (FeLV-C) causes red cell aplasia in cats,
likely through its interaction with its cell surface receptor. We
identified this receptor by the functional screening of a library of
complementary DNAs (cDNA) from feline T cells. The library, which was
cloned into a retroviral vector, was introduced into FeLV-C-resistant
murine (NIH 3T3) cells. The gene conferring susceptibility to FeLV-C
was isolated and reintroduced into the same cell type, as well as into
FeLV-C-resistant rat (NRK 52E) cells, to verify its role in viral
infection. The receptor cDNA is predicted to encode a protein of 560 amino acids with 12 membrane-spanning domains, termed FLVCR. FLVCR has
significant amino acid sequence homology with members of the major
facilitator superfamily and especially D-glucarate transporters
described in bacteria and in C. elegans. As FeLV-C impairs the
in vivo differentiation of burst-forming unit-erythroid to
colony-forming unit-erythroid, we hypothesize that this transporter
system could have an essential role in early erythropoiesis. In further
studies, a 6-kb fragment of the human FLVCR gene was amplified by
polymerase chain reaction from genomic DNA, using
homologous cDNA sequences identified in the human Expressed Sequence
Tags database. By radiation hybrid mapping, the human gene was
localized to a 0.5-centiMorgan region on the long arm of chromosome 1 at q31.3.
(Blood. 2000;95:1093-1099)
© 2000 by The American Society of Hematology.
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Introduction |
Cats naturally or experimentally infected with feline
leukemia virus-C (FeLV-C) develop profound anemia
(hematocrit = 4%-15%).1-5 There is an absence of
reticulocytes in the circulation, and the marrow lacks hemoglobinized
cells. As the maturation and number of granulocytic cells and platelets
are normal, the presentation satisfies the clinical definition of pure
red cell aplasia (PRCA).6 Burst-forming unit-erythroid
(BFU-E) can be detected in the marrow of anemic animals (and may be
increased in frequency), but colony-forming unit-erythroid (CFU-E) is
absent.4 This finding has lead to the
hypothesis that the PRCA is due to an impairment of BFU-E to CFU-E differentiation.
The exact mechanism by which FeLV-C impedes early erythropoiesis is not
known. All cats chronically viremic with FeLV-C develop PRCA. This
uniform response in an outbred population suggests that an immunologic
pathogenesis (the most common mechanism of acquired human
PRCA7) is unlikely. Similarly, in vitro
studies confirm that erythropoiesis is not suppressed by an aberrant
population of T cells or an autoantibody to erythroid progenitor cells
or to erythropoietin.8 Also, neonatal cats (which have
immature immune systems) develop PRCA more rapidly than adolescent
animals,3,9 implying that viral burden, and not immunologic
response, is the major correlate of disease onset.
By 4-6 weeks after cats are inoculated with virus derived from a
molecular clone of FeLV-C (FeLV-C/Sarma), all hematopoietic cells,
including granulocytes, monocytes, lymphocytes, BFU-E, and
colony-forming unit-granulocyte-macrophage, as well as fibroblasts are
infected.9-11 The consequence of FeLV-C/Sarma infection
therefore differs among infected cell types. Thus, the pathogenesis of
FeLV-C-induced anemia differs from the pathogenesis of anemia in human
parvovirus B19 infection. As human parvovirus B19 uniquely infects and
lyses erythroid precursor cells (the cell surface receptor is the P antigen),12 cell entry alone determines clinical phenotype.
Three subgroups (A, B, and C) of FeLV have been defined by host cell
range, neutralization, and interference assays,13-15
studies which reflect properties of the surface unit (SU) of the
retrovirus envelope protein. FeLV-A has the most restrictive host cell
range and only infects cat and some dog cells. FeLV-B and FeLV-C can infect cells of many species, including cat, dog, monkey, human, cow,
and pig. Only FeLV-C viruses can infect guinea pig cells. Also,
although the host cell range of FeLV-C is broad, this subgroup of virus
cannot infect most rodent (hamster, mouse, and rat) cells. Receptor
specificity has been inferred by the use of interference assays. The
principle of an interference assay is that a permissive cell (eg, a cat
cell) that is infected with one type of FeLV (eg, C) cannot be infected
by additional viruses of the same subgroup but can be super-infected by
viruses of other subgroups (eg, FeLV-A) that use different cell surface
receptors.16 Two mechanisms likely contribute to retroviral
interference: (1) the entrapment of the receptor within the endoplasmic
reticulum by interaction with SU protein derived from integrated
proviral DNA; and (2) the competitive inhibition by SU protein of the
binding of additional virus to residual cell surface
receptors.16 Interference assays (with human cells) have
shown that FeLV-B shares a receptor with the gibbon ape leukemia
virus.14,17 No other retrovirus interferes with the ability
of FeLV-C to infect human cells, making its receptor of interest from a
virologic, as well as a hematologic, perspective.14
FeLV-A, -B, and -C have been molecularly cloned.5,18-21
Predicted amino acid sequences differ predominately in the SU protein, and variable region 1 of 5 discrete variable regions is most divergent. Both the genetic determinants of the anemia and subgroup C phenotype map to variable region 1 in studies of chimeric viruses constructed from FeLV-A/61E (a nonpathogenic subgroup A virus) and
FeLV-C/Sarma.22,23 Thus, it appears that the 30 amino acid region of the SU protein that is required for
anemia is also required for the binding of FeLV-C to its cell
surface receptor. This observation led to the hypothesis that the
cell surface receptor for FeLV-C has an important physiologic
role in the normal maturation of BFU-E to CFU-E but is redundant or
nonessential for granulocytic differentiation. When cells are infected
by FeLV-C and receptor function or expression is impaired (via the
mechanism of envelope-mediated interference), PRCA results.
To gain insights into the mechanisms controlling early erythropoiesis,
we have cloned the feline complementary DNA (cDNA) encoding the FeLV-C
cell surface receptor (FLVCR). The experimental strategy employed a
retroviral vector cDNA library, an approach critical to the successful
cloning of cDNAs for simian immunodeficiency virus co-receptors, and
the human receptors for xenotropic (and polytropic) murine leukemia
viruses (MuLV), and the feline endogenous virus RD114.24-27
The protein that confers susceptibility to FeLV-C infection appears to
be a D-glucarate transporter and member of the major facilitator
superfamily (MFS) of transporter proteins.
Sequences homologous to the FLVCR cDNA were identified in the human
Expressed Sequence Tags (EST) database and were utilized to amplify by
polymerase chain reaction (PCR), a 6-kb fragment of the
human gene. The human FLVCR (huFLVCR) was mapped, using radiation
hybrid mapping, to chromosome 1q31.3. Of interest, rearrangement of the
distal region of chromosome 1q has been described in a patient with
Diamond-Blackfan anemia, a congenital PRCA.28
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Materials and methods |
Plasmids, viruses, and cell lines
The retroviral vector LAPSN (derived from Moloney
MuLV,29 a gift from A. D. Miller, Fred
Hutchinson Cancer Research Center, Seattle, WA) expresses human
alkaline phosphatase (AP) and neomycin phosphotransferase (neo); the
MSCV retroviral vectors30 (available from Clontech, Palo
Alto, CA) are derived from myeloproliferative sarcoma virus, and
express neo (MSCVneo), or the puromycin N-acetyl transferase gene
(MSCVpuro). The retrovirus vector pMX (a gift from T. Kitamura,
University of Tokyo, Japan) used for feline cDNA library expression, is
derived from pBabe-puro (with deletion of the selectable
marker).31
To pseudotype retroviral vectors with the FeLV-C/Sarma
envelope, we subcloned a 2.0-kb XhoI/RsRII
fragment of pFSC5 (a gift from J. Mullins, University of
Washington, Seattle, WA) into the replication defective
FeLV-A/61E-based retroviral packaging vector p61E  -A32
digested with the same enzymes to generate p61E-Cenv.
Sequence analysis of p61E-Cenv confirmed replacement of
the FeLV-A env gene by the complete coding region of FeLV-C env. The host cell range of the virus derived from
p61E-Cenv was identical to that of FeLV-C/Sarma as
assayed by transfer of the MSCVneo vector (data not shown).
The plasmid pSV-MLV-env33 (a gift from M. Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA) was used
to pseudotype retroviral vectors with the amphotropic envelope. The
plasmid p61E-LTR--gp is an FeLV-A/61E-based env-deleted
packaging vector that expresses FeLV-A/61E gag and pol
genes; this construct can be complemented in trans with expression
vectors for FeLV or MuLV envelope to generate infectious particles (C. Meiring and J. Overbaugh, unpublished). The plasmid
pCMVhph34 (a gift from M. Linial, Fred Hutchinson Cancer
Research Center, Seattle, WA) contains the hygromycin B
phosphotransferase gene under the control of a CMV promoter.
Replication defective FeLV-C particles carrying MSCV vector RNA,
MSCVneo(61E-Cenv), and MSCVpuro(61E-Cenv)
were produced by cotransfection [using the calcium-phosphate technique
(CalPhos Mammalian Transfection Kit, Clontech)] of 293T human
embryonic kidney cells35 with p61E-Cenv, and
pMSCVneo or pMSCVpuro, respectively. A stable packaging cell line able
to produce replication-defective LAPSN(61E-Cenv)
particles was generated by first transfecting D17 canine osteosarcoma
cells (ATCC CRL 6248) with pLAPSN followed by selection
(× 14d) in G418 (1000 µg/mL active,
Gibco, Gaithersburg, MD). Pools of resistant colonies were then
transfected with p61E-Cenv and pCMVHygro at a molar ratio
of 20:1 and selected (× 14d) in hygromycin B (300 µg/mL, Sigma,
St Louis, MO). Replication-competent FeLV-B subgroup virus
(FeLV-B-90Z36) and replication-defective amphotropic pseudotype retroviral vectors were produced by
cotransfection of 293T cells with pLAPSN, and pFeLV-B-90Z or
p61E-LTR-gp/pSV-MLV-env to generate LAPSN(FeLV-B-90Z) and
LAPSN(61E-LTR--gp/Ampho env), respectively. (The nomenclature
used lists the viral vector followed by the viral genome employed to
express virion proteins in parentheses.) All transient retroviral
vector supernatants were collected 48 hours posttransfection, filtered,
and applied to target cells or frozen at  80°C.
Mammalian cells, including mouse embryo fibroblasts, NIH 3T3 (ATCC CRL
1658); rat kidney cells, NRK 52E (ATCC CRL 1571); feline embryonic
fibroblasts (a gift of E. Hoover, Ohio State University, Columbus, OH);
guinea pig transformed fetal cells, GP104C1 (ATCC CRL 1405); canine D17
cells; human 293T cells; and the BOSC23 packaging cell
line35 were grown in DMEM high glucose (Gibco) supplemented
with 10% fetal calf serum (Summit, Fort Collins, CO).
Feline embryonic fibroblast cells were used to determine the infectious
titer of FeLV pseudotype vectors. The cells, plated at a density of
2 × 105 per 60-mm tissue culture dish, were exposed
after 16 hours to a dilution (10 4 to 1 mL) of viral
supernatant made up to 2 mL with media and polybrene (8 µg/mL,
Sigma). Two days later, 10% and 90% of the total cells from each dish
were replated and selected in G418 (700 µg/mL active). Seven to 10 days later, colonies were stained with Coomassie Blue and counted.
Colony-forming units (cfu) for FeLV pseudotype vectors encoding
puromycin resistance were determined using puromycin selection (2.0 µg/mL, Sigma).
Expression cloning of FLVCR cDNA
A feline cDNA library derived from 3201B cells, a feline T cell
line,37 was subcloned into the retroviral expression vector pMX, using the protocol of Kitamura et al31 (M. Anderson
and J. Overbaugh, unpublished).
The retroviral cDNA library (complexity > 3 × 106
individual clones) was pseudotyped with ecotropic MuLV envelope
proteins with the use of BOSC23 packaging cells. NIH 3T3 cells (in 10 plates at a density of 2 × 105 per 100-mm dish)
were transduced with the cDNA library at an estimated multiplicity of
infection (MOI) of 0.1 virus per cell with the use of polybrene at 8 µg/mL (an MOI of <1 was used to decrease the number of proviral
inserts per infectant26). Forty-eight hours after
transduction, NIH 3T3 cells were exposed to
MSCVneo(61E-Cenv), at an MOI of 5:1. The next
day, the cells were transferred to 150-mm tissue culture dishes and
then were selected in G418 (600 µg/mL active) for 10 days. Pools of
neomycin-resistant colonies were replated, exposed to the FeLV-C
pseudotype retroviral vector MSCVpuro(61E-Cenv) at an MOI
of 0.1, and selected in puromycin (1.5 µg/mL). Distinct colonies were
isolated at this stage.
Isolation of FeLV receptor DNA and expression in mammalian cells
DNA was extracted from reinfectable colonies with the use of the
Puregene DNA isolation kit (Gentra, Minneapolis, MN). PCR was performed
with the use of the Expand Long Template PCR kit (Boehringer Mannheim,
Indianapolis, IN) in a GeneAmp 9700 thermocycler for 35 cycles each of
94°C for 30 seconds, 65°C for 30 seconds, 68°C for 4 minutes, with a final extension at 68°C for 7 minutes. The primer
pair used (upstream, pMX#11, 5'-GTGGACCATCCTCTAGACTGC-3' and downstream, pMX#14, 5'-GAAAATAAAATAGCAGCTGGTGACACG-3')
bind within the polylinker of pMX, and pMX#14 includes 3'
sequences that overlap the BstXI site used to clone the cDNA
inserts into pMX; thus, these primers are designed to specifically
amplify the library cDNA insert. Blunt-ended PCR products were created with the use of T4 polymerase (Gibco).
pMSCV(1.8 kb)neo (also termed pMSCV(FLVCR)neo) and pMCSV(1.6 kb)neo
were constructed by ligating the 1.8-kb or 1.6-kb PCR product into
HpaI-digested pMSCVneo. These plasmids or pMSCVneo alone was
introduced into BOSC23 packaging cells, and the resulting retroviral
vector supernatant was used to transduce naive NIH 3T3 and NRK 52E
cells. Cells were selected in G418 (600 µg/mL active) for 10 days.
Pools of neomycin-resistant cells were tested for susceptibility to
LAPSN(61E-Cenv). Various dilutions of vector
(104-1 mL in 2 mL total volume) were used in these
assays so that the infectious titers (number of AP positive cells or
clusters [focus-forming units (ffu)/mL] could be reliably determined.
AP staining was performed as described.38
The 1.8-kb cDNA, FLVCR, was sequenced twice in both directions on a
PE/ABI 373 DNA Sequencer (Applied Biosystems, Foster City, CA) with the
use of Big Dye terminator sequencing chemistry (Molecular Pharmacology
Facility, Department of Pharmacology, University of Washington). The
GenBank accession number is AF192387.
Databases39-41 were searched for homologous
sequences. Predicted amino acid sequences were aligned with the use of
ClustalW (MacVector, Oxford Molecular). Hydrophobic regions of the
predicted FLVCR protein were inferred,42 and the presence
of Prosite patterns were determined with the use of the
PROSITE database.43 Exon prediction and
identification of putative extended open reading frames for homologous
human genomic sequences in GenBank were performed with the use of
Genscan software.44
Chromosomal localization of the human FLVCR gene
PCR was performed on human genomic DNA in a GeneAmp 9700 thermocycler with the use of PCR conditions recommended in the Expand Long Template PCR kit (with an annealing temperature of 60°C) and
primers (forward, Cacof255, 5'-TGTTCACATTGGCTCAAGGA-3';
reverse, w07167r144, 5'-AATTGCCGATTCTGACTGCTTGGACA-3') with
sequence common to feline FLVCR and two human ESTs (GenBank accession
nos. AA305281 and W07167). The resulting 6-kb fragment of the 3'
end of huFLVCR was subcloned into the pGEM vector (Promega, Madison,
WI) and partially sequenced. The chromosomal location of huFLVCR was
determined with the use of the Stanford Radiation Hybrid Mapping Panel
G3.45 PCR was performed on DNA from each of the 83 G3
hybrid clones with the use of primers derived from the 6-kb fragment
partial sequence (forward, w07167f16,
5'-CTTGGTCTGTGGGACTGTCA-3', and reverse, w07167r273,
5'-GCCCCTCTGTTTCAGCATTA-3'), to amplify a 277-base pair
(bp) product (details are provided at reference 46). The resulting
vector was submitted to the Stanford Human Genome Center RH Server for
chromosomal localization.
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Results |
Expression library screen for the FeLV-C receptor
To use a gene transfer approach to identify the receptor, it was
necessary to first identify a cell line resistant to FeLV-C infection.
A number of cell lines known to be poorly infectable by FeLV-C (eg,
from rodent, canine, bovine, primate, and human species13) were studied. Candidate cells were
infected with high-titer FeLV-C/Sarma pseudotype viruses carrying
MSCVneo and screened for neomycin resistance. Rodent cell lines,
including NIH 3T3 (< 3 cfu/mL) and NRK 52E cells (< 10 cfu/mL) were
identified as least susceptible to FeLV-C infection.
NIH 3T3 cells were transduced with a retroviral vector cDNA library
generated from the feline T cell line, 3201B. 3201B cells are highly
infectable by FeLV-C37 (J. Abkowitz, unpublished
observations) and thus were predicted to encode the receptor of
interest. Transduced NIH 3T3 cells were then screened to determine
whether they had acquired the ability to be sequentially infected by
retroviral vectors that were pseudotyped with FeLV-C envelope protein
and that carried selectable markers. We used a retroviral genome that
was deleted in its packaging sequence to express gag, pol, and env
proteins so that the FeLV-C/Sarma env gene would not be
transferred to NIH 3T3 cells during infection. Thus, NIH 3T3 cells
expressing the receptor would not become resistant to reinfection as a
result of interference.
NIH 3T3 cells transduced with the library were first challenged with
MSCVneo (61E-Cenv). Cell pools identified as resistant to
neomycin were challenged with the FeLV-C pseudotype vector carrying the
puromycin resistance gene, at a MOI of 0.1. A low MOI was employed in
the secondary screen to reduce the number of false-positive events. Of
a total of 150 puromycin-resistant colonies isolated, genomic DNA from
15 colonies was subjected to PCR with the use of primers specific to
the pMX vector used to generate the cDNA library. PCR products of 1.2, 1.6, and 1.8 kb were amplified in each case, suggesting that the
colonies derived from a single-cell clone. The 1.6- and 1.8-kb PCR
fragments were selected for further study as their sizes were more
consistent with cDNAs of previously isolated retroviral
receptors.25-27,47 The fragments were each subcloned into
the retroviral vector MSCVneo [denoted MSCV(1.8 kb)neo and MSCV(1.6
kb)neo, respectively]. These vectors were then overexpressed in naive
NIH 3T3 and NRK 52E cells. Cells expressing the 1.8-kb PCR fragment
were susceptible to infection by a FeLV-C pseudotype vector (see Table
1 and Figure
1). Cells transduced in parallel with the
MSCV(1.6 kb)neo (data not shown) or MSCVneo (vector alone) remained
resistant to infection. Expression of the 1.8-kb PCR fragment had no
effect either on the susceptibility of NIH 3T3 or NRK 52E cells to
infection by FeLV subgroups A or B or to infection by viruses
pseudotyped with amphotropic envelope, demonstrating that the 1.8-kb
feline cDNA specifically confers susceptibility to FeLV-C infection and
thus encodes the putative FLVCR. NRK 52E cells expressing the receptor
[NRK 52E(FLVCR)] were significantly more infectable with FeLV-C than
were NIH 3T3(FLVCR) cells. This finding suggests that
either receptor expression was suboptimal in NIH 3T3 cells or that
other cofactors not present in this cell type are required for
efficient FeLV-C infection.
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| Fig 1.
Expression of the 1.8-kb cDNA allows NRK 52E to become
susceptible to infection by a feline leukemia virus-C (FeLV-C)
pseudotype LAPSN vector.
The NRK 52E cells in panel a contain the control vector MSCVneo, cannot
be infected by the FeLV-C pseudotype LAPSN vector, thus fail to express
human alkaline phosphatase, and are white. The NRK 52E cells in panel b
contain MSCV (1.8 kb)neo, express FeLV-C cell surface receptor, and are
readily infectable by the FeLV-C pseudotype LAPSN vector, as evidenced
by their purple stain. Cells infected with the amphotropic pseudotype
LAPSN vector stain purple (panels c and d). NRK-52E cells not exposed
to virus do not express human alkaline phosphatase and are white (data
not shown). The concentration of the FeLV-C pseudotype vector was
10 × that of the amphotropic murine leukemia virus pseudotype
vector in this study. Formal titers are shown in Table 1.
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Identity of the FeLV-C receptor
The open reading frame between bases 42 and 1724 of the FLVCR cDNA
is predicted to encode a protein of 560 amino acids (Figure 2) with a molecular mass of 60 kDa. The
hydrophobicity plot suggests the presence of 12 hydrophobic
membrane-spanning domains with the N-terminus in the
cytosol.42 Canonical N-glycosylation sites occur in the
third predicted extracellular and in the sixth predicted transmembrane
domains. Prosite patterns43 compatible with sites of
protein kinase C phosphorylation, casein kinase II phosphorylation, and
N-myristoylation were identified.
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| Fig 2.
Sequence comparison of the feline leukemia virus-C
(FeLV-C) receptor (560 amino acids) and a putative D-glucarate
transporter of C. elegans, the 623 amino acid C09D4.1 gene
product (GenBank accession no. AF002196).
The receptor (aa 106-550) shares 44% sequence identity with the
putative transporter (aa 186-617). Amino acid identities are marked
with dark shading, and similarities are indicated with light shading.
Predicted transmembrane regions are shown as lines over the amino acid
sequence, and two potential N-glycosylation sites are marked by an
asterisk.
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BlastP40 comparisons with sequences available in the
GenBank databases revealed that the predicted protein has highest
homology (P < 1 × 10106) to a
hypothetical protein encoded by the gene locus C09D4.1 in C. elegans a putative D-glucarate transporter, originally
identified in bacteria (Bacillus subtilis and
Pseudomonas spp.). It also has high homology to a number of
other putative glucarate transporter proteins in C. elegans
[eg, YT45_CAEEL (GenBank accession no. Q11073) and a protein predicted
for gene locus C05G5.1 (GenBank accession no. CAA94104)
(P < 1 × 1027)].
These amino acid homologies (23%-44% identities, extending across the bulk of the proteins) and the predicted topology of 12 membrane-spanning domains (Figure 2) suggest that FLVCR is a member of
the anion : cation symporter family48 of the MFS (see
reference 49 for review). MFS permeases are single polypeptide secondary carriers, capable of transporting small solutes across membranes (eg, sugars, Krebs cycle metabolites, inorganic phosphates) in response to chemico-osmotic gradients.48,49 They occur
ubiquitously in all classifications of living organisms and comprise
3% of proteins predicted for the S. cerevisiae (yeast)
genome.50 Characteristic of the anion : cation symporter
family48 is the presence of numerous gene paralogs (at
least 15 in C. elegans). Consistent with this
description, we have noted that a portion of a bacterial artificial chromosome sequence (200 kb in length) located on human chromosome 14q23.2 may encode a protein 357 amino acids in length (predicted with the use of Genscan software44) that is
highly homologous to the feline FLVCR amino acid sequence
(P < 1093). However, the DNA sequence
match of this hypothetical protein to the FLVCR cDNA is imperfect (only
74%), suggesting that the bacterial artificial chromosome (GenBank
accession no. AC007182.2) contains a member of this gene family.
BlastN analyses41 of the FLVCR cDNA sequence against the
human EST database identified portions of the candidate huFLVCR gene.
One EST (GenBank accession no. AA305281) is an unmapped 396-bp sequence
derived from a colonic adenocarcinoma cell line (Caco 2) cDNA library
and has a portion with 92% contiguous sequence identity (bp 137-396)
to the feline FLVCR cDNA (bp 1350-1610) (P = 10102). The second EST (GenBank
accession no. W07 167), an unmapped 656-bp sequence derived from a
fetal lung cDNA library, has a region with 90% homology to the
3' end of FLVCR.
The gene encoding huFLVCR maps to human chromosome 1q31.3
A 6-kb fragment of the 3' end of the huFLVCR gene was
amplified by PCR from human genomic DNA, using primers (Cacof255, and w07167r144) derived from sequence common to FLVCR cDNA and the two
identified homologous human ESTs (GenBank accession nos. AA305281 and
W07167). The fragment was partially sequenced, allowing the
identification of intron-exon borders and the subsequent design of
further primers (w07167f16, and w07167r273). Analysis of the Stanford
Generation 3 Radiation Hybrid (G3 RH) Mapping Panel (which consists of
a set of 83 hamster/human cell lines containing radiation-fragmented human DNA such that each clonal cell line contains random fragments of
~18% of the human genome) was performed using the latter PCR primers. These primers were designed to amplify a 277-bp fragment near
the 3' end of the gene with one primer based in an intron and the
second present in an exon. These primers were specific to human DNA and
yielded no PCR product with hamster DNA alone. Screening of the RH DNA
pools with these primers yielded the following vector
1 000 0 001 0 000 0 000 0 000 1
00 0 011 1 000 0 000 0 000 0 010 0 000 0 000 0 001 0 000 1 000 0
00 0 000 0 100 0 100 001 in which hybrids containing (1) or not
containing (0) the huFLVCR sequence are indicated. This
mapping vector was analyzed with the use of the radiation hybrid
mapping server,51 which placed the huFLVCR gene within
11centiRay10 000 (cR) of marker D1S505 (SHGC 1592, AFMa127wb5) with a logarithm of odds score of 11.12 (Figure
3). D1S505 has previously been mapped to the 6-centiMorgan (cM) region on chromosome 1q31.3 flanked by D1S491
and D1S474. The closest markers 11cRay10 000 or greater
from D1S505 (8237cR) are D1S425 (8225cR) and D1S217 (8248cR). The only
known gene mapping to this 0.5-cM interval is Activating Transcription
Factor 3 (ATF3), and no additional ESTs have been mapped to
this region (NCBI GeneMap'99).
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| Fig 3.
Schematic representation of the physical location of
human FeLV-C cell surface receptor (huFLVCR) gene on human Chromosome
1.
The chromosomal location of huFLVCR was determined, by polymerase chain
reaction analysis of the Stanford Radiation hybrid panel G3, to lie
within 11 cRay10 000 of D1S505 with a
logarithm of odds score of 11.12. The only previously identified gene
mapping to this interval is the transcription factor Activating
Transcription Factor 3. For Chromosome 1 with the G3 RH panel,
1cRay10 000 is ~26 kb.
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Discussion |
There are 8 interference groups of simple retroviruses (as defined
by assays of human cells14), suggesting that 8 distinct receptors are responsible for cell entry. The cell surface receptors for the amphotropic MuLV, gibbon ape leukemia virus (and FeLV-B), and
the feline endogenous retrovirus RD114 have been identified and are
predicted to be multiple membrane-spanning proteins that function as
transporter molecules, transporting phosphate ions or neutral amino
acids.16,26,27 The receptor for the xenotropic MuLV is also
a multiple membrane-spanning protein and may have a role in G-protein
mediated signal transduction.25 Thus, these receptors are
integral membrane proteins with basic physiologic functions.
We have cloned the cDNA for the putative cell surface receptor for
FeLV-C on the basis of its ability to confer susceptibility to FeLV-C
infection when expressed in resistant rodent cells. The sequence of
FLVCR suggests that it too is a transporter molecule and a member of
the MFS (reviewed in references 48 and 49). Through sequence homology,
it appears that FLVCR is an organic anion transporter and specifically
a D-glucarate transporter molecule.
The consequence of FeLV-C infection differs from that of other simple
retroviruses. Biological data suggest that the FeLV-C envelope SU
protein may act as a dominant negative protein, inhibiting the function
or cell surface expression of FLVCR (which is the mechanism of
retroviral interference) to result in PRCA. A corollary of this
hypothesis is that the FeLV-C receptor plays a crucial role in normal
erythropoiesis. Thus, it is intriguing that FLVCR shares homology with
D-glucarate transporters of both prokaryotes and eukaryotes.
How the transport of D-glucarate, a diacid sugar derived from glucose,
may relate to erythropoiesis is unknown. D-glucarate circulates at a
high level in the serum,52 and its principal metabolite
inhibits beta glucuronidase activity in hepatocytes, resulting in
the stabilization of bilirubin.53 A comparable role in erythroid cells could impact heme metabolism. At the
stage of differentiation between BFU-E and CFU-E, globin
transcription increases, iron absorption increases (via the increased
cell surface expression of the transferrin receptor), and the
subsequent maintenance of heme is also critical for initiation of
hemoglobin synthesis.54 The identification of the
FeLV-C receptor should allow the direct study of novel
physiologic mechanisms that regulate early erythropoiesis.
In additional studies, the gene encoding the human ortholog of FLVCR
was localized to chromosome 1q31.3 with the use of radiation hybrid
mapping. Apart from a transcription factor (ATF3, Figure 3), no other
genes have been mapped to this region to date. The clinical similarity
between FeLV-C-induced PRCA in cats and human PRCA, including
congenital PRCA (Diamond-Blackfan anemia), raises the possibility that
an abnormality in the expression or structure of the huFLVCR gene could
result in erythroid marrow failure. It is, therefore, of interest that
a case report describes a patient with Diamond-Blackfan anemia and a
rearrangement of the distal region of 1q.28 Although
cytogenetic abnormalities are rare in children with Diamond-Blackfan
anemia, 10%-20% of patients have a family history of anemia, implying
a genetic basis for their disease.28,55 Recent studies have
demonstrated that the gene on chromosome 19q13.2 that encodes ribosomal
protein S19, is deleted in some,56,57 but not
all,58 families with Diamond-Blackfan anemia. The
identification of the huFLVCR gene should allow us to determine its
relevance to this disease.
The first FeLV receptor to be identified was the receptor for FeLV-B,
Pit 1.17,59 As FLVCR shares minimal structural similarity but no sequence homology with Pit 1, the relationship of the structure of these retroviral receptor proteins to their function can also be
studied. In addition, the mechanisms by which the closely related viruses FeLV-C and FeLV-B have evolved to use distinct cell surface receptors can be explored.
Note: Since the submission of this manuscript, the cDNA and predicted
amino acid sequences of huFLVCR have been published (Tailor et al, J
Virol. 1999;73:6500). There is 88% identity among base pairs of the
coding regions of the feline and human cDNAs. Feline FLVCR has 83%
amino acid identity and 89% similarity to the huFLVCR protein. The
proteins are most disparate (57% identity) in the predicted N terminal
intracellular domain (aa1-106), a region that has no homology to
CO9D4.1 (see Figure 1). HuFLVCR messenger RNA is expressed in multiple
tissues (eg, pancreas, kidney) as well as marrow, consistent with
FeLV-C infectivity data in cats.10 The ESTs we used to
construct a huFLVCR genomic probe are 99% identical to huFLVCR cDNA,
confirming the appropriateness of this probe for the chromosomal
localization studies. The structural similarity of feline FLVCR and
huFLVCR thus supports the contention that these proteins have an
equivalent function in the regulation of erythropoiesis as well as in
the regulation of retroviral entry.
| |
Acknowledgments |
The authors would like to thank Allan Dimaunahan and Zenaida Sisk for
help in preparation of the manuscript.
| |
Footnotes |
Submitted July 14, 1999; accepted September 10, 1999.
Supported by grants R01 HL31823 and CA51080 from the National
Institutes of Health. Dr Abkowitz is recipient of a Faculty Research
Award from the American Cancer Society.
Reprints: Janis L. Abkowitz, Professor of Medicine, Division of
Hematology, University of Washington, Box 357710, Seattle, WA
98195-7710.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction