From the Molecular Medicine Laboratory, International
Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy, ¶ Unit of General Pathology and Immunology,
Department of Biomedical Sciences and Biotechnology, School of
Medicine, University of Brescia, 25123 Brescia, Italy, and
Scuola Normale Superiore, 56126 Pisa, Italy
Received for publication, July 27, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Tat, the transactivator protein of human
immunodeficiency virus-1, has the unusual capacity of being
internalized by cells when present in the extracellular milieu. This
property can be exploited for the cellular delivery of heterologous
proteins fused to Tat both in cell culture and in living animals. Here
we provide genetic and biochemical evidence that cell membrane heparan
sulfate (HS) proteoglycans act as receptors for extracellular Tat
uptake. Cells genetically defective in the biosynthesis of fully
sulfated HS are selectively impaired in the internalization of
recombinant Tat fused to the green fluorescent protein, as evaluated by
both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell
surface HS proteoglycans also mediate physiological internalization of
Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells
and cells genetically impaired in proteoglycan synthesis are equally
proficient in the extracellular release of Tat, thus indicating that
proteoglycans are not required for this process. The ubiquitous
distribution of HS proteoglycans is consistent with the efficient
intracellular delivery of heterologous proteins fused with Tat to
different mammalian cell types.
The Tat protein of human immunodeficiency virus-1
(HIV-1)1 is a powerful
transcriptional activator of the integrated viral genome. The protein
binds to a highly structured RNA element located at the 5' end of all
viral transcripts (1), and from there it increases the rates of both
transcriptional initiation and elongation from the long terminal repeat
(LTR) promoter. These two functions are mediated by the interaction of
Tat with nuclear proteins possessing chromatin remodeling activity
(2-4) and with cellular kinases phosphorylating the C-terminal domain
of RNA polymerase II (5-9), respectively.
In addition to these transcriptional functions at the HIV promoter, the
Tat polypeptide exerts pleiotropic biological activities when present
in the extracellular compartment. Extracellular Tat promotes the
production of cytokines (10-15) and cytokine receptors (16-18);
modulates the survival, proliferation, and migration of different cell
types (19-24); exerts angiogenic activity in vitro and
in vivo (25-27); inhibits antigen-specific lymphocyte
proliferation (28-30); and induces neurotoxicity in the central
nervous system (31-36). Since a growing body of evidence exists that
Tat could be released by producing cells (37-40), it is likely that
some of the above-mentioned effects of extracellular Tat could have important implications for the pathogenesis of HIV disease in an
autocrine or paracrine fashion.
Besides its interaction with cell surface receptors and the consequent
activation of intracellular signal transduction pathways (41-43), most
of the activities of extracellular Tat are mediated by its unique
property of being rapidly internalized by a variety of cell types, as
originally shown more than 10 years ago (44-46). One of the
consequences of Tat internalization is the activation of cellular
transcription factor NF- The uptake, internalization, and nuclear translocation of extracellular
Tat can also be exploited as a biotechnological tool for intracellular
protein delivery. Chemical cross-linking of Tat peptides with
heterologous proteins (51) or, more efficiently, production of
recombinant proteins containing the protein transduction domain of Tat
(52, 53) facilitate the intracellular delivery of these proteins. In
particular, it has been recently reported that the intraperitoneal
injection of the 120-kDa Despite the large body of evidence available about the functions of
extracellular Tat and its recent use, a biotechnological vector for
protein transduction, the cellular mechanisms for Tat uptake and
internalization, are still largely unexplored. Inspired by the
observation that Tat can enter a variety of different cell types both
in vitro and in vivo and by the possibility of
modulating several of the biological properties of extracellular Tat by
soluble heparin (54-56), we started investigating the role of cell
surface proteoglycans in the process of Tat translocation through the plasma membrane. Here we provide genetic and biochemical evidences that
cell membrane heparan sulfate (HS) proteoglycans are the receptors for
extracellular Tat internalization.
Cell Lines--
The wild type CHO K1 and CHO K1 mutants
deficient in proteoglycan biosynthesis (57) were obtained from the
American Type Culture Collection (Manassas, VA). The pgs A-745
cell line does not produce detectable levels of proteoglycans since it
lacks xylosyltransferase, an enzyme necessary for the initiation of glycosaminoglycan (GAG) synthesis. Mutant pgs B-618 has a defect in the
galactosyltransferase-I enzyme gene and produces about 15% the amount
of proteoglycans synthesized by wild type cells. Cell line pgs E-606 is
partially deficient in HS N-sulfotransferase and produces an
undersulfated form of HS proteoglycan. The cell line pgs D-677 has a
single mutation that affects both
N-acetylglucosaminyltransferase and glucuronosyltransferase
activities, which are necessary for the polymerization of HS
disaccharide chains and does not synthesize any HS proteoglycan. This
mutant cell line also produces approximately three times more
chondroitin sulfate than wild type cells. Finally, mutant cell line pgs
C-605 has a defect in a saturable,
4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive
transport system required for sulfate uptake. Despite a dramatic
reduction in 35SO4 incorporation, the mutant
synthesizes sulfated heparan and chondroitin chains by using the
inorganic sulfate produced from oxidative metabolism of cysteine and
methionine (58).
HL3T1 cells (a HeLa derivative containing an integrated LTR-CAT
cassette) were a kind gift of B. Felber. Cell lines constitutively expressing Tat-green fluorescent protein (GFP) were obtained by selection for neomycin-resistant clones with 500 µg/ml G418 (Life Technologies, Inc.) after transfection of pCDNA3-Tat-GFP; single clones were collected and propagated.
Recombinant Proteins--
Recombinant glutathione
S-transferase (GST)-Tat containing the 86-amino acid Tat
protein of HIV-1 clone HXB2 fused to glutathione S-transferase and its mutated derivative containing alanine
substitutions at arginines 49, 52, 53, 55, 56, and 57, GST-Tat R
(49-57)A, were produced and purified by glutathione-agarose
affinity chromatography as already described (3, 48). The plasmid
expressing GST-Tat-GFP was obtained by cloning a polymerase chain
reaction-amplified fragment into the BamHI and
EcoRI sites of the commercial vector pGEX2T (Amersham
Pharmacia Biotech). The fragment was obtained by the separate
amplifications of HXB2 Tat using primers
5'-GTGGATCCATGGAGCCAGTAGATCCTA-3' and
5'-CCCTTGCTCACCATAAGCTTTTCCTTCGGGCC-3' and of enhanced green fluorescent protein (GFP) using primers
5'-GGCCCGAAGGAAAAGCTTATGGTGAGCAAGGG-3' and
5'-GGCGAATTCTCTAGAGTCGCGGCCGCTTTA-3'. Templates for amplification were
plasmid pGEX2T-Tat (3) and pEGFP-N1 (CLONTECH, Palo
Alto CA) respectively. The two amplification products contain
complementary sequences at the 3' and 5' regions of the coding strands
of Tat and GFP, respectively. They were gel purified, mixed, annealed, and amplified with the external primers to obtain a single
amplification products, which contains BamHI and
EcoRI sites at the extremities.
Tat Protein Transduction--
To study the kinetics of
recombinant Tat internalization, HL3T1 cells were seeded in 24-well or
10-cm-diameter dishes at the density of 1-2 × 104
cells/cm2 in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. After 24 h, cell cultures were
washed twice and incubated for an additional 24 h in fresh medium
containing 10% fetal calf serum, 100 µM chloroquine, and
recombinant Tat protein. Incubation in the presence of chloroquine
favors Tat uptake by modifying the pH of endolysosomal vesicles and
preventing protein degradation (46). After 24 h, the medium was
changed to Dulbecco's modified Eagle's medium, 10% fetal calf serum,
and cells were incubated for an additional 24 h. At the end of
incubation, cells were extracted, and the amount of CAT protein present
in the cell extracts was determined by the CAT enzyme-linked
immunosorbent assay kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Alternatively, cells were collected and
analyzed by FACS (see below).
Internalization of 125I-GST-Tat--
Recombinant
GST-Tat was labeled with 125I (17 Ci/mg, PerkinElmer
Life Sciences) using iodogen (Pierce) to a specific radioactivity of
400 cpm/fmol as described previously (55). HL3T1 cells were seeded in
24-well dishes at the density of 45,000 cells/cm2 in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
After 24 h, cell cultures were washed twice with Tris-buffered saline and incubated for 16 h at 37 °C in binding medium
(serum-free medium containing 0.15% gelatin and 20 mM
Hepes buffer, pH 7.5) with the addition of 20 ng/ml
[125I]GST-Tat plus 200 ng/ml unlabeled GST-Tat as a
carrier and in the presence of 100 µM chloroquine. After
incubation, medium was removed, cells were washed three times with cold
Tris-buffered saline and lysed by incubation with 0.5% Triton X-100 in
0.1 M sodium phosphate, pH 8.1. Radioactivity of the cell
lysates was measured, and nonspecific binding, determined in the
presence of a 200-fold molar excess of unlabeled GST-Tat (4 µg/ml),
was subtracted.
Immunocytochemistry--
For immunocytochemistry, HL3T1 cells
were grown to about 60% confluency on glass coverslips. The GST-Tat
protein (1 µg/ml) was added to the cell culture medium in the
presence of 100 µM chloroquine. After different time
intervals, cells were washed 6 times with PBS and fixed with a cold
acetone:methanol mixture (50:50) for 15 min. Cells were then washed 3 times with PBS containing 0.2% Triton X-100 (PBS-Triton X-100) and
then 5 times with PBS for 5 min each. Cells were then incubated with an
anti-Tat monoclonal antibody (ADP352/NT3, obtained from the Medical
Research Council repository for AIDS research) for 1 h,
washed 5 times with warm PBS (25 to 28 °C), and incubated with
rhodamine-conjugated secondary antibody (Sigma) for 30 min. Cells were
then washed three times with warm PBS/Triton X-100 and with warm PBS
for 5 min each time. For each immunostaining, one coverslip was
incubated in secondary antibody alone as a negative control for
background immunofluorescence. Nuclei were counterstained with Hoechst
33342 (10 µg/ml in PBS) for 5 min, and coverslips were washed three
times with PBS and mounted on glass slides. Slides were observed using
Zeiss Axiophot fluorescence microscope.
Flow Cytometry--
To analyze GFP-Tat internalization by cell
cytometry, cells were washed four times with PBS, trypsinized, again
washed with PBS, and analyzed with a FACScan flow cytometer (Becton
Dickinson). A total of 10,000 events per sample were considered.
Cell Treatment with Soluble GAGs--
The soluble GAGs (heparin,
from porcine intestinal mucosa; chondroitin sulfate A, from bovine
trachea; chondroitin sulfate B, from porcine intestinal mucosa; and
chondroitin sulfate C, from shark cartilage) and dextran sulfate
(molecular weight, 5000) were all purchased from Sigma. In the FACS
experiments with soluble GAG analogues, 5 × 105 CHO
K1 cells were incubated in fresh culture medium with the addition of 1 µg/ml GST-Tat-GFP protein, 100 µM chloroquine, and the
appropriate amount of GAG dissolved in PBS.
Inhibition of Tat transactivation by soluble GAGs was studied in 3 × 105 CHO K1 cells transfected with 1 µg of
pBlue-LTR-CAT (containing an LTR-CAT cassette, a kind gift of B. Berkhout) using Lipofectin (Life Technologies, Inc.). Forty-eight hours
after transfection, cells were treated with different concentrations of
GAGs along with 1 µg/ml GST-Tat-GFP and 100 µM
chloroquine in fresh culture medium for 14 h. After this time
period, cells were washed four times with PBS, and fresh culture medium
was added. CAT assays were performed after 36 h according to
standard protocols (59) and after normalization for transfection
efficiency. Each experimental point was performed in triplicate.
Cell Treatment with GAG Lyases--
Enzymatic treatment with GAG
lyases was performed as described (60). Briefly, 5 × 105 CHO K1 cells were incubated with the GAG lyases (Sigma;
dissolved in PBS) in PBS containing 0.1% bovine serum albumin, 0.2%
gelatin, and 0.1% glucose for 40 min at 37 °C in a CO2
incubator. Cells were then washed gently 6 times with PBS and incubated
in Ham's F-10 medium without serum in the presence of 1 µg/ml
GST-Tat-GFP protein and 100 µM chloroquine for 5 h.
Cells were then washed 4 times with PBS, trypsinized, washed again with
complete Ham's F-10 medium, resuspended, and used for FACS analysis.
Each experimental point was performed in triplicate.
HIV-1 LTR Transactivation Assays in Cell Mutants--
For CAT
assays, 1 × 106 cells were seeded in 10-cm-diameter
dishes. After 24 h, cells were transfected with either
pBlue-LTR-CAT (1 µg) alone or together with pCDNA3-Tat-GFP (1 µg). The latter plasmid was obtained by recovering the Tat-GFP
cassette of plasmid pGEX2T-Tat-GFP using EcoRI and
BamHI and cloning into the respective sites of vector
pCDNA3 (Invitrogen, Carlsbad, CA). In this plasmid, Tat-GFP is
expressed under the control of the cytomegalovirus promoter. After
lipofection, cells were washed twice with PBS and incubated in fresh
culture medium for additional 24 h. Cells transfected with
pBlue-LTR-CAT alone were supplied with 1 µg/ml GST-Tat-GFP in the
presence of 100 µM chloroquine and incubated for 24 h. Cells were then washed twice with PBS and incubated for additional
24 h in fresh medium. Finally, cells were scraped off the dishes
using a rubber policeman, and cell extracts were used for CAT assays.
Transcellular Transactivation--
Wild type CHO K1 cells, the
mutated pgs A-745 clone, and HeLa cells were transfected by Lipofectin
with 1 µg/ml pBlue-LTR-CAT. After 48 h, cells were trypsinized
and plated in equal number with CHO and A-745 cell clones expressing
Tat-GFP, with the combinations reported in Fig. 7A. Cells were
incubated in complete medium containing 100 µM
chloroquine. Seventy-two hours later, cells were scraped off the plate
with a rubber policeman, and cell lysates were analyzed for CAT activity.
Kinetics of Extracellular Tat Internalization--
To explore the
mechanisms of HIV-1 LTR transactivation by Tat, in the last few years
we have taken advantage of the property of a GST-Tat fusion
protein to enter cells when added to the cell culture medium (3, 48).
This protein contains the first 86 amino acids of Tat fused at the C
terminus of the GST protein (~34 kDa total). The kinetics of HIV-1
LTR transactivation by GST-Tat and by a fusion protein additionally
containing the GFP at the C terminus (~60 kDa total) are shown in
Fig. 1A. LTR transactivation starts to be observed a few hours after the addition of GST-Tat to the
culture medium of HL3T1 cells (containing an integrated LTR-CAT
reporter gene that is silent in the absence of stimulation) and peaks
at 10-15 h. The kinetics of LTR transactivation by the GST-Tat-GFP
protein is delayed by a few hours but still reaches the same levels
after 15-20 h. These data are consistent with previous findings
showing a peak of LTR-CAT mRNA expression ~5 h after the addition
of exogenous GST-Tat protein to the culture medium (48). As shown in
Fig. 1B, LTR transactivation is already appreciable at a
concentration of GST-Tat protein equal to 50 ng/ml (~1.5
nM) and reaches a plateau at 200 ng/ml. A similar dose
response curve is evident for the GST-Tat-GFP protein when considering
the molar differences between the two preparations. In contrast to
these recombinant proteins, which contain the wild type Tat amino acid
sequence, fusion of GST to the Tat R (49-57)A mutant, in which
arginines at positions 49, 52, 53, 55, 56, and 57 were mutated to
alanines (47), produces a protein that is completely inefficient in
driving LTR activation, even at the highest concentrations (Figs. 1,
A and B).
Entry of GST-Tat into the cells was also directly appreciated by
labeling the protein with [125I]iodine. As shown in Fig.
1C, the protein was rapidly internalized, reaching
significant intracellular levels already 4 h after the addition to
the medium. By staining with an antibody specific for Tat, after 2 h the protein began to be found inside the cells with a distribution
compatible with its presence inside endosomal vesicles, as already
described (Ref. 46 and data not shown). At 4 h, the anti-Tat
antibody stained the nuclei of most cells, consistent with the
transactivating activity of the protein at this time point (Fig.
1E). At 24 h, the protein could be still visualized
inside the cells, but it was mostly excluded from nuclei. Contrary to
the GST-Tat fusion protein, treatment of cells with 1 µg/ml
recombinant GST protein for 24 h did not result in any internalization of this protein that could be appreciable with anti-GST
antibody (Fig. 1E, upper panels).
Cell entry of GST-Tat-GFP was also visualized by flow cytometric
analysis of the treated cells (Fig. 1D). A time course study showed that increased cell fluorescence started to be detected 2 h
after the addition of the protein to the cell culture medium and
increased during the first 24 h. At each time point, the
relatively narrow FACS peak width indicates the uniform uptake of
exogenous Tat by all exposed cells, consistent with the uniform pattern of staining with the anti-Tat antibody of Fig. 1E. Again, a
24-h treatment with the same amount of a recombinant GST-GFP protein did not result in any appreciable increase of intracellular
fluorescence. Altogether, these data indicate that functional
recombinant Tat is able to specifically enter most treated cells with
relatively rapid kinetics and direct the entry of larger protein
cargos inside the cells.
Effect of Soluble Glycosaminoglycans on Tat
Internalization--
What is the receptor for Tat internalization?
Earlier studies indicate that cellular entry and HIV-1-LTR
transactivation activity of exogenous Tat are inhibited by heparin and
that heparin-Tat interaction involves the basic domain of Tat (25,
46, 54-56). Heparin is a close structural homologue of the HS GAG, a
major constituent of cell surface and extracellular matrix
proteoglycans (reviewed in Refs. 61-63), thus suggesting that heparin
could compete with cell surface HS proteoglycans for binding to Tat, as
observed for other heparin binding growth factors. We therefore tested whether other soluble GAGs could inhibit Tat internalization. Hamster
CHO K1 cells were incubated with recombinant GST-Tat-GFP in the
presence of different GAGs, and internalization of the fluorescent
protein was visualized by flow cytometry. The CHO K1 cell line was
chosen since mutants genetically impaired in GAGs biosynthesis
are available. As shown in Fig. 2,
heparin (average molecular mass 13.6 kDa) almost completely prevented
Tat entry at a concentration as low as 1 µg/ml. In contrast,
chondroitin sulfate B (dermatan sulfate), chondroitin sulfate A, and
chondroitin sulfate C caused only a very limited inhibition at the
highest concentrations tested.
GAGs are characterized by differences in their negative charge density
and saccharide composition. In previous experiments, we observed that
high affinity interaction with Tat is dependent on the extent of
sulfation of the heparin chain, which imparts an average charge density
higher than that of chondroitin sulfates (55). To understand whether
inhibition of Tat entry by heparin depends on specific structural
requirements of the GAG chain or simply by its ionic charge,
competitive inhibition studies were also performed using dextran
sulfate (average molecular mass 5 kDa), a homogeneous highly sulfated
polysaccharide. As shown in Fig. 2, high concentrations of dextran
sulfate (>10 µg/ml) were necessary to inhibit Tat internalization.
Thus, on a molar basis heparin appears to be a Tat antagonist at least
30 times more potent than dextran sulfate, indicating that both
saccharide composition and charge distribution of the heparin chain
play an essential role in Tat interaction.
The inhibition of Tat internalization by soluble GAGs was further
confirmed by determining the ability of different GAGs to inhibit HIV-1
LTR promoter transactivation by the recombinant Tat-GFP protein. CHO K1
cells were transiently transfected with a plasmid containing the
reporter CAT gene under the control of the HIV-1 LTR promoter, and
after 48 h, they were incubated with the Tat protein in the
presence of different concentrations of GAGs. In keeping with the flow
cytometric analysis data, heparin showed a dose-dependent
inhibition of LTR transactivation by extracellular Tat, whereas all the
chondroitin sulfates were ineffective, and dextran sulfate inhibited
LTR transactivation only at 100 µg/ml (Fig.
3). It is interesting to note that
heparin appears to be more potent in inhibiting cell internalization
(dose inhibiting 50% of uptake < 1 µg/ml) than LTR
transactivation activity (ID50 = 5 µg/ml),promoted by
saturating concentrations of extracellular Tat (1 µg/ml). This
observation is in agreement with the notion that limited amounts of Tat
are sufficient to exert a still potent transactivation activity (see
Fig. 1B).
Tat Uptake Requires Cell Surface Heparan Sulfate
Proteoglycans--
Altogether, the above-reported observations
indicate that the interaction between Tat and heparin/HS is specific
and suggest a role for cell surface HS proteoglycans in Tat uptake. A
large body of evidence indicate that cell surface HS proteoglycans are rapidly internalized through an endocytic pathway and may directly internalize ligands that bind to their GAG chains. Such a mechanism of
entry has already been described for other HS proteoglycan ligands,
including basic fibroblast growth factor, bacteria, and animal viruses
(57, 63). To test whether this was the case also for Tat, we studied
Tat internalization after removal of different cell surface GAGs by
specific lyases. CHO K1 cells were treated with the enzymes indicated
in Fig. 4, incubated with the Tat-GFP
protein, and analyzed 5 h later by flow cytometry to assess the
amount of intracellular fluorescence. Cell treatment with heparitinase
(heparinase-III), an enzyme mostly active on HS (64), impaired Tat
internalization in a dose-dependent manner. In contrast, treatment with heparinase I, known to be active on heparin but at a
much lesser extent on HS (64), showed detectable inhibition of Tat
internalization only at high concentrations. Treatments with
chondroitinase ABC, cleaving at a linkage found in all chondroitin sulfates including dermatan sulfate (chondroitin sulfate B), or with
chondroitinase AC, digesting specifically chondroitin sulfates A and C,
were completely ineffective in inhibiting Tat internalization.
To provide a final genetic proof that cell surface HS proteoglycans act
as major receptors for Tat internalization, we analyzed different
mutant cell lines originated from CHO K1 cells and defective in GAG
biosynthesis (57, 65, 66). Wild type and mutant cells were treated by
the addition of the recombinant Tat-GFP protein to the cell culture
medium, and cellular fluorescence was analyzed at different time
intervals by flow cytometry (Fig. 5). In
agreement with the data shown in Figs. 2 and 4, internalization of the
protein was clearly detectable in the wild type CHO K1 cells, and it
was proportional to the time of treatment.
In contrast, Tat entry was undetectable in pgs A-745 mutant cells,
which are defective in the enzyme xylosyltransferase, which initiates
GAG synthesis, and it was very reduced in pgs B-618 cells, which lack
galactosyltransferase-I activity catalyzing the second step in GAG
synthesis and produce about 15% of the amount of GAGs synthesized by
wild type cells (65, 66). Cell line pgs C-605, which is deficient in
sulfate transport but retains its sulfated GAG synthetic capacity (58),
behaved as wild type cells. In contrast, very little Tat-GFP
internalization was observed in the HS-deficient pgs D-677 cells,
bearing a single mutation that affects both
N-acetylglucosaminyltransferase and glucuronosyltransferase activities, which are necessary for the polymerization of HS
disaccharide chains (66). Interestingly, in these cells the defect in
HS production is paralleled by an increase in chondroitin sulfate synthesis that is, however, unable to support Tat internalization. Finally, Tat entry was severely impaired also in the mutant pgs E-606
cell line (66), which produces an N-undersulfated form of HS
due to a defect in the HS N-sulfotransferase enzyme. This observation further supports the notion that selective sulfation of HS
is an important determinant for Tat/HS recognition (55).
Next, the ability of CHO K1 mutants to support Tat-mediated LTR
transactivation was tested by transient transfection of an LTR reporter
gene construct followed either by treatment with recombinant Tat or by
co-transfection of a Tat-expressing plasmid. All cells efficiently
supported Tat functions when this was expressed as an endogenous
protein, indicating that the intracellular mechanisms mediating Tat
transactivation were intact in all these cell lines (Fig.
6B). In contrast, mutants pgs
A-745 and pgs B-618 were markedly impaired in LTR transactivation by
exogenously added Tat (Fig. 6A). The residual LTR
transactivation activity in the former mutant is most likely
attributable to the low level of xylosyltransferase activity
(
Overall, these data indicate that cell surface HS proteoglycans are the
major cellular receptors for Tat internalization. In addition, the
reduced ability of Tat entry in pgs E-606 cells suggests that the
density and distribution of sulfate groups in HS are important
determinants for the efficiency of this process.
Transcellular Transactivation--
Tat is released from expressing
cells and enters neighboring cells through an endosome-mediated
pathway. To assess the role of cell surface proteoglycans in this
physiological process, we obtained wild type CHO K1 and GAG-deficient
pgs A-745 cell clones constitutively expressing Tat-GFP. The two cell
lines were undistinguishable by fluorescent microscopic examination and
showed nucleoplasmic localization of the expressed protein (Fig.
7A). Both cell lines were
co-cultured for 72 h with wild type hamster CHO K1 or human HeLa
cells previously transfected with an LTR-CAT reporter gene construct.
In both cases, transactivation of the LTR promoter contained in the
latter cells could be readily detected (Fig. 7B). In
contrast, transactivation did not occur when the reporter cells were
the GAG-deficient pgs A-745 mutants (Fig. 7B). Thus, in
keeping with the results obtained with exogenously added recombinant Tat protein, cell surface proteoglycans mediate internalization of
endogenously expressed Tat released from producing cells. It is worth
noting that the capacity of both CHO/Tat-GFP and pgs A-745/Tat-GFP
transfectants to release significant amounts of biologically active Tat
indicates that cell surface proteoglycans are not involved in the
process of Tat export from the cell.
The results presented here provide genetic and biochemical
evidence that cell-associated HS proteoglycans function as cell surface
receptors for extracellular Tat internalization. This conclusion is
supported by the findings that Tat uptake is inhibited by heparin
but not by other soluble GAGs, that cell treatment with GAG lyases
specific for HS, but not for chondroitin sulfates, blocks Tat
internalization, and that cell lines with genetic defects in the
cellular pathway involved in the production of sulfated HS
proteoglycans fail to internalize Tat. The identification of HS
proteoglycans as cell surface receptors for Tat internalization is
consistent with the notion that Tat is able to enter into a wide
variety of human, rodent, and simian cell lines, indicating that it
utilizes a ubiquitous cell surface molecule for cell entry. This
conclusion is also in agreement with the observation that extracellular
Tat enters most of the exposed cells, as concluded by the narrow FACS
peaks observed after treatment with recombinant Tat-GFP and by the
immuno-staining results shown in Fig. 1E.
A common peptidic motif for heparin/HS binding consists of a region
rich in basic amino acids flanked by hydrophobic residues (61-63). The
arginine-rich domain of Tat (amino acids 49-57) conforms to these
characteristics. Consistently, this domain is sufficient to promote
cell internalization of Tat and of other tat-fusion proteins (46, 52,
67). We have recently observed that mutation of the arginines in this
domain or its occupancy by polysulfonated compounds prevents heparin
binding and cell internalization of GST-Tat, respectively (55).
Similar to the interaction of heparin with other cellular
macromolecules (68-70), binding of Tat to heparin/HS is most likely determined by both ionic interactions and specific structure
recognition. Other small basic proteins (having isolectric points and
sizes comparable with those of Tat) such as histone H1 and basic
fibroblast growth factor, although binding to heparan sulfate, cannot
enter the cells through this interaction nor mediate protein
transduction (71, 72). Additionally, other proteins with the same
characteristics such as cytochrome c do not even bind with
high affinity to heparan sulfates (41).
Tat/HS binding affinity is proportional to the size of heparin
oligosaccharides, with at least six saccharide residues required for
this interaction to occur (54, 56). Biochemical data also indicate that
selective 2-O-, 6-O-, or
N-desulfation/N-acetylation dramatically reduce
the capacity of heparin to bind Tat (55). This observation agrees with
the results obtained with pgs E-606 cells, which produce
N-undersulfated HS and are severely impaired in Tat
internalization. Finally, the requirement of structure recognition for
the Tat-HS interaction to occur is further supported by the observation
that several highly negatively charged molecules such as chondroitin
sulfates, dextran sulfate (see Fig. 2), and sulfated The identification of cell membrane HS proteoglycans as receptors for
extracellular Tat uptake and the observation that Tat can drive
internalization of larger protein cargos inside the cells with a
relatively rapid kinetics further support the use of Tat as a
biotechnological tool for protein transduction. This property of Tat
can be exploited either by obtaining recombinant proteins of
pharmacological interest fused to Tat or Tat peptides (52, 53) or by
expressing these fusions in vivo after appropriate gene
transfer. In this respect, it should be observed that the molecular
mechanisms that promote escape of internalized Tat from the endosomal
vesicles are still largely unexplored. The immunofluorescence data
shown in Fig. 1E indicate that at 4 h after the
addition to the cell culture medium the protein has a clear nuclear
localization. Previous experiments in which we measured the levels of
LTR-driven reporter gene mRNA in cells treated with extracellular
Tat indicated that LTR transactivation is maximal after ~5 h, whereas
it progressively decreases at later time points to become undetectable
after 12 h (48). These data are consistent with the pattern of
residual Tat localization at 24 h, when immunoreactive Tat protein
is still appreciable inside the cells but with a distribution that is
no longer nuclear and might be compatible with its localization within late endosomal-lysosomal vescicles. Similar to the exit of internalized Tat from endosomes, the molecular mechanisms responsible for Tat release from expressing cells are also poorly understood. The results
presented in Fig. 7 on the capacity of GAG-deficient pgs A-745 cells to
sustain Tat release clearly indicate that cell surface proteoglycans
are not involved in this process.
Finally, the results presented in Fig. 7 as well as other consistent
results reported in the literature (37-40) clearly indicated that the
process of Tat-mediated transcellular transactivation is not limited to
the experimental use of high doses of recombinant Tat added to the
extracellular medium but is easily observable for endogenously produced
Tat released by expressing cells. This event is even more significant
when considering that even very limited amounts of Tat are sufficient
to produce a significant level of viral LTR transactivation (45). What
is the functional significance of transcellular transactivation? It has
been suggested that under some conditions Tat might act as a viral
growth factor to stimulate viral replication in latently infected cells
(45). Alternatively, it might be envisaged that the pleiotropic
functions of Tat on the expression of several cellular genes could
stimulate the generation of a cellular environment more permissible for viral infection. Should additional experiments indicate that this process has pathogenetic significance during the course of HIV infection, the interaction between Tat and cell surface HS
proteoglycans would also constitute a novel important target for
therapeutic intervention.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B; several of the pleiotropic functions of
extracellular Tat could be mediated by this pathway (47-50).
-galactosidase protein fused to 11 amino
acids encompassing the arginine-rich region of Tat results in delivery
of the fusion protein to virtually all tissues in mice (53).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Kinetics of uptake of extracellular Tat
fusion proteins. A, time-dependent
transactivation activity of exogenous recombinant GST-Tat and
GST-Tat-GFP. The recombinant proteins (200 ng/ml), purified as GST
fusions, were added to the culture medium of HL3T1 cells containing an
integrated LTR-CAT construct. At the indicated time points, cells were
extensively washed, and fresh medium was added. CAT activity was
determined by enzyme-linked immunosorbent assay after an additional
24 h. Experiments were performed at least in triplicate; shown are
the mean value and S.D. for each measurement. As a control, the mutant
recombinant protein GST-Tat R(49-57)A (200 ng/ml) was used. This
protein contains arginine to alanine substitutions at positions 49, 52, 53, 55, 56, and 57 and is neither able to enter the cells nor to
activate transcription when expressed endogenously (47). B,
dose-dependent transactivation activity of exogenous
recombinant GST-Tat and GST-Tat-GFP. Transactivation of the LTR-CAT
construct was analyzed in HL3T1 cells after a 24-h treatment with
increasing amounts of GST-Tat and GST-Tat-GFP. C,
internalization of radiolabeled GST-Tat. HL3T1 cells were incubated in
serum-free medium containing 0.15% gelatin and 20 mM Hepes
buffer, pH 7.5, in the presence of 20 ng/ml [125I]GST-Tat
and 200 ng/ml unlabeled GST-Tat as a carrier. At different time
intervals, cells were washed and lysed, and radioactivity of cell
lysates was measured. Under these experimental conditions, up to 90%
of radioactivity remained associated with the cells after a wash with
2.0 M NaCl in sodium acetate, pH 4.0, thus demonstrating
the intracellular localization of cell-associated
[125I]GST-Tat (55). D, analysis of
internalization of GST-Tat-GFP by flow cytometry. HL3T1 cells were
incubated for 2, 4, and 24 h with recombinant GST-Tat-GFP (1 µg/ml), washed extensively, and then analyzed for intensity of
fluorescence by flow cytometry. Cellular fluorescence was specifically
due to Tat-mediated internalization of the recombinant protein, since a
GST-GFP fusion (1 µg/ml) was unable to modify cellular fluorescence
even after a 24-h treatment (gray-filled profile). The
rightmost peak shows the fluorescence of a CHO K1 cell clone
stably expressing the Tat-GFP fusion protein (see Fig. 7A).
E, subcellular localization of internalized GST-Tat. HL3T1
cells were treated with recombinant GST-Tat (1 µg/ml) for the
indicated time intervals. After treatment, cells were extensively
washed, fixed, and reacted with an anti-Tat antibody followed by
recognition with a rhodamine-labeled secondary antibody
(panels on the right side). In the same preparations, nuclei
were also visualized by reactivity to Hoechst 33342 (panels
on the left side). The two uppermost panels show cells
treated for 24 h with a recombinant GST protein (1 µg/ml) as a
control. OD, absorbance.
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Fig. 2.
Internalization of GST-Tat-GFP in cells
treated with soluble GAGs. CHO K1 cells were incubated with 1 µg/ml recombinant GST-Tat-GFP in the presence of the indicated
concentrations of heparin (Hep), chondroitin sulfate A, B,
and C (CS-A, CS-B, and
CS-C, respectively) and dextran sulfate
(DS). After 14 h, cells were extensively washed and
analyzed by flow cytometry.
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Fig. 3.
Inhibition of Tat transactivation by soluble
GAGs. CHO K1 cells were transfected with an LTR-CAT plasmid and,
after 48 h, treated with 1 µg/ml GST-Tat-GFP for 14 h in
the presence of the indicated concentrations of soluble GAGs. CAT
assays were performed after an additional 36 h.
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Fig. 4.
Effects of GAG lyases on the uptake of
extracellular Tat. CHO K1 cells were incubated with the indicated
GAG lyases for 40 min and then incubated with 1 µg/ml GST-Tat-GFP for
5 h; this short incubation time was chosen to minimize synthesis
of novel proteoglycans. After this time period, cells were extensively
washed and analyzed by flow cytometry. Hep I, heparinase I;
Hep III, heparinase III; ChonAC, chondroitinase
AC; ChonABC, chondroitinase ABC.
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Fig. 5.
Tat internalization into cells genetically
defective in GAG biosynthesis. Wild type (wt) CHO K1
cells and the indicated cell clones mutated at different steps in GAG
biosynthesis were treated with 1 µg/ml GST-Tat-GFP and analyzed by
flow cytometry after 2, 5, 9, and 24 h of treatment. In each
panel, the uppermost FACS profile shows fluorescence of CHO
K1 cells clone stably expressing Tat-GPF (see Fig. 7A). The
main recognized defect of each cell line is indicated.
normal), which is still present in these cells and might
be sufficient for the production of limited amounts of proteoglycans
(66). Consistent with the GST-Tat-GFP internalization data, the
specific role of HS proteoglycans in Tat entry was indicated by the
results obtained with pgs D-677 cells, overproducing chondroitin
sulfate, and pgs E-606 cells, producing an undersulfated form of HS. In
both cell lines, LTR transactivation by recombinant Tat was clearly
impaired. In contrast, LTR transactivation by exogenous Tat was normal
in pgs C-605 cells.
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Fig. 6.
HIV-1 LTR transactivation in cells
genetically defective in GAG biosynthesis. Wild type
(wt) CHO K1 cells and the indicated cell clones mutated at
different steps in GAG biosynthesis were transfected with an LTR-CAT
reporter plasmid (10 µg) and either treated with recombinant GST-Tat
(200 ng/ml; panel A) or co-transfected with a Tat-expressing
plasmid (1 µg; panel B). CAT assays were performed after
48 h. The results shown represent average values and S.D. obtained
in several (at least three) independent transfections.
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Fig. 7.
Transcellular transactivation.
A, fluorescence pattern of CHO clones producing Tat-GFP.
Cellular clones stably expressing Tat-GFP were obtained for wild type
CHO K1 and pgs A-745 cells impaired in proteoglycan production (left
and right sides, respectively). In both cell lines, the protein showed
predominant nuclear localization with exclusion of the nucleoli. , results of co-culture experiments. Wild type (wt)
CHO/Tat-GFP and mutated pgs A-745/Tat-GFP cells were co-cultured (1:1
ratio) for 72 h with wild type CHO K1, pgs A-745, and HeLa cells
that had been previously transfected with an LTR-CAT reporter plasmid.
The histograms indicate the average levels and S.D. of LTR-CAT
transactivation obtained in three independent experiments. As a further
control of the transcellular transactivation of the Tat-GFP protein
produced by CHO/Tat-GFP and pgs A-745/Tat-GFP cells, these were also
co-cultured with HL3T1 cells, a HeLa derivative containing an
integrated LTR-CAT construct.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin
(55) are poor Tat antagonists. This observation is in agreement with
the inability of pgs D-677 cells, which do not produce HS, to support
Tat internalization despite their overproduction of cell surface
chondroitin sulfates. Altogether, from these considerations, it may be
concluded that the interaction between Tat and heparin/HS is specific
and is determined by size, saccharide composition, and extent and
distribution of sulfation of the GAG backbone.
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ACKNOWLEDGEMENTS |
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We thank B. Boziglav for excellent technical assistance. We are grateful to B. Berkhout for plasmid pBlue-LTR-CAT and to B. Felber for the HL3T1 cell line.
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FOOTNOTES |
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* This work was supported by grants from the National Research Program on AIDS of Istituto Superiore de Sanita' (to M. G. and M. P.); from the Associazione Italiana per la Ricerca sul Cancro (to M. P.); from the National Research Council (Target Project on Biotechnology; to M. P.) and from the Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica (to M. G. and M. R.)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.
§ Supported by a pre-doctoral fellowship of the International School for Advanced Studies (ISAS) of Trieste, Italy.
** To whom correspondence should be addressed: Molecular Medicine Laboratory, ICGEB, Padriciano, 99, 34012 Trieste, Italy. Tel.: 39-040-3757.324; Fax: 39-040-226555; E-mail: giacca@icgeb.trieste.it.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M006701200
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus-1; LTR, long terminal repeat; HS, heparan sulfate; CHO, Chinese hamster ovary; GAG, glycosaminoglycan; CAT, chloramphenicol transferase; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; GST, glutathione S-transferase.
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