Previous Article | Next Article 
J Virol, January 1998, p. 593-599, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Mouse Homolog of the Bovine Leukemia Virus Receptor Is
Closely Related to the 
Subunit of Adaptor-Related Protein
Complex AP-3, Not Associated with the Cell Surface
Takako
Suzuki and
Hidetoshi
Ikeda*
Laboratory of Immunogenetics, National
Institute of Animal Health, Tsukuba, Ibaraki-ken 305, Japan
Received 18 July 1997/Accepted 28 September 1997
 |
ABSTRACT |
A mouse cDNA (mBLVR1) which was highly homologous to the bovine
cDNA of the bovine leukemia virus receptor (BLVR) gene
was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR
protein was thought to be a type I transmembrane protein, the deduced
protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine
BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding
transmembrane and cytoplasmic regions of the predicted bovine BLVR
protein. No significant hydrophobic region was found in the mouse
protein. Recently, a human cDNA which was highly
homologous (69.6% homology) to the mouse BLVR gene
was reported. The cDNA encodes the 
subunit of the human
adaptor-related protein complex AP-3, which aligned almost collinearly
with the mouse BLVR protein. AP-3 and all other related adaptor
protein complexes have been shown to be associated with intracellular
vesicles but not with the cell surface. Thus, the mouse
BLVR homolog appeared to be the mouse AP-3 subunit itself or closely related to it, but the bovine BLVR gene
seemed slightly different from the adaptor subunit gene family.
| |
INTRODUCTION |
Bovine leukemia virus (BLV) is a
member of the type C retroviruses and is the pathogen that causes
enzootic bovine leukosis (13). BLV is closely related to
human T-lymphotropic virus type 1 and type 2 and simian T-lymphotropic
virus type 1 in genomic structure (6, 35). In natural
infections, bovines and sheep develop lymphoproliferation and tumors of
B-lymphocyte lineage. In experimental infections, BLV can propagate in
various animal species, such as goats, rabbits, chimpanzees, rhesus
monkeys, deer, pigs, cats, and rats, and lead to the development of
disease in goats and rabbits (2, 13, 37). Furthermore,
tissue-cultured cells from a wider range of animal species are variable
in susceptibility to BLV infection (9, 19).
The first step in virus infection is the binding of viruses to cellular
receptors, and the susceptibilities of the cells to the viruses are
determined by the virus receptors. Several cellular receptors for
retroviruses have been identified: a novel protein as a BLV receptor
(3, 4), CD4 (7, 15) and chemokine receptors
(8, 10, 12) as human immunodeficiency virus type 1 receptors, a cationic amino acid transporter as an ecotropic murine
leukemia virus receptor (1), sodium-phosphate symporters as
amphotropic murine leukemia virus (20, 34) and gibbon ape leukemia virus receptors (22), and a low-density lipoprotein receptor as an avian leukosis virus subgroup A receptor (5). These receptors belong to different families of membrane proteins, and
no virus receptor which is not anchored to a cell membrane has been
reported.
The cDNAs of a candidate gene of the BLV receptor (BLVR)
were cloned from a bovine cell line susceptible to BLV. Based on the
sequences of two isolated cDNA clones, the gene product was supposed to
be a type I transmembrane (TM) protein which has signal peptides and a TM domain (3, 4). BLVR protein expressed in
Escherichia coli by the cDNA bound to the BLV Env
protein (3, 23), and transfection of the cDNA into mouse NIH
3T3 cells greatly increased the susceptibility of the cells to BLV
infection (3). The relationship between the BLVR
gene and the host range of BLV is not known.
We cloned a cDNA (mBLVR1) of the mouse homolog (mBLVR) of
the bovine BLVR gene. Mice are resistant to BLV infection,
whereas mouse NIH 3T3 cells are weakly susceptible to the virus
(9). The deduced protein of the mouse BLVR homolog did not
appear to be a typical type I TM protein like the bovine BLVR. Rather,
the mouse BLVR homolog was more closely related to the
recently isolated gene encoding the subunit of adaptor-related
protein complex AP-3, which has been shown to localize in the
cytoplasm (32).
| |
MATERIALS AND METHODS |
Cloning and sequencing of cDNA.
A lambda phage
library of C57BL/6 mouse spleen cDNA (Lambda ZAP II vector; Stratagene)
was screened with a 32P-labeled probe of a 1-kb fragment
derived from bovine BLV receptor cDNA clone BLVRcp1 (nucleotide [nt]
799 to 1790) (a gift from R. Kettmann) (3). Phage DNAs were
transferred to nitrocellulose membranes (Immobilon-NC; Millipore),
following denaturation with alkaline solution and neutralization. The
membranes were hybridized with the 32P-labeled probe for 12 to 20 h at 55°C in a solution containing 10% dextran sulfate, 1 M NaCl, and 1% sodium dodecyl sulfate (SDS). After hybridization, the
membranes were washed for 30 min at 55°C in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% SDS and then for 30 min at
55°C in 0.2× SSC-0.1% SDS. Hybridization and washing were carried
out in rotation in a hybridization oven. The hybridization signals were
detected by a bio-imaging analyzer (Fuji BAS 2000) by exposing the
imaging plate for 4 to 6 h. Insert cDNAs of recombinant phages
were excised as plasmid DNAs (Bluescript II vector) according to the in
vivo excision protocol with the ExAssist/SOLR system (31).
cDNAs were sequenced with a Dye Terminator FS cycle sequencing kit
(Perkin-Elmer) and DNA sequencer model 373S (Applied Biosystems). Synthetic oligonucleotide primers were used to determine the entire nucleotide sequences of both strands.
Southern blot hybridization.
Five micrograms of genomic DNAs
digested with restriction enzymes were separated on an 0.8% agarose
gel. After denaturation with alkaline solution and neutralization, DNAs
were transferred to a nitrocellulose membrane (Nitro Plus; Micron
Separations Inc.), and the same membrane was used for hybridization
with both mouse and bovine BLVR probes after stripping out the prior
probe. The mouse BLVR probes we used were the 0.6-kb
NdeI-EcoNI (nt 3159 to 3791) fragment derived
from mBLVR1 cDNA and the entire 4.7-kb mBLVR1 cDNA. The bovine BLVR
probe was the 0.6-kb BsiWI-MscI (nt 1486 to 2140)
fragment derived from bovine BLVRcp1. The membrane was hybridized with
32P-labeled probes for 18 to 20 h at 35°C in a
solution containing 50% formamide, 5× SSC, 50 mM sodium phosphate,
1× Denhardt's solution, and 0.1% SDS. After hybridization, the
membrane was washed four times for 5 min each at room temperature in
2× SSC-0.1% SDS, twice for 15 min each at 35°C in 0.1× SSC-0.1%
SDS, and finally twice for 5 min each at 35°C in 2× SSC.
Hybridization and washing were carried out in rotation in a
hybridization oven. Hybridization signals were detected by a
bio-imaging analyzer by exposing the imaging plate for 3 days.
Northern blot hybridization.
Total RNAs were isolated from
various organs of BALB/c mice by using a Quick-Prep total RNA
extraction kit (Pharmacia Biotech). Five micrograms of total RNAs were
fractionated on a 1% agarose gel containing formaldehyde. After
alkaline treatment and neutralization, the RNAs were transferred to a
nitrocellulose membrane (Nitro Plus; Micron Separations Inc.) and
hybridized with 32P-labeled probes of the entire 4.7-kb
mBLVR1 cDNA or the mouse 18S ribosomal DNA (rDNA) clone 18SA (a gift
from H. Suzuki and R. Kominami) (16). Hybridization was
carried out as in "Southern blot hybridization," above, except for
hybridization temperatures at 42°C and washing temperatures at
50°C. Hybridization signals of 18S rRNA were detected by exposing the
imaging plate for 15 min.
Nucleotide sequence accession number.
The nucleotide
sequence data of mBLVR1 has been submitted to the DDBJ/EMBL/GenBank
databases under accession no. AB004305.
| |
RESULTS |
Isolation of cDNA of the mouse BLVR homolog gene.
We screened a Lambda ZAP II phage library of C57BL/6 mouse spleen cDNA
with a probe of a portion of bovine BLV receptor cDNA, BLVRcp1, and
isolated two positive clones from about 106 phages. The
cDNA inserts were excised as plasmids termed mBLVR1 and mBLVR2. The
sizes of the cDNA inserts of mBLVR1 and mBLVR2 were 4.7 and 1.8 kb,
respectively. Partial sequencing of mBLVR2 cDNA indicated that the cDNA
is a part of the mBLVR1 cDNA (nt 1183 to 2952). The mBLVR1 cDNA was
almost equal in size to the major mRNA (about 5 kb) expressed in mouse
tissues (see below) and was therefore the mBLVR1 cDNA characterized in
this study.
The nucleotide sequence and deduced amino acid sequence are shown in
Fig.
1 and
2. The mBLVR1 cDNA is 4,730 bp in length
and
has 3,648 bp of open reading frame (ORF) (nt 170 to 3817), with
the
first methionine codon at nt 221. The flanking sequence of
the ATG
initiation codon (CCGCGATGG) is consistent with the
consensus
sequences of the efficient translation start site
(
17). The
ORF can encode a protein of 1,199 amino acids (aa)
with two potential
glycosylation sites at aa 296 and 1001. This clone
lacks a polyadenylation
signal (AATAAA) and a poly(A)
region.
View larger version (112K):
[in this window]
[in a new window]
|
FIG. 1.
Nucleotide sequences of mouse BLVR (m) and bovine BLVR
(b) (3, 4) cDNAs. Nucleotides identical to each other are
indicated by dots. The start position of the mouse BLVR ORF is
indicated by a bracket. The presumed ATG initiation codons and
termination codons are indicated by boldface underlining. The fragments
used for the probe in Southern blot analysis (Fig. 5A and B) are
indicated by boldface letters. The predicted coding region of the
bovine BLVR TM region (3) is indicated by lightface
underlining.
|
|
View larger version (84K):
[in this window]
[in a new window]
|
FIG. 2.
Deduced amino acid sequence of mouse BLVR (mBLVR) human
AP-3 (hAP-3), and bovine BLVR (bBLVR). Dots in the upper and
lower lines indicate identical amino acids between mouse BLVR and human
AP-3 and between mouse BLVR and bovine BLVR, respectively. Two
consensus N glycosylation sites of mBLVR are indicated by boldface
letters. The predicted initiation codons and the TM region of bovine
BLVR are indicated by boldface and lightface underlining,
respectively.
|
|
When the nucleotide sequences of the two bovine cDNA clones, BLVRcp1
(
3) and BLVRcp1/5' (
4), are aligned, the mouse
mBLVR1 extends about 1.7 kb toward the 5' terminus (Fig.
1 and
3). The termination codon of the mouse
mBLVR1 ORF is at nt 3818,
which is 836 bp upstream of that of the
bovine BLVRcp1 ORF. In
the ORF region of mBLVR1, an approximately
2.1-kb DNA (nt 1697
to 3817) overlaps the bovine cDNA and shows high
nucleotide homology
(79%). In contrast, the 3' untranslated region of
mBLVR1 after
the termination codon (nt 3818) has only 51% homology
with the
equivalent region of bovine BLVRcp1 cDNA (Fig.
1) and contains
33 stop codons in the three frames (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Schematic positions of ORFs in the mouse
BLVR, human AP-3 , and bovine BLVR genes. The
ORFs are indicated by open boxes, and putative methionine initiation
codons are indicated by arrows. Probes used for the Southern
hybridization analysis (Fig. 5) are indicated by shaded boxes. The
putative TM region of bovine BLVR is indicated by a black box. Numbers
are expressed as nucleotide positions relative to the mouse
BLVR. The AP-3 gene lacks a region corresponding to nt
728 to 1000 of the mouse BLVR.
|
|
Based on the sequence data of the cDNAs, the bovine BLVR protein was
proposed to be a type I TM protein containing a hydrophobic
TM region
(aa 600 to 626) and hydrophobic signal peptide sequences
(
3,
4). Because the termination codon of the mouse mBLVR
ORF is
located upstream of the predicted TM region of the bovine
BLVR, the
mouse BLVR should miss the corresponding TM and cytoplasmic
regions
(Fig.
2 and
3). The hydropathy profile of the mBLVR ORF
shows no
significant hydrophobic region (Fig.
4).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 4.
Hydropathy plot of the deduced amino acid sequence of
mBLVR. Average hydropathy values were calculated according to the
algorithm of Kyte and Doolittle (18) using a 9-aa window.
High values indicate hydrophobic regions, and low values indicate
hydrophilic regions.
|
|
The initiation codon of bovine BLVR was predicted to be at nt 453 of
the bovine BLVR. However, the ORF of the same frame extended
150 aa
further toward the N terminus from the proposed initiation
codon (Fig.
2 and
3). This region shows very high nucleotide homology
(86%) with
the corresponding region of mouse mBLVR1. We speculate,
therefore, that
the real initiation codon of bovine BLVR cDNA
may exist in the uncloned
upstream region.
Nucleotide sequence homology (79%) is seen in the overall overlapping
region of the mouse and bovine
BLVR ORFs. In contrast,
amino
acid homology is not uniformly distributed; especially low
homology
(21%) is observed in the region from aa 875 to 957 of
mBLVR1 (Fig.
2).
This region of mBLVR1 cDNA (nt 2843 to 3091)
has many insertions and
deletions compared with the homologous
region of bovine BLVRcp1 cDNA
(Fig.
1). Particularly, insertions
of 1 bp at nt 2845 and 11 bp from nt
2859 to 2869 and deletions
of 2 bp at nt 2919 and 10 bp at nt 3091 on
the mouse mBLVR1 should
cause changes in the amino acid reading frame.
These appeared
to be directly related to the low amino acid homology in
the region
from aa 875 to 957.
Recently, the human gene encoding the
subunit of the AP-3
adaptor-related protein complex was reported (
32). We found
that it is very closely related to the mouse
BLVR gene
(69.6%
nucleotide homology and 83.6% identity in 972 aa); their
nucleotide
and amino acid sequences align almost collinearly (Fig.
2),
and
their hydropathy profiles are very similar (data not shown). The
subunit belongs to a protein family including two functionally
related proteins, the
subunit of the AP-1 adaptor complex
(
28)
and the
subunit of the AP-2 adaptor complex
(
29). In contrast
to the high homology with the
subunit,
the mBLVR has lower homologies
with the mouse
subunit (21% in 626 aa) and the mouse
subunit
(21% in 596 aa), and their homologies
are prominent only in the
N-terminal half of each protein. A major
difference between the
mBLVR and the human AP-3
subunit proteins is
a 91-aa insertion
at a position 169 aa downstream of the N-terminal end
of the
subunit protein (Fig.
2 and
3). However, sequences related
to
the insertion are seen in yeast
/
adaptin (
27)
(56.9% match)
and human
adaptin (
24) (37% match), so
it could be a variation
within the gene family. A reverse
transcription-PCR method was
employed to detect a possible
insertionless counterpart in mice,
but only one fragment with the
insert was amplified from brain,
testis, heart, lung, and liver RNAs
(data not shown). The mBLVR
protein and the
subunit protein have
the same WICGEF sequence
(aa 487 to 492 in mBLVR), known as a
"WI(I/L)GEY" consensus sequence,
which is a motif of unknown
function but which is found in the
related gene family including
,
, and
subunits (
14,
21,
25,
28,
29,
32) and even in
the distantly related
-COP
subunit of the COP I adaptor-related
protein complex (
11).
Cross-hybridization of mouse and bovine BLVR probes with the same
fragments of mouse or bovine genomic DNAs.
Because of the sequence
differences, we tried to test whether the mBLVR1 cDNA we cloned was
derived from a gene directly homologous or distantly related to the
bovine BLVR gene. We carried out Southern blot hybridization
for bovine and mouse genomic DNAs digested with five restriction
enzymes. Both the mouse and bovine BLVR cDNA probes were approximately
0.6-kb fragments derived from identical regions (nt 3159 to 3791 for
the mouse probe and nt 1486 to 2140 for the bovine probe) with high
homology (84%) (Fig. 1 and 3). In most digestions, the mouse probe was
hybridized with a single fragment of bovine and mouse DNA (Fig.
5A). Two exceptions were two fragments of
HindIII-digested bovine DNA (Fig. 5A, lane 2) and four
fragments of PstI-digested mouse DNA (lane 9). There is one
HindIII site in the probe region of bovine cDNA;
therefore, it is reasonable that the mouse probe was hybridized to the
two HindIII fragments of bovine DNA. No PstI
site existed in the probe region of mouse DNA, so the reason for the
resulting four PstI fragments was not known.
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 5.
Southern blot analysis of mouse and bovine genomic DNAs
with mouse and bovine BLVR cDNA probes. Bovine (lanes 1 to 5) and mouse
(lanes 6 to 10) genomic DNAs were digested with BamHI (lanes
1 and 6), HindIII (lanes 2 and 7), EcoRI
(lanes 3 and 8), PstI (lanes 4 and 9), and KpnI
(lanes 5 and 10). Five micrograms of DNAs were fractionated on an 0.8%
agarose gel and blotted onto a nitrocellulose membrane. The same
membrane was hybridized with the 32P-labeled 0.6-kb mouse
BLVR probe (nt 3159 to 3791) (A), the 0.6-kb bovine BLVR probe (nt 1486 to 2140) (B), and the entire 4.7-kb mBLVR1 cDNA probe (C) (Fig. 2).
|
|
The same membrane was hybridized with the bovine 0.6-kb probe. The
major fragments of bovine and mouse DNAs which hybridized
with the
bovine probe also hybridized with the mouse probe (Fig.
5B). For
the bovine DNA, the bovine probe also hybridized weakly
with one
additional fragment in
BamHI,
EcoRI, and
KpnI digestions
(Fig.
5B, lanes 1, 3, and 5) and two
additional fragments in
HindIII
and
PstI
digestions (lanes 2 and 4). Likely explanations for the
additional
bands are that the bovine BLVR cDNA probe may be derived
from at least
two exons and its introns may have the five restriction
sites, or
another related gene cross-hybridizing to the bovine
probe may be
present in the bovine DNA.
Because in most digestions the mouse 0.6-kb probe hybridized with only
one fragment of mouse DNA, we tested the possibility
that the mouse
mBLVR gene is a pseudogene lacking introns. The
same
membrane was used to hybridize with a probe of the entire
4.7-kb
mBLVR1 cDNA (Fig.
5C). The probe hybridized with four,
two, one,
four, and three fragments in
BamHI,
HindIII,
EcoRI,
PstI, and
KpnI digestions of
mouse DNA, respectively (Fig.
5C,
lanes 1 to 5). The multiple
hybridizing bands suggest that the
mouse
mBLVR gene is not
an intronless pseudogene. According to
the total size of the
hybridized fragments in these digestions,
we estimate roughly that the
mouse
mBLVR gene may span more than
10 kb.
RNA expression of mBLVR in various tissues.
We examined the
tissue specificity of the mBLVR gene expression. Total RNAs
from the tissues of BALB/c mice were analyzed by Northern blot
hybridization with a probe of the full-length mBLVR1 cDNA (Fig.
6). All 13 tissue samples tested
expressed an approximately 5-kb mBLVR RNA, but the amounts were
variable. High levels of expression were observed in the cerebrum (Fig.
6, lane 1), cerebellum (lane 2), and testis (lane 12) RNAs;
intermediate levels of expression were observed in the thymus (lane 4),
lung (lane 5), heart (lane 6), kidney (lane 10), ovary plus uterus (lane 11), and muscle (lane 13) RNAs; and low levels of expression were
observed in the submaxillary gland (lane 3), liver (lane 7), spleen
(lane 8) and lymph node (lane 9) RNAs.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 6.
RNA expression of the mBLVR gene in mouse
tissues. RNAs were isolated from various tissues of BALB/c mice. Five
micrograms of total RNAs were fractionated on a 1%
formaldehyde-agarose gel, blotted onto a nitrocellulose membrane, and
hybridized with the entire 32P-labeled 4.7-kb mBLVR1 cDNA
probe. Left-side arrows indicate size markers of 28S (4.8 kb) and 18S
(1.9 kb) rRNA. The major 5.0 kb RNA was detected in all organ samples.
The minor 6.2- and 4.6-kb RNAs were detected in some organ samples. As
a control, the same membrane was rehybridized with an 18S rDNA probe.
|
|
Two other RNAs, of 4.6 and 6.2 kb, were hybridized with the probe. The
4.6-kb RNA was expressed only in the testis, although
the hybridization
signal was lower than that of the 5-kb RNA of
the same organ. This
4.6-kb signal may be an alternatively spliced
mRNA, or it may be
transcribed from other related genes. Because
the faint 6.2-kb RNA
bands detected in various tissues were also
hybridized to an 18S rDNA
probe with almost the same pattern (data
not shown), we suspect it may
be nonspecific hybridization.
| |
DISCUSSION |
We cloned and characterized the mouse cDNA, mBLVR1,
homologous to bovine cDNA of the BLVR gene. The mouse
homolog of the BLVR gene appears to be more related to the
human gene encoding the subunit of AP-3 adaptor-related protein
complex (32) than to the bovine BLVR gene. When
the mouse BLVR cDNA was compared with the bovine BLVR cDNA, several
important differences were noted. The most important difference was the
positions of the termination codons of the ORFs. The deduced bovine
BLVR protein predicted a type I TM protein with signal peptides and a
hydrophobic TM region (3, 4). The termination codon of the
mouse mBLVR1 ORF was located upstream of the predicted TM region of
bovine BLVR, suggesting that the mouse mBLVR protein misses the
corresponding TM and cytoplasmic regions. The predicted amino
acid sequence of mouse mBLVR had no significant hydrophobic region
(Fig. 4). Therefore, the mouse mBLVR gene did not appear to
code for a typical TM protein.
When the nucleotide sequences of the mouse and bovine BLVR cDNAs were
compared, homology was found to be 79% in the ORF but only 51% in the
3' untranslated region of the mouse BLVR. Similarly, in a comparison of
the AP-3 subunit cDNA and the bovine BLVR cDNA, an obvious gap in
nucleotide sequence homology was seen at the termination codon of the
AP-3 subunit cDNA. Thus, although the 3' ORF region of the bovine
BLVR gene (nt 2146 to 2981) is important because it encodes
the TM and cytoplasmic domain, it did not match the other related
genes.
The mBLVR1 ORF started from about 2 kb upstream of the predicted
initiation codon of bovine BLVR cDNA, so signal peptides deduced to be
encoded by the bovine BLVR sequence may not be applicable for the mouse
BLVR protein. The bovine BLVR had an in-frame ORF encoding 150 aa
extending upstream of the predicted initiation codon, and this upstream
ORF region was highly homologous, 86% in nucleotide homology, to the
mouse ORF. The bovine BLVR mRNA was reported to be 4.8 kb long
(3), whereas the two bovine cDNAs, BLVRcp1 and BLVRcp1/5',
cover only 3.2 kb (3, 4). Thus, we suppose that the full ORF
of bovine BLVR cDNA still has not been cloned.
The predicted differences in structure and cellular localization of the
mouse and bovine BLVR proteins might be related to their functions as
BLV receptors and might thereby reflect different susceptibilities to
BLV infection. However, further analyses are needed to resolve the
possible differences in structure and function between the mouse and
bovine BLVR gene products.
After the characterization of the mBLVR1 cDNA was finished, the human
gene encoding the subunit of the adaptor-related protein complex
AP-3 was reported (32). AP-3 is associated with
non-clathrin-coated intracellular vesicles, while the other adaptor
protein complexes, AP-1 and AP-2, are associated with clathrin-coated
vesicles. These vesicle-associated protein complexes are thought
to regulate intercellular vesicle traffic (30). The AP-1,
AP-2, and AP-3 protein complexes show structural similarities and
consist of four different subunits, each of which belongs to four
different families. The , , and subunits of the AP-1, AP-2,
and AP-3 complexes, respectively, form a gene family. There are also
other non-clathrin-associated adaptor-related protein complexes
(30), some subunits of which show sequence similarities with
the // subunit gene family. These indicate that there is a
large group of adaptor subunit genes with considerable diversity.
Our Southern blot analysis did not entirely rule out the possibility
that the bovine and mouse BLVR genes are not directly homologous but rather are distantly related. However, our previous chromosomal mapping data suggested that the mBLVR1 cDNA we cloned is
derived from the direct homolog of bovine BLVR. The mouse
homolog of the BLVR gene, termed Bolvr, was
located to mouse chromosome 10 by a PCR-single-strand conformation
polymorphism method using the 3' untranslated sequences of mouse mBLVR1
(33). The bovine BLVR gene was mapped to bovine
chromosome 7q15 by the fluorescence in situ hybridization method
(26). A comparative map between mouse and bovine
(36) indicated that the mouse chromosomal region including
the mouse Bolvr gene is homologous to the bovine chromosomal region including the bovine BLVR gene.
Although the mouse BLVR gene is highly homologous to the
AP-3 subunit gene, it is not clear how much the bovine
BLVR gene is related to the // subunit gene family.
The bovine BLVR gene may be a variant of the gene family.
The mouse BLVR cDNA and the AP-3 subunit cDNA were cloned from cDNA
libraries of total mRNAs, based on cross-hybridization with DNA probes,
and both may be representatives of the abundantly expressed mRNAs
because the cDNA sizes are almost the same as those detected by
Northern blots. In contrast, the bovine BLVR cDNA was cloned from an
expression library, based on the binding properties of recombinant
proteins expressed by E. coli to BLV Env glycoprotein on
nylon membranes. An Env-binding domain of the BLVR protein was mapped
to a region corresponding to aa 899 to 1009 of the mouse BLVR protein
(3, 23) that includes amino acids relatively unconserved
among the mouse BLVR, bovine BLVR, and human AP-3 subunit (Fig. 2).
Therefore, the origin of the BLVR gene should be
further clarified.
| |
ACKNOWLEDGMENTS |
We thank Richard Kettmann, Faculty of Agronomy Belgium, for the
gift of the BLVRcp1 cDNA clone; Hitoshi Suzuki, Hokkaido University, and Ryo Kominami, Niigata University, for the gift of the 18S rDNA
clone; and Hiroshi Sentsui, National Institute of Animal Health, for
valuable discussion.
This study was supported in part by grants from the Science and
Technology Agencies of Japan and the Ministry of Agriculture, Forestry
and Fisheries of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Immunogenetics, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki-ken 305, Japan. Phone: 81-298-38-7757. Fax:
81-298-38-7880. E-mail: hikeda{at}niah.affrc.go.jp.
| |
REFERENCES |
| 1.
|
Albritton, L. M.,
L. Tseng,
D. Scadden, and J. M. Cunningham.
1989.
A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.
Cell
57:659-666[Medline].
|
| 2.
|
Altanerova, V.,
D. Portetelle,
R. Kettmann, and C. Altaner.
1989.
Infection of rats with bovine leukaemia virus: establishment of a virus-producing rat cell line.
J. Gen. Virol.
70:1929-1932[Abstract/Free Full Text].
|
| 3.
|
Ban, J.,
D. Portetelle,
C. Altaner,
B. Horion,
D. Milan,
V. Krchnak,
A. Burny, and R. Kettmann.
1993.
Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.
J. Virol.
67:1050-1057[Abstract/Free Full Text].
|
| 4.
|
Ban, J.,
A. T. Truong,
B. Horion,
C. Altaner,
A. Burny,
D. Portetelle, and R. Kettmann.
1994.
Isolation of the missing 5'-end of the encoding region of the bovine leukemia virus cell receptor gene.
Arch. Virol.
138:379-383[Medline].
|
| 5.
|
Bates, P.,
J. A. T. Young, and H. E. Varmus.
1993.
A receptor for subgroup A Rous sarcoma virus is related to the low density lipoprotein receptor.
Cell
74:1043-1051[Medline].
|
| 6.
|
Coffin, J. M.
1992.
Structure and classification of retroviruses, p. 19-49. In
J. A. Levy (ed.), The Retroviridae, vol. 1.
Plenum Press, New York, N.Y.
|
| 7.
|
Dalgleish, A. G.,
P. C. L. Beverley,
P. R. Clapham,
D. H. Crawford,
M. F. Greaves, and R. A. Weiss.
1984.
The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.
Nature
312:763-767[Medline].
|
| 8.
|
Deng, H.,
R. Liu,
W. Ellmeier,
S. Choe,
D. Unutmaz,
M. Burkhart,
P. Di Marzio,
S. Marmon,
R. E. Sutton,
C. M. Hill,
C. B. Davis,
S. C. Peiper,
T. J. Schall,
D. R. Littman, and N. R. Landau.
1996.
Identification of a major co-receptor for primary isolates of HIV-1.
Nature
381:661-666[Medline].
|
| 9.
|
Derse, D., and L. Martarano.
1990.
Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.
J. Virol.
64:401-405[Abstract/Free Full Text].
|
| 10.
|
Dragic, T.,
V. Litwin,
G. P. Allaway,
S. R. Martin,
Y. Huang,
K. A. Nagashima,
C. Cayanan,
P. J. Maddon,
R. A. Koup,
J. P. Moore, and W. A. Paxton.
1996.
HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature
381:667-673[Medline].
|
| 11.
|
Duden, R.,
G. Griffiths,
R. Frank,
P. Argos, and T. E. Kreis.
1991.
Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin.
Cell
64:649-665[Medline].
|
| 12.
|
Feng, Y.,
C. C. Broder,
P. E. Kennedy, and E. A. Berger.
1996.
HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.
Science
272:872-877[Abstract].
|
| 13.
|
Kettmann, R.,
A. Burny,
I. Callebaut,
L. Droogmans,
M. Mammerickx,
L. Willems, and D. Portetelle.
1994.
Bovine leukemia virus, p. 39-81. In
J. A. Levy (ed.), The Retroviridae, vol. 3.
Plenum Press, New York, N.Y.
|
| 14.
|
Kirchhausen, T.,
K. L. Nathanson,
W. Matsui,
A. Vaisberg,
E. P. Chow,
C. Burne,
J. H. Keen, and A. E. Davis.
1989.
Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.
Proc. Natl. Acad. Sci. USA
86:2612-2616[Abstract/Free Full Text].
|
| 15.
|
Klatzmann, D.,
E. Champagne,
S. Chamaret,
J. Gruest,
D. Guetard,
T. Hercend,
J.-C. Gluckman, and L. Montagnier.
1984.
T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.
Nature
312:767-768[Medline].
|
| 16.
|
Kominami, R.,
Y. Urano,
Y. Mishima, and M. Muramatsu.
1981.
Organization of ribosomal RNA gene repeats of the mouse.
Nucleic Acids Res.
9:3219-3233[Abstract/Free Full Text].
|
| 17.
|
Kozak, M.
1986.
Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
Cell
44:283-292[Medline].
|
| 18.
|
Kyte, J., and R. F. Doolittle.
1982.
A simple method for displaying the hydropathic character of a protein.
J. Mol. Biol.
157:105-132[Medline].
|
| 19.
|
Milan, D., and J.-F. Nicolas.
1991.
Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus.
J. Virol.
65:1938-1945[Abstract/Free Full Text].
|
| 20.
|
Miller, D. G.,
R. H. Edwards, and A. D. Miller.
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[Abstract/Free Full Text].
|
| 21.
|
Newman, L. S.,
M. O. McKeever,
H. J. Okano, and R. B. Darnell.
1995.
-NAP, a cerebellar degeneration antigen, is a neuron-specific vesicle coat protein.
Cell
82:773-783[Medline].
|
| 22.
|
O'Hara, B.,
S. V. Johann,
H. P. Klinger,
D. G. Blair,
H. Rubinson,
K. J. Dunn,
P. Sass,
S. M. Vitek, and T. Robins.
1990.
Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus.
Cell Growth Differ.
1:119-127[Abstract].
|
| 23.
|
Orlik, O.,
J. Ban,
J. Hlavaty,
C. Altaner,
R. Kettmann,
D. Portetelle, and G. A. Splitter.
1997.
Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.
J. Virol.
71:3263-3267[Abstract].
|
| 24.
|
Peyrard, M.,
I. Fransson,
Y. G. Xie,
F. Y. Han,
M. H. Ruttledge,
S. Swahn,
J. E. Collins,
I. Dunham,
V. P. Collins, and J. P. Dumanski.
1994.
Characterization of a new member of the human beta-adaptin gene family from chromosome 22q12, a candidate meningioma gene.
Hum. Mol. Genet.
3:1393-1399[Abstract/Free Full Text].
|
| 25.
|
Ponnambalam, S.,
M. S. Robinson,
A. P. Jackson,
L. Peiperl, and P. Parham.
1990.
Conservation and diversity in families of coated vesicle adaptins.
J. Biol. Chem.
265:4814-4820[Abstract/Free Full Text].
|
| 26.
|
Popescu, C. P.,
J. Boscher,
H. C. Hayes,
J. Ban, and R. Kettmann.
1995.
Chromosomal localization of the BLV receptor candidate gene in cattle, sheep, and goat.
Cytogenet. Cell Genet.
69:50-52[Medline].
|
| 27.
| Robinson, L. C., H. M. Engle, and H. R. Panek. 1995. EMBL database submission. Accession no. U36858.
|
| 28.
|
Robinson, M. S.
1990.
Cloning and expression of -adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus.
J. Cell Biol.
111:2319-2326[Abstract/Free Full Text].
|
| 29.
|
Robinson, M. S.
1989.
Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins).
J. Cell Biol.
108:833-842[Abstract/Free Full Text].
|
| 30.
|
Schekman, R., and L. Orci.
1996.
Coat proteins and vesicle budding.
Science
271:1526-1533[Abstract].
|
| 31.
|
Short, J. M.,
J. M. Fernadez,
J. A. Sorge, and W. D. Huse.
1988.
 ZAP: a bacteriophage expression vector with in vivo excision properties.
Nucleic Acids Res.
16:7583-7600[Abstract/Free Full Text].
|
| 32.
|
Simpson, F.,
A. A. Peden,
L. Christopoulou, and M. S. Robinson.
1997.
Characterization of the adaptor-related protein complex, AP-3.
J. Cell Biol.
137:835-845[Abstract/Free Full Text].
|
| 33.
|
Suzuki, T.,
H. Yonekawa, and H. Ikeda.
1996.
Localization of mouse homolog of the bovine leukemia virus receptor gene on mouse chromosome 10.
Mamm. Genome
7:708-709[Medline].
|
| 34.
|
van Zeijl, M.,
S. V. Johann,
E. Closs,
J. Cunningham,
R. Eddy,
T. B. Shows, and B. O'Hara.
1994.
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family.
Proc. Natl. Acad. Sci. USA
91:1168-1172[Abstract/Free Full Text].
|
| 35.
|
Watanabe, T.,
M. Seiki,
H. Tsujimoto,
I. Miyoshi,
M. Hayami, and M. Yoshida.
1985.
Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I.
Virology
144:59-65[Medline].
|
| 36.
|
Womack, J. E., and S. R. Kata.
1995.
Bovine genome mapping: evolutionary inference and the power of comparative genomics.
Curr. Opin. Genet. Dev.
5:725-733[Medline].
|
| 37.
|
Wyatt, C. R.,
D. Wingett,
J. S. White,
C. D. Buck,
D. Knowles,
R. Reeves, and N. S. Magnuson.
1989.
Persistent infection of rabbits with bovine leukemia virus associated with development of immune dysfunction.
J. Virol.
63:4498-4506[Abstract/Free Full Text].
|
J Virol, January 1998, p. 593-599, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Suzuki, T., Matsubara, Y., Kitani, H., Ikeda, H.
(2003). Evaluation of the {delta} subunit of bovine adaptor protein complex 3 as a receptor for bovine leukaemia virus. J. Gen. Virol.
84: 1309-1316
[Abstract]
[Full Text]
-
Gatot, J.-S., Callebaut, I., Van Lint, C., Demonte, D., Kerkhofs, P., Portetelle, D., Burny, A., Willems, L., Kettmann, R.
(2002). Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo. J. Virol.
76: 7956-7967
[Abstract]
[Full Text]
-
Sommerfelt, M. A.
(1999). Retrovirus receptors. J. Gen. Virol.
80: 3049-3064
[Full Text]
Top
Abstract
Introduction
