From the Laboratoire de Virologie, Faculté de Médecine, 06107 Nice Cedex 2, France
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ABSTRACT |
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We have previously demonstrated hyposialylation
of the two major CD45 and leukosialin (CD43) molecules at the surface
of latently human immunodeficiency virus type 1-infected CEM T cells
(CEMLAI/NP), (Lefebvre, J. C.,
Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P.,
Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265-274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio,
A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609-1617). Searching to clarify
mechanism(s) of hyposialylation, we observed two sulfated secreted
glycoproteins (molecular mass ~47 and ~40 kDa) (P47 and P40), which
were differentially sulfated and/or differentially secreted in the
culture supernatants of CEMLAI/NP cells when compared with
parental CEM cells. A hybridoma clone (7H1) resulting from the fusion
between CEMLAI/NP and human embryonic fibroblasts MRC5
cells produced very large amounts of P47 that was purified using
Jacalin lectin (specific for O-glycans) and microsequenced.
Cloning of P47 was achieved using a CEMLAI/NP cDNA
library screened with a degenerate oligonucleotide probe based on its
NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated
protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking -helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal
portion is rich in glutamic acids with a predicted pI of 3.9. In
addition, a hinge region with numerous condensed potential sites for
O-glycan side chains, which are also the most likely
sulfation sites, is located between the RGD and leucine zipper domains.
Transcripts were detected in lymphoid tissues (notably bone marrow) and
abundantly in T and B lymphoblastoid but very faintly in monocytoid
cell lines.
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INTRODUCTION |
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The pathophysiology of acquired immune deficiency syndrome is
usually understood as multifactorial (1, 2). There is a general
consensus as to the important role played by an overactivation state of
the immune system observed in
HIV1-infected individuals. It
is known that modifications of glycosylation are associated with
activation of immune cells (3-5). We have described an altered
glycosylation status due mainly to hyposialylation of the two major
lymphocyte molecules CD43 and CD45 in HIV-1-infected T cells (6, 7),
and autoantibodies directed to CD43 with an altered glycosylation
status have been found in HIV-1+ individuals (8, 9).
Although the level of hyposialylation might be very important in
HIV-1-infected T cells and seems to mostly affect O-glycans
(10), the activity of Gal1-3GalNAc
-2,3-sialyltransferases types I and II (hST3Gal I and
hST3Gal II), which are the two principal sialyltransferases that act on O-glycan cores, appears to be conserved (11).
Among other pathways examined to explain the mechanism(s) of hyposialylation, secretion of two O-glycosylated and sulfated proteins (P47 and P40) has been observed in culture supernatants of latently HIV-1-infected CEMLAI/NP cells (6, 7, 11).
Here, we report molecular cloning and initial characterization of the P47 protein, which we propose to name LSLCL (lymphocytic secreted long form of C-type lectin).
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Tissue Culture-- CEM, MOLT-4, HUT 78, Jurkat, SUP-T1, Raji, Daudi, HL-60, THP-1, U-937, and MRC5 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 or basal medium Eagle (Life Technologies, Inc.) containing 5% fetal calf serum (Whittaker M.A. Bioproducts), supplemented with glutamine (2 mM), penicillin (100 units/ml), and streptomycin (50 µg/ml). CEMLAI/NP are CEM cells infected by HIV-1 LAI strain. They harbor HIV-1 provirus but are latently infected and virus-nonproducing (6). The 7H1 clone was obtained as follows2: hypoxanthine aminopterin thymidine-sensitive CEMLAI/NP cells were first selected as cells resistant to 8-azaguanine (15 µg/ml) after mutational treatment with ethyl methanesulfonate. Using polyethylene glycol, resistant cells were fused with human embryonic fibroblasts MRC5. Hybrid clones were selected as hypoxanthine aminopterin thymidine-resistant cells.
Preparation of Rabbit Serum Anti-P47-- Rabbits were immunized with the oligopeptide ARGAEREWEGGWGGAQEE (Arga), coupled to KLH protein (Neosystem Laboratoire, Strasbourg, France). The sequence of the peptide was determined by NH2-terminal microsequencing of purified P47 as described below. A rabbit serum (Argara) was shown to precipitate two proteins (P47 and P40) from lysates and culture supernatants of secreting cells.
35SO42
Metabolic-labeling Assay and Precipitations--
Cells (4 × 106/ml) were washed and incubated for 18 h in
SO42
-free RPMI 1640 medium containing
SO42
(ICN Biomedicals) (500 µCi/ml)
and 5% dialyzed fetal calf serum. Cells were sedimented, and culture
supernatants added with phenylmethylsulfonyl fluoride (1 mM, final concentration) (Sigma), aprotinin (10 mg/ml) (Sigma), and leupeptin (10 µg/ml) (Boehringer Mannheim). Supernatants were then mixed with 10 µl of immobilized streptavidin beads (Pierce) previously coated with 2 µg of the various biotinylated lectins, PNA
(peanut agglutinin from Arachis hypogaea), Jacalin (from
Artocarpus integrifolia), or GNA (from Galanthus
nivalis), all purchased from EY Laboratories. 10 µl of protein
A-Sepharose 4B beads (Sigma) precoated with 10 µl of rabbit serum
were added for immunoprecipitations. After 2 h of agitation,
precipitates were collected by brief centrifugation. The pellets were
washed three times in 50 mM HEPES buffer, pH 7.5, containing 1% Nonidet P-40, 150 mM NaCl, 1 mM
EGTA, and the antiprotease mixture as described above, and washed once
with the same buffer containing 1 M NaCl. They were
resuspended in SDS-polyacrylamide gel electrophoresis sample buffer
(2% SDS, 1.7 M 2-mercaptoethanol) and boiled for 3 min.
Samples were resolved by SDS-polyacrylamide gel electrophoresis (7.5%
acrylamide). After intensification with Amplify (Amersham), the gels
were vacuum dried and exposed to XAR5 Kodak films.
Purification and Microsequencing of the P47 Glycoprotein-- The culture of 7H1 cells was amplified up to one liter of cell suspension in RPMI 1640 medium containing 5% fetal calf serum. Then the cells were resuspended in RPMI 1640 without fetal calf serum and maintained at 37 °C for two days. After centrifugation of cells, supernatants were added with the mix of antiproteases as described above and with streptavidin-immobilized beads (1 ml/liter) precoated with biotinylated Jacalin. After 2 h of agitation, precipitates were collected by brief centrifugation. The pellets were washed 3 times in lysis-buffer and once with the same buffer containing 1 M NaCl. They were resuspended in SDS-polyacrylamide gel electrophoresis sample buffer (2% SDS, 1.7 M 2-mercaptoethanol), boiled for 3 min, and resolved by SDS-polyacrylamide gel electrophoresis (7.5% acrylamide). For NH2-terminal sequencing, proteins were transferred to Problott membranes (Applied Biosystems). The peptides were detected by staining with 0.003% amido black, then the P47 band was carefully excised and treated on an Applied Biosystems model 470A sequencer. Internal sequences were obtained from peptides separated by high performance chromatography following cleavage of purified P47 with trypsin.
cDNA Cloning and Sequence Analysis--
A
CEMLAI/NP cell line cDNA library was constructed using
pcDNA3 vector plasmid and a cDNA synthesis kit (both from
Invitrogen) with an oligo(dT) (NotI) primer according to the
manufacturer's recommendations. Approximately 4 × 106 colonies of Escherichia coli DH5
transfected with this library were isolated and screened by using the
oligonucleotide probe 5
-GAGTGGGAGGGIGGITGGGGIGGIGCICAGGAGGAG-3
, where
inosine were incorporated to limit degeneracy. The sequence of this
probe was based on the peptide sequence EWEGGWGGAQEE established by
NH2-terminal sequencing of P47. This probe was 5
-end
radiolabeled using T4 polynucleotide kinase
(CLONTECH) and [
-32P]ATP (ICN
Biomedicals). Membrane prehybridization was carried out at 60 °C for
3 h in 20 mM phosphate buffer, pH 7.5, containing 5 × SSC, 7% SDS, 10 × Denhardt's solution, and 1% salmon
sperm DNA. Hybridization was achieved overnight at 60 °C. Then
membranes were washed twice in 2 × SSC, 2% SDS solution for 20 min at 20 °C, once in 0.1 × SSC, 0.1% SDS solution for 20 min
at 20 °C, and once in 0.1 × SSC, 0.1% SDS solution for 30 min
at 48 °C, and thereafter exposed to XAR5 Kodak film for 1 day at
70 °C. Several types of recombinants were selected and sequenced
on both strands by the dideoxynucleotide chain-termination method (12),
using an ABI PRISMTM dye terminator cycle sequencing ready
reaction kit with AmpliTaq® DNA polymerase, FS (Perkin
Elmer), and an ABI PRISM 310 genetic analyzer (Perkin Elmer). Analysis
was started on each strand using SP6 and T7 primers (Invitrogen) and
extended with successive overlapping primers (20-base oligonucleotides)
that were synthesized using an Applied Biosystem 381A DNA synthesizer.
Sequences were analyzed using MacDNASIS (Hitachi Software).
Northern Blot Analysis-- Total RNA were extracted according to the guanidine isothiocyanate technique, described by Davis et al. (13). Poly(A)+ RNA were selected from 200 µg of total RNA by incubating with 100 mg oligo(dT) cellulose (Pharmacia Biotech Inc.) in 5 ml of TL buffer containing 20 mM Tris, pH 7.5, 0.5 M LiCl, 1 mM EDTA, and 0.1% SDS at room temperature with gentle shaking for at least 30 min. The beads were collected and washed twice with TL buffer and twice with the same buffer containing 0.15 M LiCl. The beads were then deposited on a Spin-X column (Costar, Cambridge, MA) for extensive washings with TL buffer containing 0.15 M LiCl, and thereafter the poly(A)+ RNA were eluted with 200 µl of 10 mM Tris buffer, pH 7.5 (containing 1 mM EDTA, 0.05% SDS) and precipitated with 2.5 vol of ethanol.
For Northern blot analysis, 5 µg of poly(A)+ RNA were electrophoresed on denaturing 1.2% agarose gel and transferred to a Hybond N nylon membrane (Amersham) with a 20 × SSC transfer buffer. Premade Northern blots of poly(A)+ RNA from different human tissues (CLONTECH) were also used. The P47 probe was aComputer Analysis-- Data base searches for nucleotides and amino acids sequences alignments were achieved using the BLAST program of the National Center for Biotech. Protein sequence analysis, motif search, and hydrophobicity were established with MacDNASIS programs.
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RESULTS AND DISCUSSION |
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Since the alterations of sialylation that we have observed on
HIV-1-infected CEM cells were principally involving
O-glycans attached to the two major lymphocytic surface
glycoproteins CD45 (6) and CD43 (7), we verified the presence of
possible O-glycosylated proteins in the culture supernatants
by using the lectins PNA, specific for the O-glycan core 1 nonsialylated disaccharides Gal1-3GalNAc (14) and Jacalin, specific
for sialylated and nonsialylated O-glycans (15). In
addition, an 35SO42
metabolic incorporation assay was used to display secreted
glycoproteins. Jacalin only was able to precipitate a protein doublet
(molecular mass, 47 and 40 kDa) (P47, P40) from supernatants of
parental CEM cells culture (Fig. 1,
lane 2), whereas this was achieved by both Jacalin or PNA
from supernatants of HIV-1-infected CEMLAI/NP cells culture
(Fig. 1, lanes 7 and 8). This fact could be
explained by hyposialylation status of CEMLAI/NP cells that
affects multiple O-glycosylated proteins, whereas parental
cells are normally sialylated. The following has to be pointed out: (i)
the nonreactivity of the lectin GNA (Fig. 1, lanes 3 and
9) that preferentially binds terminal mannose (16); (ii) a
weak upward of the CEM doublet (Fig. 1, lane 2) probably due
to the more numerous sialic acid molecules beared by the P40 and P47
proteins of these cells as confirmed by the nonreactivity of the lectin
PNA (Fig. 1, lane 1); and (iii) a much more pronounced
doublet P40-P47 from CEMLAI/NP cells when compared with
parental CEM doublet although equal quantities of total protein were
deposited in each lane of the gel. This interesting result was further
confirmed on several occasions and was also observed when culture
supernatants from CEMBCL cells (virus-producing CEM cells
infected by HIV-1 strain BCL) (6) were compared with supernatants from
parental CEM cells culture (not shown). This is currently being
investigated in terms of increased activity of sulfotransferases and/or
secretion regulation.
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To obtain a sufficient material to allow sequencing of these
proteins, hybrid cells were raised by fusion between
CEMLAI/NP cells and embryonic human fibroblasts MRC5
by using polyethylene glycol as described under "Experimental
Procedures."2 It was found that the clone 7H1 secreted
very large amounts of P47 (Fig. 1, lanes 4 and 5)
that was then purified using Jacalin lectin and then microsequenced.
Thus, the sequences of an NH2-terminal and two internal
peptides were obtained (Fig. 2,
underlined sequences). Thereafter, the cloning of P47 was
achieved by screening a CEMLAI/NP cDNA library
constructed in pcDNA3 vector using a degenerate oligonucleotide based on the NH2-terminal amino acid sequence. The longest
isolated recombinant (1.4 kb) yielded a single open reading frame of
972 nucleotides encoding a protein of 323 amino acids (Fig. 2). Apart from an NH2-terminal hydrophobic region of 21 residues
located just upstream from the known microsequenced
NH2-terminal part of P47 (Figs. 2 and
3), no other potential hydrophobic
transmembrane domain was found (Fig. 3). Thus this region very likely
corresponded to the signal peptide of preprotein P47, which was
purified from culture supernatants, and then appeared as a secreted
protein with a Gly21Ala22 signal peptidase
cleavage site. As shown on Fig. 2, several other domains were
identified on P47: (i) a glutamic acid-rich NH2-terminal domain localized between amino acids 22 and 90 with a stretch of 6 successive identical residues (Glu84-89); (ii) an RGD
triplet (Arg61-Gly-Asp63) in the middle of the
glutamic acid-rich domain; (iii) a leucine zipper consisting of six
heptads, spanning Val108-Ala149; and (iv) a
C-terminal C-type lectin domain that is known as a carbohydrate
recognition domain (CRD) (17) spanning
Gly176-Phe323 and including most of the amino
acids conserved for this type of functional domain, in particular the
six cysteines (Fig. 2, closed arrows) found in the long form
CRDs (18). In addition, a (Thr-Pro-Ser)-rich domain with 13 Thr/Pro/Ser
out of 14 residues was found between amino acids 91 and 104 (Fig.
2).
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The leucine zipper of P47 is particularly prone to form coiled coil
structures (19), since it is composed of six heptads with a Leu/Val
residue at structural position a and an Ile/Leu residue at
position d (Fig. 4). The
structure of P47 could thus be related to that of collectins, which are
secreted proteins containing a CRD connected to a collagen-like
-helix suitable to form coiled coils (17). The
NH2-terminal portion of P47, spanning
Ala22-Ala90, is rich in glutamic acid (30% of
residues) and has a predicted pI of 3.95. This is reminiscent of the
secretory eosinophil major basic protein, which also contains a
C-terminal C-type lectin and a glutamic acid-rich
NH2-terminal part with a pI of 3.9 (20) that is sequestered
into secretory granules before release.
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At the time we were cloning P47, there was no significant similarity to
any other protein accessible from data banks. The best alignments were
found to be 29.2% with tetranectin-like protein (21) and 24.8% with
tetranectin (22), provided by the restricted CRD sequence
Cys177-Arg228 of P47 (Fig. 4). Tetranectin is a
plasminogen kringle 4-binding protein with a CRD joined to an
-helical coiled coil structure (23, 24). During preparation of this
manuscript, Hiraoka et al. (25), using a differential
technique with subtractive probe, published the cloning of SCGF, a
protein that very closely matched P47. However, SCGF and P47 appear to
be different because SCGF cDNA lacks 234 nucleotides encoding a
peptide of 76 amino acids located in the CRD found on P47. In
particular, the Cys204 of P47 is absent from SCGF (Fig. 2).
Surprisingly, these authors did not mention any of the protein domains
(RGD, leucine zipper, O-glycan sites, or CRD) that are
present on SCGF and P47 (Fig. 2).
Sulfation of P47 was not likely to occur on tyrosine residues. Indeed, tyrosyl protein sulfotransferase is known to recognize tyrosines in exposed protein domains containing acidic amino acids (26, 27) that were not found in the surroundings of the 6 Tyr residues present on P47 (Fig. 2, open arrows).
P47 did not show any potential N-glycosylation sites (Asn-Xaa-Ser/Thr). This fact correlated well with the nonreactivity of P47 with the lectin GNA (Fig. 1). On the other hand, the (Thr-Pro-Ser)-rich domain is particularly suited for O-glycans attachment (28) and could explain the binding of P47 with the lectins Jacalin and PNA (Fig. 1). In this context, and since sulfation of P47 did not implicate tyrosine residues nor N-glycan, the O-glycans remained the most probable or even the sole possible sulfation sites, as it has been demonstrated on other proteins such as the cell surface mucin of mammary adenocarcinoma cells (29), gpMEL-14 (LECAM-1) (30), and leukosialin (CD43) (31, 32). The molecular mass of P47, calculated on the basis of its amino acid sequence subtracted the signal peptide, was 33,532 Da. We conclude that O-glycan side chains added with sulfate residues account for approximately 13,500 Da in the mature secreted form of P47, which thus could be regarded as a mucin-like protein.
It was tempting to compare P47 to a bifunctional molecule with an RGD
domain located in the very hydrophilic NH2-terminal portion
and known to interact with integrins (33), and a C-terminal C-type
lectin domain well known to bind glycoconjugates (17), separated from
each other by a neck region consisting of an extended O-glycosylated hinge domain and a leucine zipper. In this
context it seemed interesting to select, among numerous proteins
harboring a CRD, the two proteins tetranectin (22) and low affinity
receptor for IgE (CD23) (34) for alignment with P47. Indeed, these
molecules showed a neck leucine-rich region, upstream of their CRD,
suitable for coiled coil associations. As shown on Fig.
5, there are several conserved amino
acids, especially the six cysteines and surrounding residues in CRD,
but also numerous aliphatic amino acids in the portion corresponding to
the leucine zipper of P47 that is aligned with the leucine zipper of
CD23 as well as with the E2 -helix that governs the coiled coil
formation of tetranectin (24).
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Northern blot analysis showed a single band at approximately 1.42 kb (Fig. 6, A and C), even when a better resolution was obtained with mRNA deposits in 1-cm large wells (Fig. 6B). Among the various cell lines tested, all T and B lymphoblastoid (CEM, MOLT-4, HUT 78, Jurkat, SUP-T1, Raji, Daudi) cells highly expressed P47, whereas monocytoid U-937 and THP-1 expressed very faintly, and embryonic fibroblasts MRC5 did not (Fig. 6A). Tissue-specific expression was analyzed using commercially prepared Northern blots. A very weak band was seen in most tissues (Fig. 6C, lanes 7-14). Lymphoid tissues, notably bone marrow, appeared to better express P47 (Fig. 6C, lanes 1-6). Since the sequence studied in this report is likely of lymphoblast/lymphocyte origin, it is quite possible that the weak Northern signals seen in essentially all tested tissues is the result of contaminating blood that it is difficult to entirely remove during preparation of tissues.
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The transcription rate of P47 appeared to be identical in both
HIV-1-infected CEMLAI/NP and parental CEM cells, as
confirmed on several occasions (Fig. 6 A and B).
This result is important to consider while compared with the apparent
differential expression of P47 when examined after
35SO42 incorporation (Fig.
1). This phenomenon is currently being investigated in terms of
increased activity of sulfotransferases and/or secretion regulation.
Several discrepancies were noted between P47 (this study) and SCGF (25). Whereas expression of SCGF was found in fibroblasts, P47 was not detected in similar cells (Fig. 6A). On the contrary, SCGF mRNAs were not detected in MOLT-4 cells, although we did find P47 in these cells (Fig. 6A). We showed two closely migrating sulfated proteins with a molecular mass of 47 and 40 kDa that were precipitated from culture supernatants by lectins (Fig. 1). The 40-kDa species is very likely a derivative of P47, since the two species were precipitated by rabbit serum Argara raised against an NH2-terminal peptide of P47 (not shown). Although a single mRNA species (1.42 kb) was seen after probing with a full-length cDNA corresponding to P47 (Fig. 6), it is tempting to think that P40 could correspond to SCGF. However, SCGF was claimed to have a molecular mass of 29 kDa according to (i) calculation from its amino acid sequence and (ii) partial purification of SCGF monomer by chromatography as an elution peak of 29 kDa (25). Nevertheless, it has to be pointed out that SCGF contains the same Thr/Pro/Ser-rich domain as P47 and that it is very likely O-glycosylated, thus leading SCGF to a molecular mass >29,000 Da. In addition, it is difficult to consider SCGF as a spliced form of P47. Indeed, it can be deduced from comparison of gene structures of C-type CRDs (35) that the most probable exon that might be removed from P47 to generate SCGF could correspond to the internal peptide Glu221-Phe262 and not to the differential peptide Ala198-Gln275 present on P47 only. Therefore, P40 is very likely a processed form of P47, and there is no obvious explanation for differential transcription of LSLCL (P47) and SCGF.
SCGF has been shown to exhibit burst promoting activities when associated with growth factors such as erythropoietin or granulocyte/macrophage colony-stimulating factor (25). The RGD domain present on P47 and SCGF might explain such properties, since this type of motif is known to interact with integrins that activate signal transduction pathways coordinated with responses to growth factors (36). However, Hiraoka et al. (25) claimed the activities of SCGF to be accounted for by a protein purified from culture supernatants as an elution peak of 29 kDa. They have cloned SCGF using a differential technique with subtractive probe and claimed this protein to be the active species of 29 kDa. However, analogy between P47 and SCGF is sufficient to allow similar molecular masses that might be of 40-47 kDa rather than 29 kDa. In additon, the various functional domains (C-type lectin, leucine zipper, and RGD) are reminiscent of collectins that are known as anti-infectious agents (17) rather than of growth factors. Hiraoka et al. (25) conclude their paper as follows: "It is presently unclear, however, whether KPB-M15-CM contains another SCGF-like factor." We think that cloning of SCGF might be fortuitous and that the growth factor activity of culture supernatants from KPB-M15 cells remains to be determined.
Since P47 is composed of several domains that allow for prediction of various interactions, counterparts of this complex molecule remain to be identified to understand its exact role(s).
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ACKNOWLEDGEMENTS |
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Special thanks to J. d'Alayer and M. Davi (Institut Pasteur, Paris, France) for peptides sequencing.
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FOOTNOTES |
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* This work was supported by the Agence Nationale de Recherches sur le SIDA (ANRS) Grant 96012 (to J. C. L.) and the Association pour le Développement du Diagnostic des Maladies Virales (ADDMV).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF020044.
To whom correspondence should be addressed. Tel.:
33-4-93-37-76-78; Fax: 33-4-93-81-54-84; E-mail:
lefebvre{at}unice.fr.
1 The abbreviations used are: HIV, human immunodeficiency virus; GalNAc, N-acetylgalactosamine; LSLCL, lymphocytic secreted long C-type lectin; PNA, peanut agglutinin; GNA, Galanthus nivalis agglutinin; RGD, Arg-Gly-Asp; CRD, carbohydrate recognition domain; SCGF, stem cell growth factor; kb, kilobase(s).
2 J. Lesimple, S. Bannwarth, V. Giordanengo, and J.C. Lefebvre, manuscript in preparation.
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REFERENCES |
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