1 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
2 Gulbenkian Institute of Science, Oeiras, Portugal
3 Department of Biochemistry and Molecular Immunology, University of Bielefeld, D-33615 Bielefeld, Germany
Correspondence
Linda Dixon
linda.dixon{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Department of Cellular Biochemistry, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.
Present address: Oxford Biomedica, Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford OX4 4GA, UK.
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INTRODUCTION |
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The EP402R gene encodes a protein with similarity to the T-cell adhesion molecule CD2, termed CD2v. CD2v contains a signal peptide, a predicted transmembrane domain and a 147 amino acid cytoplasmic tail. CD2v causes erythrocyte haemadsorption around cells infected with certain ASFV strains and the adhesion of virions to erythrocytes, linked to ASFV dissemination within the host (Rodriguez et al., 1993; Borca et al., 1994
). ASFV infection of cultured peripheral blood mononuclear cells reduces the mitogen-dependent proliferation of lymphocytes within this culture, which do not become infected. Deletion of the gene encoding CD2v abrogates this effect (Borca et al., 1998
).
Cellular CD2 participates in many inter- and intracellular signalling events (Selvaraj et al., 1987; Bierer et al., 1988
). Proline-rich regions within its cytoplasmic tail interact with the adaptor protein CD2AP, which is involved in receptor patterning, cytoskeletal polarization, integrin-linked signalling complex formation and dynamic actin organization (Dustin et al., 1998
; Kirsch et al., 1999
).
SH3 domains occur in different types of protein including enzymes, proteins associated with the cytoskeleton and adaptor proteins. Their interactions with proline-rich motifs regulate many functions including enzyme activity, ligand targeting and complex nucleation (reviewed in Pawson & Schlessinger, 1993; Pawson, 1995
; Mayer & Gupta, 1998
).
In this report we demonstrate that CD2v binds to the adaptor protein SH3P7 (also named mAbp1 or HIP55). SH3P7 is the mammalian homologue of the yeast actin-binding protein Abp1. SH3P7 contains an SH3 domain, an actin depolymerization factor homology (ADF-H) domain (Lappalainen et al., 1998) and two tyrosines, which are phosphorylated following T- and B-cell receptor stimulation (Larbolette et al., 1999
; Kessels et al., 2000
, 2001
). SH3P7 is linked to endocytosis, vesicle trafficking through the Golgi and signal transduction (Warren et al., 2002
; Fucini et al., 2002
; Mise-Omata et al., 2003
; J. Wienands and others, unpublished results). We have shown that CD2v and SH3P7 co-localize around the virus factories of infected cells, in perinuclear regions derived from the Golgi network. The implications of these findings on the functions of SH3P7 and CD2v during ASFV infection are discussed.
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METHODS |
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Yeast strain Y190 was doubly transformed with pGBT9CD2vct and a porcine macrophage cDNA library in the pACT2 vector (Clontech) (Miskin et al., 1998). Clones expressing interacting proteins were selected by HIS3 and lacZ reporter gene assays, by growth in the absence of histidine and by filter lift assays for
-galactosidase expression. Colonies were transferred to nitrocellulose membranes and frozen in liquid nitrogen. The nitrocellulose was then placed on filter paper soaked in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 0·27 %
-mercaptoethanol, 1 mg X-Gal ml-1) and incubated at 30 °C. Positive colonies became blue. Plasmids were isolated from positive colonies and retransformed into yeast strain Y190 with pGBT9CD2vct or a control plasmid of the ASFV A238L gene in pGBT9 to confirm interactions.
Sequence analysis.
Sequencing was carried out using a Pharmacia Biotech ALFexpress automatic sequencer, according to the manufacturer's instructions.
PCR.
RNA was purified from tissues ground under liquid nitrogen using Trizol (Sigma), and poly(A)+ RNA was extracted using the oligotex mRNA midi kit (Qiagen). RT-PCR was carried out using RT-PCR beads (Pharmacia) according to the manufacturer's instructions. Primers amplified either 270 bp of the unique sequence in clones SH3P7ii and SH3P7iii (primers 5'-GTTCCCTGTGGCGGGGCCAGGCTGTG-3' and 5'-GCCCTGGGTAGTGGTCAACGTGTGCC-3') or the C-terminal 400 bp of the SH3P7i clone (primers 5'-GGCAAGCTGAGGAGCCCCTCC-3' and 5'-TTACTCAATGAGCTCCACGTAGTT-3').
The Gene Racer Kit (Clontech) was used to amplify the 5' end of the porcine SH3P7 gene using a primer from within the SH3P7 clone. A PCR product of 900 bp was obtained and cloned into the pGEMT vector (Promega).
In vitro binding studies.
The complete predicted cytoplasmic tail of CD2v from the Malawi LIL20/1 isolate encoding amino acids 221375 was amplified from plasmid pGBT9CD2vct using the primer pair 5'-CGGGATCCCGAAAAAGAAAAAAACATGTTGAA-3' and 5'-CCGGAATTCTTAAATAATTCTATCTACGTG-3' containing BamHI and EcoRI sites and ligated into these sites in the pET21a(+) vector (Novagen) to give plasmid T7CD2vct. Expression of the T7-tagged protein in the BL21(DE3) strain of E. coli was induced with IPTG at 30 °C for 2 h. The protein was purified on T7-antibody agarose (Novagen), according to the manufacturer's instructions. The SH3P7ii insert was amplified from the pACT2SH3P7ii library clone using the primer pair 5'-CGGGATCCCGGCAAGCTGAGGAGCCCC-3' and 5'-CGGAATTCGTCTTTACTCAATGAGCTCCAC-3' containing BamHI and EcoRI sites and ligated into these sites in the pGEX2TK vector to give plasmid GSTSH3P7ii. The full-length mouse SH3P7 cDNA was cloned in the pRP261 vector. These two SH3P7 proteins were expressed as glutathione S-transferase (GST) fusions in the BL21(DE3) strain of E. coli, at 30 °C, following induction with IPTG for 2 h. The fusion proteins were purified and eluted from glutathioneSepharose (Pharmacia), according to the manufacturer's instructions.
The GST fusion proteins or GST, immobilized on glutathioneSepharose, were incubated with purified T7CD2vct for 2 h at 4 °C in 0·025 % NP-40 in PBS, then washed three times with 0·05 % deoxycholate, 0·5 % NP-40, 150 mM NaCl, 50 mM Tris/HCl, pH 7·5. Bound proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and detected by Western blotting using a mouse monoclonal primary antibody to the T7 epitope (Novagen) diluted 1 : 10 000, followed by a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary antibody diluted 1 : 2000.
Cell cultures.
Vero cells were cultured in Dulbecco's modified Eagle's medium containing 10 % FBS.
Immunofluorescence.
A fragment containing the CD2v gene from the Malawi LIL20/1 isolate, with its stop codon removed and a 350 bp upstream sequence containing the promoter region, was amplified by PCR from clone LMw8 (Dixon, 1988). Primers used were 5'-ATAAAGCTTGGGATCATTATATGACATGTAAC-3' (forward) and 5'-ATAGGATCCAATAATTCTATCTACGTGAATAAGCG-3' (reverse). This fragment was cloned into the HindIII and BamHI sites in the pcDNA3 vector (Clontech). A double-stranded oligonucleotide encoding the influenza virus haemagglutinin epitope tag (HA) and including a 3' stop codon, with upstream BamHI and downstream XbaI sites, was cloned downstream and in frame with the CD2v gene. The oligonucleotides were 5'-ATAGGATCCATGGCTTACCCATACGACGTACCAGACTACGCATCACTATGATCTAGAATA-3' and 5'-TATTCTAGATCAGTTTAGGACTTCTTCTGATATTAGTTTTTGTTCCATGGATCCTAT-3'. This plasmid was called CD2vHA.
A plasmid containing the mouse SH3P7 gene as a 3' fusion with the enhanced green fluorescent protein (EGFP) gene has previously been described (Larbolette et al., 1999). This gene fusion was amplified by PCR incorporating BamHI and XbaI sites at the 5' and 3' ends and cloned in the pGEM-T vector. A 47 bp double-stranded DNA oligonucleotide containing the ASFV VP72 promoter was inserted upstream as a BamHISph I fragment to give plasmid VP72GFPSH3P7.
These constructs were transformed into Vero cells using Lipofectin (Invitrogen) according to the manufacturer's instructions and cells were infected with the BA71V tissue-culture adapted strain of ASFV. At various times after infection, cells were fixed with 3 % paraformaldehyde in PBS and permeabilized with 0·1 % Triton X-100 in PBS for 15 min. CD2v was detected using a rabbit antibody against the HA epitope (Santa Cruz) diluted 1 : 200, followed by a goat anti-rabbit Alexa Fluor 568 secondary antibody (Molecular Probes) diluted 1 : 200. The virus capsid protein VP72 was detected using mouse monoclonal antibody 4H3 (Cobbold et al., 1996) at a concentration of 1 : 10. Anti-mouse secondary antibodies labelled with Alexa Fluor 488 or 633 were used at a concentration of 1 : 200 to detect bound antibodies. Cell nuclei and virus factories were stained with 4,6-diamidino-2-phenylindole (DAPI) at 0·4 µg ml-1. Cells were imaged on a Leica TCS NT confocal microscope. Labelling controls included omission of primary antibody and adjustment of confocal imaging parameters to avoid all risk of cross talk between fluorophores.
Analysis of protein glycosylation.
Vero cells in six-well dishes were infected with the BA71V ASFV isolate and transfected with CD2vHA using Lipofectin (Invitrogen). Cells were incubated with or without tunicamycin (10 µg ml-1) for 16 h and harvested in lysis buffer containing 0·2 % NP-40 (500 µl per well). Cell extracts from three wells were immunoprecipitated using 30 µl of a 50 % slurry of anti-HA beads (Roche) and the beads were washed three times in PBS. For endoglycosidase F (endo-F) digestions, immunoprecipitates were washed in 0·1 M Tris/HCl, pH 7·5, then resuspended in 50 µl 0·1 M Tris/HCl, pH 7·5, and 1 unit endo-F added. After overnight incubation at 37 °C, 50 µl 2x SDS sample buffer was added and samples were separated by SDS-PAGE followed by immunoblotting using an HRP-conjugated anti-HA antibody (1 : 800). Endoglycosidase H (endo-H) digestions were carried out similarly except that 0·1 M sodium citrate, pH 5·5, was used with 0·005 units of endo-H.
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RESULTS |
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CD2v localizes near virus factories in ASFV-infected cells
To follow CD2v localization during ASFV infection, CD2vHA was expressed in ASFV-infected Vero cells. Cells were fixed at 4, 12, 16 and 20 h post-infection and CD2vHA (shown in red) was detected by indirect immunofluorescence using a rat monoclonal antibody against the HA epitope and a fluorescent-labelled goat anti-rat secondary antibody. CD2v is expressed late in infection and therefore no expression could be detected at 4 h. From 12 h post-infection, a punctate pattern was detectable around the virus factories (Fig. 2D and F).
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SH3P7 co-localizes with actin filaments at membrane ruffles and in the periplasm (Larbolette et al., 1999; Kessels et al., 2000
). SH3P7 also associates with the Golgi network (Warren et al., 2002
; Fucini et al., 2002
). To determine whether CD2vHA was present in membrane areas derived from the Golgi, we infected cells with the ASFV BA71V isolate and transfected them with the CD2vHA plasmid. Cells were fixed 16 h post-infection and stained with an antibody against a Golgi protein (Harrison-Lavoie et al., 1993
) (Fig. 2H
, green) and with anti-HA antibody to detect CD2vHA (Fig. 2G
, red). Virus factories were stained with DAPI (blue) and with a monoclonal antibody against the virus capsid protein VP72 (Fig. 2I
, white). The anti-Golgi antibody stained the areas around the virus factories where CD2vHA was located. CD2vHA did not co-localize with this Golgi protein (Fig. 2I
) suggesting that the proteins are in the same structures but do not interact. The Golgi distribution differed in ASFV-infected cells compared with uninfected cells, as previously described (McCrossan et al., 2001
) (Fig. 2H
). Uninfected cells did not express CD2vHA.
Sequence analysis and tissue-specific expression of porcine SH3P7
The inserts in clones SH3P7i, SH3P7ii and SH3P7iii isolated in the yeast two-hybrid screen were sequenced (Fig. 5). The SH3P7i clone encoded 93 amino acids of a protein with 90 % nucleotide identity and 83 % amino acid identity to the human SH3P7 sequence. The SH3 domain of SH3P7i comprised 59 amino acids with 98 % amino acid identity to the human SH3P7 SH3 domain. A tyrosine residue, which is phosphorylated in the mouse protein and also conserved in human, rat and mouse SH3P7 sequences, was present in SH3P7i.
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We used RACE PCR to clone the 5' end of the porcine SH3P7 gene. RNA from porcine macrophages was reverse transcribed using primers that read from within the sequence from clones SH3P7ii and -iii towards the 5' end of the gene. The cDNA was amplified by PCR using this primer and a primer that binds to the cap structure. The resulting fragment was cloned and sequenced. The predicted sequence of porcine SH3P7 was 430 amino acids long and shared 88 % and 83 % amino acid identity with the human and mouse proteins, respectively. The most variable regions of the protein were between residues 220 and 253 and residues 297 and 369 outside of the ADF-H and SH3 domains. We failed to obtain a full-length clone for the variant form of SH3P7 encoded by clones SH3P7ii and -iii.
To investigate porcine SH3P7 expression, we designed primers to amplify either the unique region in clones SH3P7ii and -iii or a conserved region of the gene from SH3P7 database sequences by RT-PCR. Fragments of the expected size (230 bp for the variant sequence and
450 bp for the conserved sequence) were obtained from mRNA from a wide variety of tissues including brain, heart, muscle, liver and bone marrow (Fig. 6
). Hybridization with a probe from the SH3P7 gene confirmed that these fragments contained SH3P7 sequences (data not shown). Smaller fragments amplified in some samples did not hybridize with the probe showing that they were non-specific (data not shown). These results suggest that the wild-type and variant forms of SH3P7 are expressed in a wide variety of porcine tissues. Poly(A)-containing RNA was used in these experiments, suggesting that these are mature mRNA sequences rather than primary transcripts. Previous analysis of mouse tissues showed that SH3P7 was expressed in B cells, testis, brain, heart, lung, thymus and spleen but not muscle or ovary (Larbolette et al., 1999
). Human SH3P7 transcripts were found in all tissues tested including muscle (Ensenat et al., 1999
). Database searches identified SH3P7 cDNA sequences in many tissues including brain, mammary gland, thymus, gonad, eyeball, pituitary, bladder, bone, heart, lung and stomach.
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DISCUSSION |
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We observed co-localization of CD2v with SH3P7 around perinuclear virus factories in areas that were stained with an antibody to a Golgi protein. We also demonstrated that CD2v is partially resistant to endo-H digestion showing that it is transported beyond the endoplasmic reticulum, at least to the Golgi network. CD2v expression is required for the haemadsorption of erythrocytes around ASFV-infected cells, indicating that some CD2v is present at the cell surface. However, both our study and a previous study have shown, using immunofluorescence, that most CD2v is inside cells (Ruiz-Gonzalvo & McColl, 1993). CD2v may either be retained within membrane compartments as it is transported to the cell surface, or may be retrieved to these compartments from the cell surface.
CD2v interacts with SH3P7 via an area of proline repeats. Binding is to the SH3 domain of SH3P7, since no other region is common to both the intact SH3P7 protein and the SH3P7ii variant, which both bind CD2v in vitro. SH3 domains contain two perpendicular -sheets, forming a cleft, which the polyproline helices of their ligands fit into. The interaction is stabilized by a salt bridge at one end of the cleft between a basic residue in the ligand and an acidic residue in the SH3 domain. Ligands can bind along the cleft in either orientation: class I ligands have the basic residue at their N terminus and class II ligands at the C terminus (Yu et al., 1994
; Lim et al., 1994
; Feng et al., 1994
). SH3P7 was found in screens using class II ligands (Sparks et al., 1996a
, b
). SH3 domains usually bind only to sets of related ligands (Feng et al., 1994
; Sparks et al., 1996b
). The class II consensus sequence, from the N to the C terminus, is X3-P-p-X2-P-p-X1, where X3 and X2 are hydrophobic and X1 is the stabilizing basic residue. P represents a critical proline, which interacts with the SH3 domain, and p represents a proline residue that maintains the structure of the polyproline II helix. The repeats in the CD2v protein consist of variable numbers of repeats of PCPPPK. As this sequence is repeated, the actual binding site may consist of a greater-than-unit-length sequence such as KPCPPPK, which is closest to the class II ligand consensus. The presence of repeated SH3 binding domains increases their local concentration and may make interactions with SH3 domains more likely (Feng et al., 1994
; Sparks et al., 1996a
). The SH3 domain of yeast Abp1 binds to unusual ligands containing two positive side-chains separated by seven or eight residues including a PXXP motif (Fazi et al., 2002
).
Several short polymorphisms within SH3P7 have been described, four from the rat protein and two from the mouse protein, apparently produced by alternative splicing. An additional putative SH3 domain binding motif is present in one rat isoform, but the functions of these forms are unknown (Yamazaki et al., 2001; Kessels et al., 2001
). We identified two porcine SH3P7 homologues and showed that both are widely expressed in porcine tissues. The variant form of SH3P7 encodes the SH3 domain with an additional 237 bp and an upstream frame shift. The two phospho-tyrosine residues are not present (Larbolette et al., 1999
). A putative SH3 domain binding sequence is disrupted, but a new PXXP SH3 domain binding motif is introduced. The presence of motifs at the N terminus of this variant SH3P7, which we have not cloned and sequenced, cannot yet be determined. The function of this SH3P7 variant form is unknown, although the alterations in the variant form of domains involved in protein interactions are intriguing. A regulatory role in which the two forms of SH3P7 compete for ligand binding seems possible.
CD2v has several functions. It is required for erythrocyte haemadsorption around ASFV-infected cells and for adsorption of ASFV particles to the surface of erythrocytes. These properties may be important for virus dissemination around the host (Rodriguez et al., 1993; Borca et al., 1994
; Wardley & Wilkinson, 1977
; Thomson et al., 1979
; Borca et al., 1998
). CD2v also has an immunomodulatory role. ASFV infection of mononuclear cells greatly reduces the mitogen-dependent proliferation of uninfected lymphocytes, and expression of CD2v is required for this inhibition (Borca et al., 1998
). The interaction of CD2v with SH3P7 may be related to these effects, or to an uncharacterized function. The link between SH3P7 and JNK1 signalling could be involved in these immunomodulatory effects, via changes in transcription. Alternatively the interaction of CD2v with SH3P7 may modulate protein trafficking resulting in the inhibition of protein transport through the Golgi network to the cell surface, or modulation of endocytosis could affect the internalization of cell-surface molecules. Many virus-encoded proteins subvert host defence systems by altering receptor trafficking. Alterations in protein trafficking could alter signals delivered by infected macrophages to bystander lymphocytes. Actin filaments and the Golgi are rearranged in ASFV-infected cells (Carvalho et al., 1988
; Ferreira, 1996
; Esteves et al., 1986
; McCrossan et al., 2001
) and SH3P7 could be involved in these rearrangements as it is linked to dynamic actin and to Golgi vesicles in other cell types. However, CD2v is non-essential for virus replication and any effects of its interaction with SH3P7 must therefore also not be required. The functions of SH3P7 and CD2v in ASFV-infected macrophages remain to be determined, but may provide insights into many aspects of ASFV replication and macrophage function.
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ACKNOWLEDGEMENTS |
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Received 18 June 2003;
accepted 22 September 2003.