African Swine Fever Virus Encodes a CD2 Homolog Responsible for the Adhesion of Erythrocytes to Infected Cells
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JOURNAL OF VIROLOGY, Sept. 1993, p. 5312-5320 Vol. 67, No. 9
0022-538X/93/095312-09$02.00/0
Copyright 0 1993, American Society for Microbiology
African Swine Fever Virus Encodes a CD2 Homolog
Responsible for the Adhesion of Erythrocytes to
Infected Cells
JAVIER M. RODRIGUEZ, RAFAEL J. YANEZ, FERNANDO ALMAZAN, ELADIO VINUELA,
AND JOSE F. RODRIGUEZ*
Centro de Biologia Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Cientificas- Universidad
Aut6noma de Madrid), Facultad de Ciencias, Universidad Aut6noma, Cantoblanco, 28049 Madrid, Spain
Received 19 March 1993/Accepted 7 June 1993
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We have identified an open reading frame, EP402R, within the EcoRI E' fragment of the African swine fever
virus genome that encodes a polypeptide of 402 amino acid residues homologous to the adhesion receptor of T
cells, CD2. Transcription of EP402R takes place during the late phase of virus replication. The disruption of
EP402R, achieved through the replacement of a 354-bp-long fragment from within EP402R by the marker gene
lacZ, does not affect the virus growth rate in vitro but abrogates the ability of the virus to induce the adsorption
of pig erythrocytes to the surface of infected cells. This result demonstrates that the protein encoded by EP402R
is directly involved in the hemadsorption phenomenon induced by the infection of susceptible cells with African
swine fever virus.
African swine fever was first described by Montgomery in protein might interfere with some fundamental aspects of the
1921 (25) as a peracute infectious disease affecting the host's immune response.
domestic pigs imported to Kenya. Subsequent studies dem-
onstrated that the etiological agent of African swine fever is
a large icosahedral desoxyvirus, known as African swine MATERIALS AND METHODS
fever virus (ASFV), which is currently considered to be the
only known member of an as yet unnamed family of animal Cells and viruses. Vero cells, obtained from the American
viruses. ASFV infects soft ticks of the genus Ornithodoros Type Culture Collection, and alveolar pig macrophages,
and different species of swine (reviewed in references 10 and obtained as described previously (9), were grown in Dulbec-
37). In its natural swine host, the warthog (Phacochoerus co's modified Eagle's medium (DMEM) containing 10% fetal
aethiopicus), ASFV causes a mild disease with few clinical bovine serum. ASFV strain BA71V was propagated and
signs. However, the presence of infectious virus particles in titrated as described previously (17). Virus inoculations of
the blood of animals long after their apparent recovery cell cultures were carried out at 4°C for 2 h. For this, virus
suggests that ASFV is able to establish a persistent infection stocks were diluted in DMEM lacking sodium bicarbonate
in this host. By contrast, the infection of domestic pigs (Sus and supplemented with 25 mM N-[2-hydroxyethyl]pipera-
scropha) with unattenuated ASFV strains leads to a devas- zine-N'-[2-ethanesulfonic acid] (HEPES; pH 7.0). After in-
tating disease with a very high mortality rate (reviewed in oculation, the cell cultures were washed twice with normal
reference 39). These dramatic differences in the outcome of DMEM containing 2% fetal bovine serum and then incu-
the infection indicate that the virus-host interaction is de- bated at 37°C under 7% CO2.
stroyed when the natural host is replaced by a different Preparation of RNA. RNA was obtained as described
species of swine. previously (30) from Vero cell cultures infected with 25 PFU
The failure of conventional approaches to generate an per cell. After removal of the medium, cells were scraped,
ASFV vaccine has led to speculation that the virus possesses resuspended in chilled phosphate-buffered saline, and spun
mechanisms to avoid the host's immune surveillance similar at low speed for 5 min. The cell pellets were resuspended in
to those found in other virus systems (reviewed in references lysis buffer (4 M guanidinium thiocyanate, 0.1 M Tris-HCl
19 and 26). In an attempt to identify ASFV genes that might [pH 7.5], 1% 3-mercaptoethanol), layered on top of a cush-
be involved in such mechanisms, we have undertaken the ion of 5.7 M cesium chloride containing 10 mM EDTA, and
analysis of the complete nucleotide sequence of the virus spun at 300,000 x g for 18 h. The RNA was resuspended in
genome. In this report, we describe an ASFV gene coding H20 pretreated with 0.2% diethyl pyrocarbonate containing
1% sodium dodecyl sulfate, ethanol precipitated twice, re-
for a protein homologous to the T-cell adhesion receptor
CD2 and demonstrate that this virus-encoded CD2 homolog suspended in diethyl pyrocarbonate-treated H20 at a con-
is directly responsible for the hemadsorption phenomenon centration of 2 mg/ml, and stored at -70°C.
induced by the infection of susceptible cells with ASFV (24). Purification and analysis of DNA. Virus DNA was purified
In view of the important role played by CD2 molecules in as described previously (18). Purification of plasmid DNA,
both T-cell and natural killer cell activities (reviewed in endonuclease restriction analysis, agarose gel electrophore-
references 3 and 36), it seems possible that this ASFV sis, DNA cloning, DNA hybridizations, and preparation of
radioactive probes were performed by standard protocols
(30).
DNA sequencing. The ASFV genomic EcoRI E' fragment
(Fig. 1A) was sequenced at random, using a shotgun ap-
*
Corresponding author. proach as follows. The plasmid clone pBR325-RE' (23) was
5312VOL. 67, 1993 AFRICAN SWINE FEVER VIRUS CD2 HOMOLOG 5313
A
0 SO 100 150 170 kb
I
L U,X',V,Y,Z,U',X,
F K E' M C N B G C' OTN' D S P H RQQ'E I D'
I I 111111 I I
I I II
I I I
I I I II
I
II II II 11I I"I II11
III Ta
11s 11 I I
A
I
EcoRI I I
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I I I
= EP402R
lkb
B
sExzMW5Rm5W WSTLNQTVFLNNIFTINDTYGGLFWNTYYDNNRSNFTYCG 60
IAGNYCSCCGHNISLYNTTNNCSLIIFPNNTEIFNRTYELVYLDKKINYTVKLLKSVDSP 120
TITYNCTNSLITCKNNNGTNVNIYLIINNTIVNDTNGDILNYYWNGNNNFTATCMINNTI 180
SSLNETENINCTNPILKYQNYLSTLE=X=zZ§BEZggUNXXW&SIRRKRKKHVEE 240
IESPPPSESNEEDISHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPL 300
360
SKPCPSPESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRII
C
4
3
O
2
..-1
PC -2
4-
Residue number
FIG. 1. (A) Location of the EP402R ORF within the ASFV genome. Positions and nomenclature of the EcoRI restriction fragments of the
virus genome are shown. The relative position of the EP402R ORF, represented as a solid box, within the EcoRI E' fragment is shown. The
arrow represents the position of the transcriptional initiation site of EP402R detected by primer extension, and the circles represent locations
of the runs of thymidylate residues potentially associated with the 3'-end formation of EP402R transcripts. (B) Deduced amino acid sequence
of pEP402R. Predicted leader and transmembrane domains are shown in outlined lettering, potential glycosylation sites (NXT/S except where
X is P) in the predicted extracellular domain are underlined, and the region consisting of the repeated sequence (PPPKPC) is shown in
boldface. (C) Superimposition of the hydropathy profiles of pEP402R (solid line) and mouse CD2 (dashed line). Calculations were performed
by the method of Kyte and Doolittle (22), using a window of 11 amino acid residues.5314 RODRIGUEZ ET AL. J. VIROL.
sheared by sonication and end repaired with Si nuclease and A B
Klenow polymerase, and fragments 300 to 600 bp long were _U C A 1. U C A L I)NA lADDER
cloned into SmaI-cut M13mpl0. Single-stranded DNA from
the recombinants was sequenced by the dideoxynucleotide U
-
chain termination method (31), using the M13 universal
primer. -
XS_
Plasmid construction. The deletion vectors pACD2d and
pACD2r were constructed as follows. A 3.3-kb HindIII -0 < -K 2.2 Kh
S
-
fragment obtained from plasmid pBR325-RE', containing the in* i6h
4 h__-
EP402R open reading frame (ORF), was cloned into HindIII-
cut pUC119 to generate plasmid pBA71V-CD2. A 3.3-kb
XbaI fragment containing the lacZ gene fused to the virus
promoter p72 was obtained from plasmid pINS,Bgal (29).
This fragment was treated with Klenow enzyme and cloned
into pBA71V-CD2 which had been previously cut with NdeI,
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treated with Klenow enzyme, and then digested with FIG. 2. Transcriptional analysis of EP402R. Autoradiograms of
EcoRV. Thus, two plasmids, pACD2d and pACD2r, with a RNA hybridization (A) and primer extension analysis (B) of four
354-bp NdeI-EcoRV deletion and the p72-lacZ gene inserted types of RNA, mock infected (lanes U) and ASFV virus-induced
in opposite orientations were obtained. immediately-early (lanes C), early (lanes A), and late (lanes L), are
Hemadsorption assays. Pig macrophages grown on cover- shown. In panel A, positions of the 28S and 18S rRNAs and sizes of
the most prominent early and late transcripts are shown. The
slips were infected at a multiplicity of infection of 0.5 PFU samples in panel B were electrophoresed alongside an irrelevant
per cell. After inoculation, the cultures were washed twice DNA sequencing reaction (DNA ladder) used as a size marker.
with DMEM, and then DMEM containing 10% fetal bovine Numbers at the left correspond to the sizes (bases [b]) of the
serum and supplemented with 2.5 x 106 pig erythrocytes per relevant DNA fragments.
ml was added. At different times postinfection, coverslips
were thoroughly washed with phosphate-buffered saline,
incubated for 2 min in 95% methanol at -20°C, and mounted
on microscope slides by using 40% glycerol. The cells were transcript of approximately 2.2 kb synthesized during the
observed by phase-contrast microscopy. early phase of the infection, and two mRNAs of approxi-
Nucleotide sequence accession number. The GenBank ac- mately 1.6 and 2.1 kb in the virus-induced late RNA sample.
cession number for EP402R is L16864. Primer extension products over 500 nucleotides in length
were observed in the samples corresponding to virus-in-
RESULTS duced early and immediate-early RNA (data not shown).
The sizes of these extended products indicate that they
Identification of the EP402R ORF. The nucleotide se- result from hybridization of the primer to read-through
quence of the EcoRI E' genomic fragment of the BA71V transcripts, most likely generated by the transcription of an
strain of ASFV was determined by sequencing an M13 ORF located upstream of EP402R. The extension of the
library generated by shotgun cloning of DNA fragments. hybrids formed after incubation of late RNA to the 32P-end-
One of the ORFs identified, designated EP402R (Fig. 1A), is labeled primer resulted in the generation of a cluster of
the subject of this report. EP402R consists of 1,206 nucle- radioactive bands corresponding to DNA fragments with
otides and encodes a polypeptide, designated pEP402R, of sizes ranging from 112 to 116 nucleotides as well as two faint
402 amino acid residues with a predicted molecular size of bands corresponding to shorter (105- and 106-nucleotide),
45.33 kDa (Fig. 1B). The hydropathy profile of pEP402R DNA fragments (Fig. 2B). The most prominent bands within
(Fig. 1C) resembles those of typical class III transmembrane the cluster correspond to fragments of 113 and 114 nucle-
proteins; consequently, it is predicted that this polypeptide otides, thus demonstrating the existence of a major late
contains four differentiated domains: (i) a hydrophobic transcriptional initiation site 22 to 23 nucleotides upstream of
leader of 20 amino acids in the N-terminal region of the the first nucleotide of EP402R. The shorter DNA fragments
protein; (ii) a hydrophylic region of 183 amino acid residues might reflect the existence of a weaker transcriptional initi-
with 16 putative sites for the attachment of N-linked carbo- ation site at a short distance, 6 to 7 nucleotides, from the
hydrates; (iii) a hydrophobic stretch formed by 25 residues major one or, alternatively, the formation of incomplete
that could act as a transmembrane domain; and (iv) a cDNA molecules by premature termination during reverse
C-terminal region of 174 amino acids which contains 11 transcription of the RNA templates.
imperfect direct repeats of the hexapeptide PPPKPC. The sizes of the two late transcripts detected by RNA
Transcriptional analysis of EP402R. The transcription of hybridization would correspond to those of the mRNAs
EP402R was analyzed by RNA hybridization and primer initiating immediately upstream of EP402R if, as expected,
extension, using a 32P-end-labeled oligonucleotide (5'- their 3' ends map at sequence motifs formed by runs of seven
TTAAAAATACAGT'ITGAT'ITAAAGTACTCC-3') com- consecutive thymidylate residues (7T) (Fig. 1A). The 7T
plementary to the region between nucleotides 62 and 91 of sequence motif has been recently found to be associated
the coding strand of EP402R. For these experiments, total with the 3' end formation of both early and late ASFV
RNA from mock-infected Vero cells (uninfected RNA) or mRNAs (1, 2).
virus-infected cultures maintained in the presence of either pEP402R is a homolog of the adhesion receptor CD2. A data
cycloheximide (immediate-early RNA) or arabinofuranosyl- base search carried out with the FASTA program (27) to
cytosine (early RNA) isolated 8 h postinfection, and RNA compare the amino acid sequence of pEP402R with se-
isolated at 18 h postinfection from untreated virus-infected quences in the SwissProt data bank gave very high scores
cultures (late RNA), were used. Three major virus-induced with sequences of CD2 molecules. The highest optimized
mRNA species were recognized by this probe (Fig. 2A): a FASTA score (193, corresponding to 21.3% identity and 60%VOL. 67, 1993
MWSE
RAT
HUMAN
EP402R
MOSE
RAT
EHMAN
EIP402R
MOSE
RAT
EUMAN
EP402R
1
91
GDLKIN
NRCZF1GS
MRCZFLGS
181F8
N
LTIKKPIQIISGT
LTRDDSGT
ADCEE
SREADCRDSG
e
MSFPCZFUASFLLIFNVSSKQkV I
MIIIVIrC
IRE LKTDDQ IY KVS
CSLIIFPrn1T
E rA
SIN
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GI
AFRICAN SWINE FEVER VIRUS CD2 HOMOLOG
ID=ERCTLVA
P D
GI lIDEVIERGS
* I
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N
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I
Z
TEST
KKZIA
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TLVA EFP ZMKPFLKSGARFI
QFKKETFKKDTYKFS
*
tVTVQVQa.1LLVLAI'I RF
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C
270
0
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SI~M~Y~TRKRKQR:RENDRiH
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LSQRVITHL
ii.be
5315
90
Downloaded from http://jvi.asm.org/ on January 11, 2021 by guest
271 360
--_
MWSE > KPPAM PCAA SV
RAT
HUMaN
1PH BjQASALSaQ V
TSGESSEPHVH
A Q MPA w
EP402R c-w7%3:slDTriE;E IPYSyRYOYNTPIYYMRPSTOPLNPFPL2PK3PCPPP hsRCIP C PPCR
361 427
RAT TV
EP402R P CPPP SIPLLPNIPPLSTQNISLIHVDRII
FIG. 3. Amino acid alignment of pEP402R and human, mouse, and rat CD2 molecules. The alignment was generated by using the program
PILEUP. Residues in pEP402R identical to those in any of the CD2 molecules are boxed. Circles above the sequence indicate the
CD2-conserved cysteine residues predicted to form intramolecular disulfide bonds.
conservation) was obtained with the sequence of mouse CD2 hemadsorption. Most ASFV strains are capable of inducing a
(42). These results prompted us to further investigate the phenomenon known as hemadsorption, which is character-
relationship between pEP402R and CD2 molecules. ized by the adhesion of pig erythrocytes to the surface of
CD2 molecules are classical transmembrane proteins with infected cells (24). The molecular basis of this phenomenon
a glycosylated extracellular N-terminal region of 185 to 196 has not been established, but the fact that CD2 molecules are
amino acids, which is involved in the binding of the molecule responsible for the adhesion of sheep and human erythro-
to its specific ligands, and a proline-rich cytoplasmic C cytes to the surface of human T cells (33) led us to speculate
terminus of 116 to 126 amino acid residues. The hydropathy that pEP402R might be involved in the hemadsorption in-
profile of pEP402R is strikingly similar to those of the CD2 duced by ASFV.
molecules (Fig. 1C), indicating that the topology of this To analyze this hypothesis, two ASFV recombinants,
virus-encoded protein might be similar to that of these vACD2d and vACD2r, were generated by in vivo homolo-
adhesion receptors. A multiple alignment of the amino acid
sequences of pEP402R and the three known (mouse, rat [41],
and human [34]) CD2 molecules, generated with the program TABLE 1. Statistical analysis of the similarity between pEP402R
PILEUP (13) (Fig. 3), showed that the similarity of pEP402R and CD2 moleculesa
to these molecules extends throughout the length of the virus
polypeptide. Significantly, the four conserved cysteine resi- pEP402Rb
dues in the CD2 molecules, which form two intramolecular CD2 Complete Extracellular Cytoplasmic
disulfide bonds (21), are present in almost identical positions protein region region
in pEP402R. Furthermore, with only one exception (phen-
ylalanine 178 in mouse CD2), the CD2-conserved aromatic Humanc -0.13 12.29 -0.80
residues are either perfectly conserved or replaced by an- Moused 12.15 11.14 -1.35
other aromatic residue in the pEP402R sequence. The simi- Rate 7.85 10.05 -1.43
larity of the complete amino acid sequence as well as both a The complete amino acid sequence of pEP402R and the amino acid
the extracellular and cytoplasmic domains of pEP402R with sequences of the predicted extraceliular and cytoplasmic regions of the
those of the known CD2 molecules was assessed by using protein were compared with their counterparts in CD2 molecules, using the
RDF2G program. The comparisons were performed by using a local shuffle
the RDF2G program (27). This algorithm facilitates the routine with a window of 10 amino acid residues to generate 200 shuffled
evaluation of the significance of the similarity between two proteins. Results are the optimized scores shown as the number of standard
test sequences, avoiding possible artifacts caused by the deviations from the mean score of the shuffled proteins. Values of greater than
3 are considered statistically significant.
presence of locally biased amino acid composition in the b Extracellular region is from amino acids 21 to 204; cytoplasmic region is
proteins under scrutiny. The results of this analysis (Table 1) from amino acids 229 to 402.
demonstrate that the similarity of the extracellular region of c Extracellular region is from amino acids 25 to 209; cytoplasmic region is
pEP402R to its counterparts in the three CD2 molecules is from amino acids 236 to 351.
d Extracellular region is from amino acids 23 to 202; cytoplasmic region is
statistically significant. In contrast, the scores obtained after from amino acids 230 to 344.
comparison of the cytoplasmic regions were very poor. ' Extracellular region is from amino acids 23 to 202; cytoplasmic region is
Expression of EP402R is required for the induction of from amino acids 229 to 344.5316 RODRiGUEZ ET AL. J. VIROL.
EcoRI BamHI EcoRI BamHI EcoRI
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vACD2d I I -Ar ALI -Al..l I
p72 Lac Z
EcoRI BamHI EcoRI BamHI EcoRI
. .1i?)
vACD2r I qL,%, %.V N.. V V
Lac Z p72
lKb
FIG. 4. Predicted genomic structure of the ASFV recombinants vACD2d and vACD2r. Homologous recombination between the virus
genome and the plasmid vector pACD2d or pACD2r should lead to insertion of the lacZ gene (striped box) fused to the virus promoter p72
(dotted box) into the EP402R ORF (solid box) and deletion of a 354-bp NdeI-EcoRV fragment from EP402R. Positions of relevant
endonuclease restriction sites are indicated.
gous recombination. The plasmid vectors pACD2d and lane 7) and vACD2r (Fig. 5, lane 8), respectively. These
pACD2r were constructed as described in Materials and results demonstrate that the genomic structures of the re-
Methods. These vectors were designed to facilitate the combinant viruses are as predicted (Fig. 4). To ensure that
replacement of a genomic DNA fragment of 354 bp with the EP402R was not involved in ASFV multiplication in vitro,
marker gene lacZ fused to the ASFV promoter p72 (Fig. 4). the growth rates of the parental virus and the recombinant
This would disrupt the EP402R ORF and eliminate from the viruses vACD2d and vACD2r in Vero cell or pig macrophage
virus genome most of the sequence (from Tyr-37 to Asn-158)
of the predicted extracellular domain of pEP402R. In
vACD2d, the lacZ gene is transcribed in the same direction 4 5 6 7 8 9
as EP402R, while in vACD2r, the marker gene is transcribed Kh
in the opposite direction. DNA from the parental virus,
BA71V, and the recombinant viruses, vACD2d and vACD2r,
was purified from infected cultures, digested with the restric-
tion enzyme EcoRI, and then subjected to agarose gel 8.9 _.
electrophoresis. As shown in Fig. 5, the EcoRI restriction 8.4
6.5 -
-
-
patterns of vACD2d and vACD2r are different from that of 5.3 -
BA71V. In the parental virus (Fig. 5, lane 3), the EcoRI E'
fragment has a size of 8.9 kb and comigrates with the EcoRI 35.4 -
E fragment. However, in vACD2d (Fig. 5, lane 1), the EcoRI
E' fragment is not present but there are two new fragments
of 8.4 and 3.4 kb. Similarly, with vACD2r, the EcoRI E'
fragment is absent and two new fragments of 6.5 and 5.3 kb
are seen (Fig. 5, lane 2). The origin of the fragments
identified by agarose gel electrophoresis was confirmed by
DNA hybridization using two 32P-labeled probes: (i) a 3.3-kb FIG. 5. Genomic analysis of wild-type and recombinant viruses
HindIII subfragment of the EcoRI E' fragment which spans vACD2d and vACD2r. DNA purified from cells infected with
the EP402R ORF (Fig. 4) obtained from plasmid pBR325- vACD2d (lanes 1, 4, and 7), vACD2r (lanes 2, 5, and 8), or wild-type
RE'; and (ii) a 3-kb BamHI-EcoRI fragment obtained from ASFV (lanes 3, 6, and 9) were digested with EcoRI and subjected to
plasmid pINS,gal (29) which corresponds to the lacZ gene agarose gel electrophoresis. The DNA fragments generated were
visualized under UV light after staining with ethidium bromide
(Fig. 4). As expected, the 3.3-kb HindIII probe hybridized to (lanes 1 to 3). Identically prepared gels were blotted onto nitrocel-
all of the EcoRI fragments described above (Fig. 5, lanes 4 to lulose ifiters and hybridized with a 35P-labeled probe obtained either
6), while the lacZ probe recognized EcoRI fragments of 8.4 from the EcoRI E' fragment (lanes 4 to 6) or from the lacZ gene
and 6.5 kb in the samples corresponding to vACD2d (Fig. 5, (lanes 7 to 9). The sizes of relevant DNA fragments are indicated.VOL. 67, 1993 AFRICAN SWINE FEVER VIRUS CD2 HOMOLOG 5317
cultures were compared and found to be similar (data not Ig-related domains; domain 1 (residues 23 to 121 in rat CD2)
shown). is related to the Ig V-set domains, and domain 2 (residues
The ability of the recombinant viruses vACD2d and 122 to 196 in rat CD2) belongs to the Ig C2 set (14). To
vACD2r to induce the hemadsorption phenomenon was investigate whether pEP402R could be included as a new
analyzed by using pig macrophages. While the parental virus member of the IgSF, the sequence of its extracellular region
induced the adsorption of a large number of erythrocytes to was divided into two discrete domains encompassing resi-
the surface of the infected macrophages (Fig. 6B), neither dues 21 to 114 and 115 to 191 which, according to the
vACD2d (Fig. 6C) nor vACD2r (Fig. 6D) was capable of multiple alignment (Fig. 3), should match domains 1 and 2 of
inducing the rosetting of erythrocytes. The ability of another CD2 molecules, respectively. The similarity of the pEP402R
recombinant virus, v72p-gal, which contains the lacZ gene selected domains to different Ig sequences was analyzed by
inserted into the thymidine kinase gene of the ASFV genome using the ALIGN program (12) because it is accepted that a
(29), to induce hemadsorption was also tested and found to repeated good ALIGN score against different Ig sequences
be similar to that of the parental virus (Fig. 6E). This finding indicates that a test sequence contains an Ig-related domain
ruled out the possibility that expression of the lacZ gene has (40). The results of this analysis (data not shown) showed
an inhibitory effect on the induction of hemadsorption. In that while the selected regions of pEP402R have a statisti-
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conclusion, these results demonstrate that the disruption of cally significant degree of similarity to their counterparts in
EP402R leads to the loss of the ability of ASFV to induce the CD2 molecules, this is not the case with other Ig sequences
adsorption of erythrocytes to the surface of infected cells. from the V set, Cl set, or C2 set. Accordingly, pEP402R
cannot be classified as a member of the IgSF. However, it is
DISCUSSION important to point out that the pEP402R sequence analyzed
in this report is that of the Vero-adapted BA71V strain of
The EP402R ORF, detected within the EcoRI E' fragment ASFV, which was obtained by serial passage of a field virus
of the ASFV genome, encodes a polypeptide, pEP402R, isolate in Vero cells (17). Therefore, it would be interesting
predicted to be a transmembrane protein. The presence of 16 to determine the sequence of the corresponding gene in other
putative sites for the attachment of N-linked carbohydrates virus strains.
within the predicted extracellular region of pEP402R indi- CD2 is an adhesion receptor present in the cell membrane
cates that the mature polypeptide is likely to be highly of immature thymocytes, mature T cells, and natural killer
glycosylated. An intriguing feature of the cytoplasmic do- cells (6, 7). Two CD2 natural ligands have been identified: (i)
main of this protein is the presence of 11 imperfect consec- CD58, a 26-kDa glycoprotein (32) present in the cell mem-
utive repeats of the hexapeptide PPPKPC. A search on the brane of a wide variety of cell lineages, including target cells
data bank PROSITE (4) did not reveal the presence of this for both cytotoxic T cells and natural killer cells and antigen-
sequence motif in other proteins. Interestingly, the region of presenting cells for T helper cells (reviewed in reference 16);
the protein which is made up of this repeated sequence is and (ii) CD59, a widely distributed, 18- to 20-kDa membrane
predicted to form a large domain composed of 3 turns. The glycoprotein (11) that restricts the lysis of human erythro-
possible role of this region is currently under investigation. cytes and leukocytes by human complement (38). The CD2-
EP402R is transcribed during the late phase of the infec- CD59 interaction, described very recently, is thought to be
tious cycle. It is important to note the presence of a 7T motif involved in T-cell adhesion and activation (20). The CD2-
within the 5' end of the EP402R ORF (nucleotides 17 to 23). CD58 interaction has been extensively characterized and is
Although, in view of its position, this 7T motif might be known to play an important role both in the alternative,
involved in the 3'-end formation of the mRNAs from up- antigen-independent, and antigen-specific pathways of T-cell
stream ORFs, it is likely that it reduces the expression of activation and in natural killer cell activity (reviewed in
EP402R. references 6 and 7). In addition, the CD2-CD58 interaction is
Data base searches indicated that pEP402R might be responsible for the formation of rosettes between human T
related to the adhesion receptor CD2. CD2 molecules are cells and both autologous and heterologous erythrocytes
classical transmembrane proteins belonging to the immuno- (15). The results presented in this report demonstrate that
globulin (Ig) superfamily (IgSF) (14, 36, 40). The mature the disruption of EP402R abrogates the ability of ASFV to
form of such molecules contains two differentiated regions: induce the specific adhesion of pig erythrocytes to infected
(i) an N-terminal extracellular region, related to the IgSF, cells, thus showing that pEP402R is directly responsible for
which is responsible for the interaction with the specific this phenomenon. This result constitutes strong evidence
ligands (20, 28); and (ii) a C-terminal cytoplasmic region supporting the belief that pEP402R is a functional adhesion
which appears to be involved in the transduction of activa- molecule capable of interacting with a specific ligand on the
tion signals (reviewed in references 6 and 7). As described surface of erythrocytes. Experiments to characterize the
above, pEP402R is predicted to behave as a transmembrane ligand of pEP402R are under way.
protein, and the relationship between the predicted extracel- Although the hemadsorption induced by ASFV has been
lular region of pEP402R and those of known CD2 molecules extensively used for diagnostic purposes (reviewed in refer-
was confirmed by the results of the statistical analysis using ence 39), little is known about its physiological relevance. It
the RD2FG program. In contrast, in spite of the very high has been recently shown that erythrocytes bind to infected
proline content found in the cytoplasmic region of pEP402R cells in tissues of ASFV-infected pigs (35). The use of the
(one of the most prominent features of the cytoplasmic hemadsorption-negative mutants described here may shed
region of CD2 molecules), the similarity between this region some light on the possible contribution of this phenomenon
of pEP402R and the cytoplasmic regions of known CD2 to the pathological processes of ASFV infection. There are
molecules was not statistically significant. In view of these numerous examples of virus-encoded proteins with homol-
results, pEP402R can be considered a CD2 homolog which ogy to important elements of the immune system which are
has maintained a high level of similarity only in its extracel- able to counteract different defense mechanisms of the host
lular region. (reviewed in references 19 and 26), and as has been observed
The extracellular region of CD2 molecules contains two for EP402R, most genes encoding such proteins are not5318 RODRIGUEZ ET AL. J. VIROL.
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FIG. 6. Effect of the deletion of EP402R on the hemadsorption phenomenon induced by ASFV. The ability of wild-type and mutant
ASFVs to induce the rosetting of pig erythrocytes around the surface of infected macrophages was analyzed as described in Materials and
Methods. (A) Uninfected cells; (B) wild-type ASFV-infected cells; (C) vA&CD2d-infected cells; (D) vACD2r-infected cells; (E) v7213-gal-
infected cells. The arrows in panels B and E indicate positions of the rosettes.
essential for virus multiplication in vitro. It is tempting to hemadsorption is only an artifact of the expression of this
speculate that pEP402R, a functional ASFV-encoded CD2 protein. An exciting possibility is that the expression of
homolog, is primarily involved in a mechanism used by EP402R could be used by the virus to reduce the number of
ASFV to avoid the host's immune surveillance and that the CD58 and/or CD59 molecules available on the cell mem-VOL. 67, 1993 AFRICAN SWINE FEVER VIRUS CD2 HOMOLOG 5319
brane, thus decreasing the ability of T cells and/or natural 1991. Structure of domain 1 of rat T lymphocyte CD2 antigen.
killer cells to interact with ASFV-infected cells. This could Nature (London) 353:762-765.
occur via a mechanism similar to that used by human 15. Dustin, M. L., M. E. Sanders, S. Shaw, and T. A. Springer. 1987.
cytomegalovirus to prevent the expression of mature major Purified lymphocyte function-associated antigen 3 binds to CD2
histocompatibility complex (MHC) class I molecules on the and mediates T lymphocyte adhesion. J. Exp. Med. 165:677-
surface of infected cells. Human cytomegalovirus encodes a 692.
16. Dustin, M. L., and T. A. Springer. 1991. Role of lymphocyte
homolog (5) of the heavy chain of the MHC class I protein adhesion receptors in transient interactions and cell locomotion.
that binds the nascent chain of 02-microglobulin in the Annu. Rev. Immunol. 9:27-66.
endoplasmic reticulum, thus preventing its binding to the 17. Enjuanes, L., A. L. Carrascosa, M. A. Moreno, and E. Vifiuela.
MHC heavy chain (8). 1976. Titration of African swine fever virus (ASF) virus. J. Gen.
The characterization of ASFV genes, such as EP402R, Virol. 32:471-477.
with potential to interfere with the immune system will 18. Esposito, J. J., R. C. Condit, and J. Obijeski. 1981. The
hopefully lead to a better understanding of the virus-host preparation of orthopoxvirus DNA. J. Virol. Methods 2:175-
179.
relationship that may provide new means to control the 19. Gooding, L. R. 1992. Virus proteins that counteract the host
disease. immune defenses. Cell 71:5-7.
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20. Hahn, W. C., E. Manu, A. L. M. Bothwell, P. J. Sims, and B. E.
ACKNOWLEDGMENTS Bierer. 1992. Overlapping but nonidentical binding sites on CD2
for CD58 and a second ligand CD59. Science 256:1805-1807.
We thank Maria L. Nogal, Luis Yuste, Carlos Enriquez, and 21. Jones, Y., S. J. Davis, A. F. Williams, K. Harlos, and D. I.
Antonio Varas for skillful technical assistance, Mariano Bautista for Stuart. 1992. Crystal structure at 2.8 A resolution of a soluble
photomicrographs, Chamorro Somoza and Neil Barklay for helpful form of the cell adhesion molecule CD2. Nature (London)
advice, and Katherine Law for critical reading of the manuscript. 360:232-239.
This work was supported by grants from Comisi6n Interministe- 22. Kyte, J., and R. F. Doolittle. 1982. A simple method for
rial de Ciencia y Tecnologia, Communidad de Madrid, Junta de displaying the hydropathic character of a protein. J. Mol. Biol.
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institutional grant from Fundaci6n Ram6n Areces. 23. Ley, V., J. M. Almendral, P. Carbonero, E. Vifiuela, and A.
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