Evaluation of the {delta} subunit of bovine adaptor protein complex 3 as a receptor for bovine leukaemia virus

Takako Suzuki1, Yutaka Matsubara2, Hiroshi Kitani4 and Hidetoshi Ikeda3

1 Department of Immunology, National Institute of Animal Health, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan
2 Department of Planning and Coordination, National Institute of Animal Health, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan
3 Department of Infectious Diseases, National Institute of Animal Health, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan
4 Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Correspondence
Hidetoshi Ikeda
hikeda{at}affrc.go.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A candidate gene of the bovine leukaemia virus (BLV) receptor (BLVR) was cloned previously and predicted to encode a transmembrane protein. Subsequent cloning of related genes from other organisms indicated that the candidate gene is related, but unique, to a gene family of the {delta} subunit of the adaptor protein (AP) complex 3, AP-3. Therefore, bovine cDNAs (boAP3{delta}) that are highly homologous to the candidate gene were cloned and sequenced. The nucleotide sequences suggested that the boAP3{delta} cDNA encodes the {delta} subunit of boAP3 without transmembrane domains. Part of the AP3{delta} cDNA isolated from the lymph node, spleen and MDBK cells, from which the BLVR candidate cDNA was derived, has almost the same nucleotide sequences as the boAP3{delta} cDNA. A boAP3{delta} protein tagged with green fluorescent protein was localized in the cytoplasm and incorporated into AP-3 in bovine cells. Unlike the previous report about the candidate gene, the boAP3{delta} gene introduced into murine NIH 3T3 cells did not increase the susceptibility of the cells to BLV infection. Many small insertions and deletions of nucleotides could generate the predicted transmembrane and cytoplasmic regions of the BLVR protein from the prototypic boAP3{delta} gene.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many retroviruses bind to specific receptors on the target cell surface to initiate infection and the receptors are one of the important factors determining the host range of retroviruses. Many receptors for retroviruses have been identified that are either multiple or single membrane-spanning proteins (Sommerfelt, 1999). BLVRcp1, a cDNA encoding the candidate protein of the bovine leukaemia virus (BLV) receptor (BLVR), was cloned from an expression library of the bovine cell line MDBK, susceptible to BLV infection based on the binding property of bacterially expressed protein to BLV gp51 (Ban et al., 1993). According to the nucleotide sequence, the candidate protein was predicted to be a type I transmembrane protein with a single transmembrane domain and transfection of a BLVRcp1 expression plasmid into mouse and human cells increased the susceptibility of cells to recombinant BLV infection (Ban et al., 1993).

We cloned previously a mouse cDNA (mBLVR1) homologous to bovine BLVRcp1 (Ban et al., 1993) and BLVRcp1/5', missing the 5'-part of BLVRcp1 (Ban et al., 1994) (BLVRcp) based on cross-hybridization, and found that mBLVR1 encodes a protein without the typical hydrophobic transmembrane region and is closely related to the {delta} subunit of the adaptor protein (AP) complex 3, AP-3 (Suzuki & Ikeda, 1998). In humans and mice, there are four different AP complexes, AP-1, -2, -3 and -4, and all of them mediate intracellular protein transport (Boehm & Bonifacino, 2002; Robinson & Bonifacino, 2001). The AP complexes consist of four different subunit proteins, each of which belongs to the {gamma}/{alpha}/{delta}/{varepsilon}, {beta}, µ and {sigma} gene families, respectively, and AP-3 has the {delta}, {beta}3, µ3 and {sigma}3 subunits (Boehm & Bonifacino, 2002; Robinson & Bonifacino, 2001). Humans and mice appear to carry only one {delta} gene expressed ubiquitously (Boehm & Bonifacino, 2001). Although BLVRcp is clearly related to the {gamma}/{alpha}/{delta}/{varepsilon} subunit family at the nucleotide sequence level, the predicted protein structure is unique in the family because none of the other members has any transmembrane domains. To address the questions of whether BLVRcp is a representative of the bovine AP3{delta} homologue (boAP3{delta}) and if not, how BLVRcp was different from the AP3{delta} family, we recloned bovine BLVRcp-related cDNAs from the brain, lymph node and spleen and from MDBK cells, from which BLVRcp1 was isolated originally. We then characterized their encoding proteins for their potential to interact with other boAP3 subunits. We also tested the susceptibility of the cells transfected with the cloned cDNA to BLV infection.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
BT cells (ATCC, #CRL1390) (McClurkin et al., 1974) were derived from bovine turbinate; MDBK cells (ATCC, #CCL-22) were derived from bovine kidney; NIH 3T3 cells (ATCC, #CCL-92) were derived from mouse embryo; CC81 cells were derived from a cat (Fischinger et al., 1974) and used for BLV titration. The bat BLV-Bat2cl1 cells produce high amounts of infectious BLV (Graves & Ferrer, 1976). All cells were cultured with Dulbecco's modified Eagle's medium (DMEM) (Nissui) containing 0·25 % HEPES, 50 µg kanamycin ml-1 and 5–10 % foetal calf serum. The BLV-Bat2cl1 cells were stably transfected with plasmid pBLV-SVNEO, encoding a defective BLV with a neomycin-resistant gene (Derse & Martarano, 1990) (a gift from D. Derse, National Cancer Institute–Frederick Cancer Research Facility, Maryland, USA) and the culture supernatant was used as infectious virus (BLV-neo).

Cloning of boAP3{delta} cDNAs.
A {lambda} phage library of bovine brain cDNA ({lambda}ZAP II vector, a gift from M. Sakurai, National Institute of Agrobiological Sciences, Japan) was screened with a 32P-labelled probe of the 1 kb EcoRI–HindIII fragment, nt 5–996, derived from the bovine BLVRcp1 clone (Ban et al., 1993) (a gift from R. Kettmann, Faculty of Agronomy, Belgium), as described previously (Suzuki & Ikeda, 1998). The brain cDNA library was constructed for other purposes (Kubota et al., 1994). To isolate longer cDNA clones, the DNAs of 90 independent pools of phages obtained from a single area of about 5–10 plaques in the first screening were analysed for the size of a 5' region of cDNAs by PCR using primers of mBLVR1 (mo-920RV, 5'-ATGAGGACCCAGTTGTTG-3') and vector (M13-20, 5'-GTAAAACGACGGCCAGT-3', or M13-RV, 5'-AACAGCTATGACCATG-3'). PCR products were subjected to electrophoresis through agarose gel followed by Southern blot hybridization with a mBLVR1 probe (nt 1–883) (Suzuki & Ikeda, 1998). Two phage clones, which produced the largest PCR fragments, were isolated and their insert cDNAs were excised as plasmid DNAs (pboAP3{delta}1 and pboAP3{delta}2) (pBluescript II vector) (Stratagene), according to the in vivo excision protocol using the Exassist/SOLR system (Short et al., 1988). The entire nucleotide sequences of the two clones were determined with a Dye Terminator Cycle Sequencing FS kit (Perkin-Elmer) and a DNA sequencer 373S (Applied Biosystems). The nucleotide sequence of boAP3{delta}1 was registered in DDBJ/EMBL/GenBank under accession no. AB015979.

Total RNAs from the lymph node and spleen of a cow and MDBK cells were isolated using a QuickPrep Total RNA Extraction kit (Amersham Pharmacia) and about 2 kb of the cDNAs (nt 2351–4298 of boAP3{delta}1) was amplified by RT-PCR using a RNA PCR kit (AMV), version 2.1 (Takara). PCR primers (FW, 5'-GTGGACATCGTCACCGA-3', and RV, 5'-ACAGACCTGCAGAGCATCCA-3') perfectly match both boAP3{delta}1 and BLVRcp1 sequences. A 1·8 kb region of these PCR products (nt 2390–4198) was sequenced directly (Hokkaido System Science).

Construction and transfection of expression plasmids.
The boAP3{delta} expression plasmid pCMV-{delta} and the BLVRcp expression plasmid pCMV-BLVR were constructed by inserting a 4·7 kb NotI fragment of pboAP3{delta}1 or a 2·3-kb EcoRI fragment of pBLVRcp1 into the NotI site of a derivative of the pCMV-{beta} expression vector (Clontech Laboratories) in which the {beta}-galactosidase gene of the original pCMV-{beta} vector was removed. For pCMV-BLVR, all ends derived from the restriction were blunt-ended and ligated. The green fluorescent protein (GFP) expression plasmid pCMV-GFP was constructed by inserting a 750 bp Bsp120I–NotI fragment encoding EGFP (enhanced GFP variants) derived from pEGFP-1 (Clontech Laboratories) into the NotI site of the pCMV-{beta} derivative. pCMV-GFP/{delta}, the expression plasmid of the GFP-boAP3{delta} fusion protein (GFP/{delta}), was generated by ligation of two blunt-ended fragments, a 4 kb XhoI–AvrII fragment from pboAP3{delta}1 (nt 7–4021) and a BsrGI–NotI fragment from pCMV-GFP, so that the entire coding region of the boAP3{delta}1 gene was placed at the 3' end of the EGFP gene of pCMV-GFP.

The boAP3{delta}, BLVRcp, GFP and GFP/{delta} proteins were expressed in the bovine BT cells or the mouse NIH 3T3 cells by either stable or transient transfection with the expression plasmids via the Lipofectamine Plus reagent (Life Technologies). To establish stable transformants, cells were cotransfected with pSV2-hph (ATCC, #37647) and the transformants were selected with medium containing 100 µg hygromycin B ml-1 (Wako). The expression of GFP and GFP/{delta} in transfected cells was analysed by flow cytometry. The expressions of boAP3{delta}, BLVRcp and GFP/{delta} were verified by RT-PCR 24 h after transfection with boAP3{delta}1- and BLVRcp1-specific primers; the transduced boAP3{delta} protein could not be distinguished from the endogenous mouse AP3{delta} by the anti-{delta} subunit antibody (Simpson et al., 1997) (a gift from M. S. Robinson, University of Cambridge, UK).

Immunoprecipitation.
BT cells and BT cells stably transfected with the pCMV-GFP/{delta} expression plasmid (BT-GFP/{delta}) were metabolically labelled with [35S]methionine and [35S]cysteine (Express Protein Labelling mix; NEN Life Science) for 7 h. After washing with cold medium (DMEM), the cells were lysed with TNE buffer (10 mM Tris/HCl pH 7·8, 1 % NP-40, 0·15 M NaCl and 1 mM EDTA) containing the protease inhibitor cocktail (Complete; Roche) at 4 °C. The labelled cell lysates (3x106 c.p.m. per lane) were incubated with protein G–Sepharose 4 Fast Flow (Amersham Pharmacia) for 17 h at 4 °C to remove the nonspecific binding to protein G. The precleared lysates were incubated with protein G–Sepharose (control) or protein G–Sepharose preabsorbed with 5 µg of the anti-GFP antibody (clone 3E6; Wako) or anti-MHC class I antibody (clone IL-A88; a gift from J. Naessens, International Livestock Research Institute, Kenya) (Toye et al., 1990) for 1 h at 4 °C. After washing five times with TNE, the precipitates were boiled in sample loading buffer (130 mM Tris/HCl pH 6·8, 6 % SDS, 20 % glycerol, 10 % 2-mercaptoethanol and 0·005 % bromophenol blue) and fractionated by SDS-PAGE on a 7 % gel. Isotope signals were detected using a bio-imaging analyser (BAS 2000; Fujifilm) by exposing an imaging plate for 5 days.

Fluorescence microscopy.
After transfection of BT cells with pCMV-GFP or pCMV-GFP/{delta} and pSV2-hph, and selection of the transformed cells with medium containing 100 µg hygromycin B ml-1, the resulting 10–20 hygromycin-resistant colonies were mixed and passaged several times. The mixed cell populations were analysed for expression of GFP fluorescence by fluorescence microscopy. BT-GFP/{delta} and BT-GFP cells, BT cells transfected with pCMV-GFP, were plated on an 8-well chamber slide at a density of 1x105 cells per well. After 14 h, the cells were washed with cold PBS and fixed with PBS containing 4 % paraformaldehyde for 30 min at 4 °C. After washing with cold PBS, the slide was mounted with Aqua Poly/Mount reagent (Polysciences). The cells were observed by fluorescence microscopy and differential interference microscopy using a LEITZ DMRD microscope (Leica) and a digital CCD camera (Hamamatsu Photonics).

Immunoblotting.
BT and BT-GFP/{delta} cells were lysed with digitonin buffer (1 % digitonin, 10 mM triethanolamine/HCl pH 7·8, 150 mM NaCl, 10 mM iodoacetoamide and 1 mM EDTA) containing the protease inhibitor cocktail at 4 °C. Cell lysates (2·5x107 cells per lane) were preabsorbed with protein G–Sepharose 4 Fast Flow and a control mouse antibody (anti-His antibody; Amersham Pharmacia), which was the same isotype as the anti-GFP antibody, and then immunoprecipitated with the mouse anti-GFP antibody (clone 3E6), as described above. Immunoprecipitates were boiled in sample loading buffer (187·5 mM Tris/HCl pH 6·8, 6 % SDS, 30 % glycerol and 0·03 % phenol red), fractionated by SDS-PAGE on a 6·5 % or a 12 % gel and transferred onto a PVDF membrane (Immobilon; Millipore). The membranes were incubated in PBS containing 5 % ECL blocking agent (Amersham Pharmacia) and 0·05 % Tween 20, and then probed with either the rabbit anti-{delta} or anti-{sigma}3 subunit antibodies (Simpson et al., 1997) (gifts from M. S. Robinson) followed by treatment with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Zymed). The HRP-mediated chemiluminescent reaction was performed with ECL Plus Western Blotting Detection reagents (Amersham Pharmacia). The same membranes were reprobed with rabbit anti-{beta}3 or anti-µ3 subunit antibodies (Simpson et al., 1996) (gifts from M. S. Robinson) after stripping of the first probed antibody with Restore Western Blot Stripping buffer (Pierce).

Infection of cells with recombinant BLV.
NIH 3T3 cells were plated at a density of 1·2x106 cells per 10 cm diameter dish and on the following day transfected with pCMV-{delta}, pCMV-BLVR or pCMV-GFP/{delta}. After 14 h, the cells were trypsinized and replated on a new 10 cm dish at a density of 5x105 cells per dish. At the same time, other cells were also plated at the same cell density. After 9 h, polybrene (Nacalai Tesque) was added to the culture medium at a final concentration of 20 µg ml-1 and the cells were incubated for a further 1 h. The cells were then infected with BLV-neo (Derse & Martarano, 1990) with polybrene for 1 h. After a 2 day culture, the medium was replaced with a culture medium containing 0·8 mg geneticin ml-1 (G418 sulfate) (Sigma) and the developed G418-resistant colonies were counted after 10–16 days of culture. Expressions of transfected genes at the time of the BLV-neo infection were verified by RT-PCR.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of boAP3{delta} cDNA
We screened a cDNA library derived from bovine brain (Kubota et al., 1994) using a probe from a portion of BLVRcp1 (Ban et al., 1993). The two largest cDNAs were cloned into plasmids and sequenced completely. They were 4683 and 4256 bp in size and tentatively termed boAP3{delta}1 and boAP3{delta}2. They had identical sequences except for two regions: the shorter clone, boAP3{delta}2, lacked 440 bp at the 5' end and had five additional bases adjacent to the poly(A) tail. Therefore, we analysed the larger boAP3{delta}1 clone and used this for further experiments.

boAP3{delta}1 cDNA encoded a protein of 1207 aa that shared many characteristics with human AP3{delta} (hAP3{delta}) (Ooi et al., 1997; Simpson et al., 1997) and the murine BLVR homologue protein (mBLVR) (Suzuki & Ikeda, 1998). The positions of the first ATG codon at nt 32 and the termination codon at nt 3652 were equivalent to those of hAP3{delta} and mBLVR1 (Fig. 1). The encoded protein showed an 81·8 % (Simpson et al., 1997) or 86·7 % (Ooi et al., 1997) identity with the two hAP3{delta} clones and 88·3 % identity with mBLVR (Suzuki & Ikeda, 1998). A hydrophobicity profile of the encoded protein also resembled those of hAP3{delta} and mBLVR (data not shown). Therefore, the cloned cDNAs appeared to encode a bovine homologue of the {delta} subunit of AP-3.



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Fig. 1. Schematic representation of boAP3{delta}1, BLVRcp, mBLVR1 and hAP3{delta} cDNAs. Grey boxes show ORFs of the cDNAs. Initiation codons (Met) of each clone are shown by arrows. The hatched box shows the predicted transmembrane (TM) domain of BLVRcp (Ban et al., 1993). Numbers are expressed as nucleotide positions relative to boAP3{delta}1. Nucleotide identities (%) between boAP3{delta}1 and BLVRcp are also shown.

 
The boAP3{delta}1 cDNA has almost the same size as the AP3{delta} mRNAs of humans (Ooi et al., 1997; Simpson et al., 1997) and mice (Suzuki & Ikeda, 1998), but BLVRcp (Ban et al., 1993, 1994) was 1·5 kb shorter than boAP3{delta}1 (Fig. 1). The nucleotide sequences of boAP3{delta}1 and BLVRcp show a very high identity throughout the overlapping region: 99·6 % in the protein-encoding region and 95·7 % in the 3' noncoding region of boAP3{delta}1 (Fig. 1), but their deduced protein structures are quite different. boAP3{delta}1 has a termination codon at nt 3653, which is located upstream of the transmembrane domain of BLVRcp and does not seem to encode a transmembrane protein (Figs 1 and 2). The overlapping region of the two protein sequences shows a high identity (99·6 %), except for a block from aa 906 to 964, in which the two proteins have the same number of amino acids but no significant identity (Fig. 2A). The nucleotide sequences of this region are identical, except for both ends of the region: BLVRcp has a one base deletion at nt 2745 and a one base insertion at nt 2922 of boAP3{delta}1 (Fig. 2B). This lack of amino acid identity should be due to frameshifts caused by the insertion and deletion. The TGA sequence at nt 3653 of boAP3{delta}1 is used as a stop codon, while this sequence is used for amino acids in BLVRcp (Fig. 2B). This difference is probably due to a frameshift by a one base insertion in BLVRcp at nt 3641 of boAP3{delta}1, 13 bases upstream of the boAP3{delta}1 stop codon. The open reading frame (ORF) of BLVRcp extends a further 836 bases to the 3' terminus but the corresponding region of boAP3{delta}1 includes 21 stop codons in the three frames. These stop codons were also used as amino acids because of the 12 one base, five two base and one four base insertions or deletions in BLVRcp.



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Fig. 2. Comparison between amino acid and nucleotide sequences of the two bovine cDNAs, boAP3{delta}1 and BLVRcp. (A) Deduced amino acid sequences of the overlapping region of the two clones. Asterisks and dots between the sequences show identical and relative amino acids. Underlining indicates the region lacking sequence identity. (B) Nucleotide sequences of boAP3{delta}1 and BLVRcp in the 3' half of boAP3{delta}1. Single underlining shows the region that lacks amino acid sequence identity, shown by the underlining in (A). Double underlining indicates the predicted transmembrane domain of BLVRcp (Ban et al., 1993). Grey boxes show the termination codons of both clones. Arrows indicate insertions and deletions of BLVRcp in comparison to boAP3{delta}1.

 
We amplified about 2 kb of the cDNAs (nt 2351–4298 of boAP3{delta}1) by RT-PCR from the bovine lymph node and spleen and MDBK cells using primers that are accordant with boAP3{delta}1 and BLVRcp1. A 1·8 kb region (nt 2390–4198), including the insertions and a deletion causing the lack of amino acid identity and skipping of the stop codon in BLVRcp (Fig. 2), were sequenced directly. Between boAP3{delta}1 and MDBK cDNA, only one nucleotide variation was observed at nt 3291 and this does not change the amino acid sequence. The cDNA sequences of the spleen and lymph node derived from the same animal were a mixture of both types. The expression of BLVRcp-type mRNA-carrying transmembrane region was not detected in MDBK cells.

Expression and localization of the GFP-boAP3{delta} fusion protein
All of the reported AP complexes are localized in the cytoplasm and mediate protein transport (Boehm & Bonifacino, 2002; Robinson & Bonifacino, 2001). AP-3 is associated at the trans-Golgi network in the cytoplasm (Dell'Angelica et al., 1997; Simpson et al., 1997). However, some intracellular proteins, such as heat shock proteins, are expressed occasionally on the cell surface (Multhoff & Hightower, 1996). Therefore, we established a BT-GFP/{delta} cell line expressing boAP3{delta} tagged with GFP and investigated the cellular localization of the protein.

The expression of GFP/{delta} was verified by immunoprecipitation of BT-GFP/{delta} cells (Fig. 3) and flow cytometry (data not shown). Lysates of BT and BT-GFP/{delta} cells metabolically labelled with [35S]methionine and [35S]cysteine were immunoprecipitated with the anti-GFP antibody. As a control, the anti-bovine MHC class I antibody was used because MHC class I molecules are expressed in a wide range of cells. The anti-MHC class I antibody precipitated a protein with the expected molecular mass (about 45 kDa) from both parental and transfected cells (Fig. 3, lanes 2 and 5). In contrast, the anti-GFP antibody precipitated two major proteins of about 190 and 100 kDa, in addition to a minor 50 kDa protein only from BT-GFP/{delta} cells but not from BT cells (Fig. 3, lane 6). A Western blot analysis of an unlabelled BT-GFP/{delta} cell lysate also detected an identical major 190 kDa band in addition to several bands ranging from 70 to 160 kDa with the anti-{delta} subunit antibody (see Fig. 5, lane 2). The molecular mass of GFP/{delta} calculated by the amino acid content was 163 kDa from the sum of the 136 kDa boAP3{delta} and the 27 kDa GFP but hAP3{delta} showed an apparent molecular mass of 160 kDa, despite its calculated molecular mass being between 125 and 130 kDa (Ooi et al., 1997; Simpson et al., 1997). Therefore, we speculate that the 190 kDa band would be an entire GFP/{delta} protein and the other smaller bands might be immature or degradation products of GFP/{delta}.



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Fig. 3. Expression of GFP/{delta} in BT-GFP/{delta} cells. BT and BT-GFP/{delta} cells were metabolically labelled with [35S]methionine and [35S]cysteine and lysed. Labelled proteins were immunoprecipitated with the anti-MHC class I antibody (lanes 2 and 5), anti-GFP antibody (lanes 3 and 6) or without antibody (lanes 1 and 4) and separated by SDS-PAGE under reducing conditions on a 7 % gel. Arrows show the proteins precipitated with the anti-GFP antibody. The open arrowhead shows the protein precipitated with the anti-MHC class I antibody.

 


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Fig. 5. Coprecipitation of the bovine AP-3 subunits with GFP/{delta}. BT and BT-GFP/{delta} cells were lysed with digitonin lysis buffer. Cell lysates were immunoprecipitated with the anti-GFP antibody and separated by SDS-PAGE on a 6·5 % (lanes 1–4) or 12 % (lanes 5–8) gel. Immunoprecipitated proteins were transferred onto PVDF membranes and detected by immunoblotting with antibodies to either of the {delta}, {beta}3, µ3 or {sigma}3 subunits of AP-3. The membranes used for the anti-{delta} or anti-{sigma}3 antibodies were re-used for the anti-{beta}3 or anti-µ3 antibodies after stripping of the first probed antibodies. Arrows in the blots with the anti-{beta}3, µ3 and {sigma}3 antibodies show the proteins with sizes equivalent to the respective human subunits. The arrow in the blot with the anti-{delta} antibodies indicates the speculated entire GFP/{delta} protein (see text). Open arrowheads are probably the anti-GFP antibodies cross-reacting with the HRP-labelled anti-rabbit IgG.

 
Cellular localization of GFP/{delta} in BT-GFP/{delta} cells was observed using a fluorescence microscope (Fig. 4). Granular fluorescence was observed in the cytoplasm but not on the cell surface (Fig. 4C). This pattern of localization was similar to that of endogenous AP-3 in bovine MDBK cells (Simpson et al., 1997). On the other hand, in BT-GFP cells expressing GFP alone, green fluorescence was located preferentially in the nucleus and diffusely in the cytoplasm, as expected (Fig. 4A).



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Fig. 4. Cellular localization of GFP/{delta}. BT-GFP (A, B) and BT-GFP/{delta} (C, D) cells were cultured in an 8-well chamber slide, fixed with 4 % paraformaldehyde and observed for GFP-derived fluorescence under a fluorescence microscope (A, C). The identical fields were shown by differential interference microscopy (B, D). Arrows indicate localization of GFP/{delta} in the BT-GFP/{delta} cytoplasm. Bar, 10 µm.

 
Inclusion of GFP-boAP3{delta} within AP-3
To investigate whether the protein encoded by the boAP3{delta}1 cDNA is the bovine AP3{delta} itself, we tested the possible incorporation of the constructed GFP/{delta} protein into the AP-3 of bovine cells. Lysates of BT and BT-GFP/{delta} cells were prepared with digitonin buffer, which generally leaves protein complexes intact, and were immunoprecipitated with the mouse anti-GFP antibody. The precipitated protein complexes were washed extensively, electrophoresed under nonreducing conditions and blotted onto membranes. The membranes were then probed with each of the rabbit polyclonal antibodies to the {delta}, {beta}3, µ3 or {sigma}3 subunits of the human or rat AP-3 (Simpson et al., 1996, 1997). These antibodies reacted with the subunits of AP-3 but did not cross react with the components of AP-1, such as {gamma}, {beta}1, µ1 and {sigma}1 (Simpson et al., 1997). The anti-{delta} antibody detected a 190 kDa protein and several proteins ranging from 70 to 160 kDa (Fig. 5, lane 2). The anti-{beta}3, -µ3 and -{sigma}3 antibodies reacted with a protein of approximately 140 kDa (Fig. 5, lane 4), a 45 kDa protein (lane 6) and a 20–23 kDa protein (lane 8), respectively, all of which were the equivalent sizes of the human proteins (Ooi et al., 1997). A Western blot experiment after fractionation under reducing conditions showed the same pattern of protein bands as the experiment under nonreducing conditions (data not shown). Strong signals of about 30 kDa shown on the blots with anti-µ3 and anti-{sigma}3 antibodies (Fig. 5, lanes 5–8) were also reactive with the HRP-labelled anti-mouse IgG antibody (data not shown), indicating a cross reaction of the mouse anti-GFP antibody used for the first immunoprecipitation to the HRP-labelled anti-rabbit IgG antibody used for immunoblots. Thus, coprecipitation of GFP/{delta} with the other subunits of AP-3 indicated the inclusion of GFP/{delta} in bovine AP-3.

Effect of boAP3{delta} expression on susceptibility of cells to BLV infection
As the transfection of mouse NIH 3T3 and human Hep-2 cells with the bovine BLVRcp1 increased the susceptibility of cells to infection by the recombinant pseudotype BLV carrying the lacZ gene by about 3- to 100-fold (Ban et al., 1993), we evaluated the effects of our boAP3{delta} upon BLV infection. Because, despite our repeated attempts, we could not obtain a stable transformant of NIH 3T3 cells expressing boAP3{delta}, we performed transient transfections of NIH 3T3 cells with pCMV-{delta}, pCMV-BLVR or pCMV-GFP/{delta} and infections with a recombinant BLV-neo pseudotype virus carrying a neomycin-resistant gene (Derse & Martarano, 1990). G418-resistant colonies were counted 10–16 days later (Table 1). Neither of the NIH 3T3-{delta} cells expressing boAP3{delta} and NIH 3T3-GFP/{delta} cells expressing GFP/{delta} showed increased susceptibility to BLV infection compared to the parental NIH 3T3 cells. In spite of a previous report, the expression of BLVRcp in NIH 3T3 cells had no effect on the susceptibility of the cells to BLV infection. BLV is known to infect cells of various animal species. In our infectivity assay, two bovine cells (MDBK and BT) and CC81 cells were highly susceptible, whereas the BLV-Bat2cl1 cells persistently infected with BLV produced very few colonies, probably via a receptor interference mechanism (Table 1).


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Table 1. G418-resistant colonies after infection of cells with recombinant BLV

The indicated cells were infected by the BLV-neo virus (a culture supernatant from BLV-Bat2cl1 cells stably transfected with pBLV-SVNEO). At 2 days after infection, the culture medium was replaced with medium containing G418 and the subsequent G418-resistant colonies were counted. Data are expressed as the mean±SD of five experiments.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We cloned the almost full-length cDNA of the bovine boAP3{delta} gene, whose nucleotide sequences are collinearly aligned with that of the bovine cDNA BLVRcp of the BLVR candidate gene (Ban et al., 1993, 1994). It is apparent that the boAP3{delta} cDNAs were derived from a bovine AP3{delta} gene or its closely related gene because of high amino acid identities with the human (Ooi et al., 1997; Simpson et al., 1997) and probably mouse (Suzuki & Ikeda, 1998) AP3{delta} genes. In addition, GFP/{delta} was incorporated into AP-3 in bovine cells (Fig. 5). We also isolated the three cDNAs of the 3'-half of the boAP3{delta} gene from bovine lymph node and spleen, which are target organs of BLV infection, and MDBK cells from which the candidate gene was cloned originally. Their nucleotide sequences were almost identical with that of the boAP3{delta} cDNA but clearly differed from that of the BLVR candidate gene.

The major difference between boAP3{delta} and BLVRcp is their predicted proteins. The BLVR candidate gene encodes a protein with a transmembrane domain (Ban et al., 1993, 1994), whereas boAP3{delta} encodes a protein with no obvious hydrophobic region, as in the case of hAP3{delta} and its probable mouse homologue (Ooi et al., 1997; Simpson et al., 1997; Suzuki & Ikeda, 1998). Little is known about the physiological function, biochemical properties or cellular localization of the BLVR candidate gene product. When the nucleotide sequences are compared, the identities are high in both the protein-encoding region (99·6 %) and the 3' noncoding region (95·7 %) of boAP3{delta}. No large sequence gap is observed. Instead, many small insertions and deletions scattered at various positions could cause the several crucial differences in the proteins. First, the portion (aa 906–964) of boAP3{delta} representing about 10 % of the overlapping region lacks identity with BLVRcp. This appears to be due to the one base insertion and the one base deletion (Fig. 2A, B). Secondly, the stop codon of the boAP3{delta} ORF appears to be skipped in BLVRcp by a frameshift caused by a one base insertion at the position 13 bases upstream from the stop codon (Fig. 2B). Lastly, the BLVRcp ORF extends 836 bp from the 3' end of the boAP3{delta} ORF. This can be explained also by many small insertions and deletions leading to skips of the many stop codons lying in the boAP3{delta} 3' noncoding region (Fig. 2B).

The origin of the BLVRcp cDNA is unknown, although the close relationship between BLVRcp and AP3{delta} is indicated clearly by the nucleotide sequence identities even in the noncoding region, as described already. A few possibilities can be considered, such as cloning artefacts, the allelic variant or an unidentified AP3{delta}-related gene. Cloning artefacts are probable because no other gene encoding the BLVRcp-like protein has ever been identified, either by others or by us. The three BLVRcp cDNA clones in the original study (Ban et al., 1993) might be amplified from one clone because they have identical inserts. A variant or mutant allele at the bovine AP3{delta} locus is possible and it might be unique to the MDBK cells or BLV-permissive cells in the animal. However, we could not detect BLVRcp-like cDNA in the MDBK cells, lymph node or spleen even if we used PCR primers that should amplify both cDNAs. The existence of an unidentified AP3{delta}-related gene cannot be ruled out. The adaptor subunit gene family and the related gene family are thought to be derived from a common ancestral gene (Boehm & Bonifacino, 2001; Schledzewski et al., 1999). Considerable variations have been found within these gene families, such as naturally occurring mutations, pseudogenes, alternatively spliced mRNAs and isoforms encoded by distinct genes (Boehm & Bonifacino, 2001). In the AP3{delta} gene family, the deletion mutant gene mocha was found in mice (Kantheti et al., 1998) and two AP3{delta} cDNAs with an internal deletion or insertion were reported in humans (Ooi et al., 1997; Simpson et al., 1997). However, our previous Southern blot hybridization of bovine DNA with a BLVRcp probe did not positively support the existence of an additional AP3{delta}-related locus in the bovine genome (Suzuki & Ikeda, 1998). Nevertheless, the cloning and analysis of the respective chromosomal genes should clarify this point.

We could not establish any stable transformant of AP3{delta}-expressing NIH 3T3 cells, although no obvious cell damage was observed several days after transfection if the cells were cultured without antibiotic selection; the reason for this is unknown. In our transient transfection experiments, neither the AP3{delta} cDNA nor the BLVRcp cDNA conferred susceptibility to the BLV-neo virus in NIH 3T3 cells, in contrast to the successful induction of BLV-susceptibility in stable and transient transformants of the NIH 3T3 cells via the introduction of a BLVRcp expression vector (Ban et al., 1993). We do not know the reason for this discrepancy. Differences in the many experimental materials and methods, such as expression vectors, length and sequence of the ORFs inserted into vectors and infected viruses, are noted. More studies are required to reevaluate the significance and generalization of the BLV receptor candidate gene.


   ACKNOWLEDGEMENTS
 
We thank Dr Richard Kettmann for providing the plasmid BLVRcp1, Dr David Derse for the plasmid pBLV-SVNEO, Dr Michiharu Sakurai for the bovine cDNA library, Dr Jan Naessens for the monoclonal antibody clone IL-A88 and Dr Margaret S. Robinson for the antibodies against AP-3 subunits. We thank Dr Shozo Arai and Dr Manabu Yamada for their technical support. This study was supported in part by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 8 August 2002; accepted 14 January 2003.



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