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
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ABSTRACT |
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INTRODUCTION |
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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
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
/
/
/
,
, µ and
gene families, respectively, and AP-3 has the
,
3, µ3 and
3 subunits (Boehm & Bonifacino, 2002
; Robinson & Bonifacino, 2001
). Humans and mice appear to carry only one
gene expressed ubiquitously (Boehm & Bonifacino, 2001
). Although BLVRcp is clearly related to the
/
/
/
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
homologue (boAP3
) and if not, how BLVRcp was different from the AP3
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.
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METHODS |
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Cloning of boAP3 cDNAs.
A phage library of bovine brain cDNA (
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 EcoRIHindIII fragment, nt 5996, 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 510 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 1883) (Suzuki & Ikeda, 1998
). Two phage clones, which produced the largest PCR fragments, were isolated and their insert cDNAs were excised as plasmid DNAs (pboAP3
1 and pboAP3
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
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 23514298 of boAP31) 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
1 and BLVRcp1 sequences. A 1·8 kb region of these PCR products (nt 23904198) was sequenced directly (Hokkaido System Science).
Construction and transfection of expression plasmids.
The boAP3 expression plasmid pCMV-
and the BLVRcp expression plasmid pCMV-BLVR were constructed by inserting a 4·7 kb NotI fragment of pboAP3
1 or a 2·3-kb EcoRI fragment of pBLVRcp1 into the NotI site of a derivative of the pCMV-
expression vector (Clontech Laboratories) in which the
-galactosidase gene of the original pCMV-
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 Bsp120INotI fragment encoding EGFP (enhanced GFP variants) derived from pEGFP-1 (Clontech Laboratories) into the NotI site of the pCMV-
derivative. pCMV-GFP/
, the expression plasmid of the GFP-boAP3
fusion protein (GFP/
), was generated by ligation of two blunt-ended fragments, a 4 kb XhoIAvrII fragment from pboAP3
1 (nt 74021) and a BsrGINotI fragment from pCMV-GFP, so that the entire coding region of the boAP3
1 gene was placed at the 3' end of the EGFP gene of pCMV-GFP.
The boAP3, BLVRcp, GFP and GFP/
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/
in transfected cells was analysed by flow cytometry. The expressions of boAP3
, BLVRcp and GFP/
were verified by RT-PCR 24 h after transfection with boAP3
1- and BLVRcp1-specific primers; the transduced boAP3
protein could not be distinguished from the endogenous mouse AP3
by the anti-
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/ expression plasmid (BT-GFP/
) 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 GSepharose 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 GSepharose (control) or protein GSepharose 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/ and pSV2-hph, and selection of the transformed cells with medium containing 100 µg hygromycin B ml-1, the resulting 1020 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/
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/ 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 GSepharose 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-
or anti-
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-
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-, pCMV-BLVR or pCMV-GFP/
. 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 1016 days of culture. Expressions of transfected genes at the time of the BLV-neo infection were verified by RT-PCR.
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RESULTS |
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boAP31 cDNA encoded a protein of 1207 aa that shared many characteristics with human AP3
(hAP3
) (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
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
clones and 88·3 % identity with mBLVR (Suzuki & Ikeda, 1998
). A hydrophobicity profile of the encoded protein also resembled those of hAP3
and mBLVR (data not shown). Therefore, the cloned cDNAs appeared to encode a bovine homologue of the
subunit of AP-3.
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Expression and localization of the GFP-boAP3 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/
cell line expressing boAP3
tagged with GFP and investigated the cellular localization of the protein.
The expression of GFP/ was verified by immunoprecipitation of BT-GFP/
cells (Fig. 3
) and flow cytometry (data not shown). Lysates of BT and BT-GFP/
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/
cells but not from BT cells (Fig. 3
, lane 6). A Western blot analysis of an unlabelled BT-GFP/
cell lysate also detected an identical major 190 kDa band in addition to several bands ranging from 70 to 160 kDa with the anti-
subunit antibody (see Fig. 5
, lane 2). The molecular mass of GFP/
calculated by the amino acid content was 163 kDa from the sum of the 136 kDa boAP3
and the 27 kDa GFP but hAP3
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/
protein and the other smaller bands might be immature or degradation products of GFP/
.
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Effect of boAP3 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
upon BLV infection. Because, despite our repeated attempts, we could not obtain a stable transformant of NIH 3T3 cells expressing boAP3
, we performed transient transfections of NIH 3T3 cells with pCMV-
, pCMV-BLVR or pCMV-GFP/
and infections with a recombinant BLV-neo pseudotype virus carrying a neomycin-resistant gene (Derse & Martarano, 1990
). G418-resistant colonies were counted 1016 days later (Table 1
). Neither of the NIH 3T3-
cells expressing boAP3
and NIH 3T3-GFP/
cells expressing GFP/
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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DISCUSSION |
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The major difference between boAP3 and BLVRcp is their predicted proteins. The BLVR candidate gene encodes a protein with a transmembrane domain (Ban et al., 1993
, 1994
), whereas boAP3
encodes a protein with no obvious hydrophobic region, as in the case of hAP3
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
. 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 906964) of boAP3
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
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
ORF. This can be explained also by many small insertions and deletions leading to skips of the many stop codons lying in the boAP3
3' noncoding region (Fig. 2B
).
The origin of the BLVRcp cDNA is unknown, although the close relationship between BLVRcp and AP3 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
-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
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
-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
gene family, the deletion mutant gene mocha was found in mice (Kantheti et al., 1998
) and two AP3
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
-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-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
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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 August 2002;
accepted 14 January 2003.
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