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. 1998 Jan;72(1):593–599. doi: 10.1128/jvi.72.1.593-599.1998

The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the δ Subunit of Adaptor-Related Protein Complex AP-3, Not Associated with the Cell Surface

Takako Suzuki 1, Hidetoshi Ikeda 1,*
PMCID: PMC109412  PMID: 9420263

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 δ 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 δ subunit itself or closely related to it, but the bovine BLVR gene seemed slightly different from the adaptor subunit gene family.

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 δ 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.

FIG. 1.

FIG. 1

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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.

FIG. 2.

FIG. 2

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Deduced amino acid sequence of mouse BLVR (mBLVR) human AP-3 δ (hAP-3δ), and bovine BLVR (bBLVR). Dots in the upper and lower lines indicate identical amino acids between mouse BLVR and human AP-3 δ 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).

FIG. 3.

FIG. 3

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Schematic positions of ORFs in the mouse BLVR, human AP-3 δ, 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 δ 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).

FIG. 4.

FIG. 4

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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 δ 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 δ subunit belongs to a protein family including two functionally related proteins, the γ subunit of the AP-1 adaptor complex (28) and the α subunit of the AP-2 adaptor complex (29). In contrast to the high homology with the δ subunit, the mBLVR has lower homologies with the mouse γ subunit (21% in 626 aa) and the mouse α 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 δ subunit proteins is a 91-aa insertion at a position 169 aa downstream of the N-terminal end of the δ subunit protein (Fig. 2 and 3). However, sequences related to the insertion are seen in yeast α/γ adaptin (27) (56.9% match) and human β 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 δ 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 α, β, and γ subunits (14, 21, 25, 28, 29, 32) and even in the distantly related β-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.

FIG. 5.

FIG. 5

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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.

FIG. 6.

FIG. 6

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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 δ 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 δ subunit cDNA and the bovine BLVR cDNA, an obvious gap in nucleotide sequence homology was seen at the termination codon of the AP-3 δ 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 δ 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 γ, α, and δ 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 γ/α/δ 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 δ subunit gene, it is not clear how much the bovine BLVR gene is related to the γ/α/δ subunit gene family. The bovine BLVR gene may be a variant of the gene family. The mouse BLVR cDNA and the AP-3 δ 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 δ 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.

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