New Insights into the Functionality of a
Virion-Anchored Host Cell Membrane
Protein: CD28 Versus HIV Type 1
This information is current as
of June 18, 2017.
Jean-François Giguère, Jean-Sébastien Paquette, Salim
Bounou, Réjean Cantin and Michel J. Tremblay
J Immunol 2002; 169:2762-2771; ;
doi: 10.4049/jimmunol.169.5.2762
http://www.jimmunol.org/content/169/5/2762
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References
The Journal of Immunology
New Insights into the Functionality of a Virion-Anchored Host
Cell Membrane Protein: CD28 Versus HIV Type 11
Jean-François Giguère,2 Jean-Sébastien Paquette,2 Salim Bounou, Réjean Cantin, and
Michel J. Tremblay3
E
nveloped retrovirus particles are formed by extrusion
through the host cell membrane. During this process, the
newly formed viral entities become coated with a lipid
bilayer derived from the cell membrane. The specificity of this
process is questioned by the observation of viral pseudotyping.
Pseudotyping with heterologous viral glycoproteins has been observed on many occasions, e.g., murine leukemia virus envelope
proteins in HIV type 1 (HIV-1)4 particles (1), envelope proteins
from human T cell leukemia virus types 1 and 2 in vesicular stomatitis virus (2), influenza virus hemagglutinin in Rous sarcoma
virus particles (3), human T cell leukemia virus-1 envelope glycoprotein in HIV-1 (4), and vesicular stomatitis virus G protein in
both murine retroviral and lentivirus vector particles (5). The low
specificity of the process governing glycoprotein incorporation
into retroviral particles is most likely responsible for the observaCentre de Recherche en Infectiologie, Centre Hospitalier de l’Université Laval, Centre Hospitalier Universitaire de Québec, and Faculté de Médecine, Département de
Biologie Médicale, Université Laval, Sainte-Foy, Québec, Canada
Received for publication February 19, 2002. Accepted for publication June 28, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by Grant no. HOP-14438 from Canadian Institutes of
Health Research HIV/AIDS Research Program (to M.J.T.). J.-F.G. is supported by a
PhD Fellowship from the Fonds de la Recherche en Santé du Québec/Fonds pour la
Formation de Chercheurs et l’Aide à la Recherche, while S.B. holds a PhD Fellowship
from the Canadian Institutes of Health Research. M.J.T. is the recipient of a Tier 1
Canada Research Chair in Human Immuno-Retrovirology.
2
J.-F.G. and J.-S.P. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Michel J. Tremblay, Laboratoire
d’Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709,
Centre Hospitalier de l’Université Laval, Centre Hospitalier Universitaire de Québec,
2705 Boulevard Laurier, Sainte-Foy, Québec G1V 4G2, Canada. E-mail address:
[email protected]
4
Abbreviations used in this paper: HIV-1, HIV type 1; MDM, monocyte-derived
macrophage.
Copyright © 2002 by The American Association of Immunologists, Inc.
tion that retroviruses have been found to incorporate certain cellderived proteins in their envelope. For example, HIV-1 has been
reported to acquire a considerable number of cell surface proteins
including CD3, CD11a (LFA-1), CD11b (Mac-1), CD18, CD25,
CD43, CD44, CD54 (ICAM-1), CD55, CD59, CD63, CD71
(transferrin receptor), HLA-DR, HLA-DP, and HLA-DQ (6).
Several studies have scrutinized the functionality of some virion-anchored host proteins with respect to the biology of HIV-1.
The physical presence of host-encoded CD55 and CD59 was found
to protect the virus from complement-mediated virolysis (7).
Castilletti and coworkers (8) have reported that virus infectivity is
increased following treatment of U937 monocytoid cells with
IFN-␥ due to an enhancement of virion-bound host ICAM-1 and
HLA-DR proteins. MHC class-II (MHC-II) proteins on purified
HIV-1 particles can present super Ag to human T cells (9). In
addition, a synergy in virus neutralization was demonstrated to
occur between an anti-LFA-1 mAb and polyclonal anti-HIV-1 Abs
(10). The incorporation of ICAM-1 rendered HIV-1 virions less
susceptible to Ab-mediated neutralization (11, 12). Data from
time-course and infectivity experiments revealed that the kinetics
of infection was more rapid for virions bearing host-derived
MHC-II glycoproteins than for HIV-1 particles devoid of host
MHC-II (13). Additional experiments revealed that the presence of
host-derived HLA-DR1 and ICAM-1 on HIV-1 led to an enhancement of virus infectivity (1.6- to 2.3-fold increase for HLA-DR1;
4.6- to 9.8-fold increase for ICAM-1) by accelerating the kinetics
of virus entry and/or increasing the efficiency of the early steps in
the viral life cycle (14, 15). Other studies revealed that surface
expression of LFA-1 in its high-affinity state for ICAM-1 markedly
enhanced susceptibility of human cells to infection by ICAM-1bearing HIV-1 particles (11, 16). Recently, infectivity of primary
and laboratory T tropic isolates of HIV-1 was increased upon incorporation of foreign MHC-I molecule (17).
0022-1767/02/$02.00
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It is now well established that the HIV type 1 (HIV-1) incorporates a vast array of host-encoded molecules in its envelope during
the budding process. Interestingly, it was demonstrated that the attachment process is accentuated by supplementary interactions
between virion-anchored host molecules and their cognate ligands. Such an enhancement of the viral attachment process was
found to result in an increase of infectivity for both T and macrophage-tropic strains of HIV-1. Given that previous work indicates
that HIV-1 is budding at the site of cell-to-cell contact, a location rich in the costimulatory CD28 glycoprotein, we investigated
whether CD28 could be efficiently acquired by HIV-1. We have been able to generate progeny viruses bearing or not bearing on
their surfaces host-derived CD28 using our previously described transient transfection and expression system. The physical
presence of CD28 was found to markedly increase virus infectivity in a CD28/B7-dependent manner following infection of two
human lymphoid cell lines expressing high levels of surface B7-1/B7-2, two natural ligands of CD28. The physiological significance
of CD28 incorporation was provided by the observation that an anti-CD28 Ab decreased replication in primary human mononuclear cells of clinical isolates of HIV-1 propagated in such cells. A virus precipitation assay revealed that M-, T-, and dual-tropic
clinical strains of HIV-1 produced in primary human mononuclear cells do indeed incorporate CD28. These results show for the
first time that HIV-1 can incorporate CD28 and the acquisition of this specific host surface glycoprotein modulates the virus life
cycle. The Journal of Immunology, 2002, 169: 2762–2771.
The Journal of Immunology
Materials and Methods
Cells
RAJI is an EBV-carrying B cell line that has been reported to express very
high levels of cell surface MHC-II molecule (26). Moreover, RAJI cells
have been shown to express similar high levels of B7-1 (CD80) and B7-2
(CD86) (27). The RAJI cell line was rendered susceptible to HIV-1 infection by stable transfection with a cDNA encoding for human CD4 (i.e.,
RAJI-CD4) (13). Flow cytometric analyses indicate that RAJI and RAJICD4 cell lines express comparable levels of CXCR4 and are both negative
for CCR5 (data not shown). The LuSIV cell line was derived from the
CEM ⫻ 174 cells and was stably transfected with the SIVmac239 long
terminal repeat (⫺225 3 ⫹149) cloned upstream of the firefly luciferase
reporter gene (kindly supplied by Dr. J. E. Clements, Johns Hopkins University School of Medicine, Baltimore, MD). LuSIV cells are highly sensitive to infection by HIV and SIV, resulting in a Tat-mediated expression
of the reporter gene, which correlates with viral infectivity (28). FACS
analysis revealed that LuSIV cells express high levels of both B7.1 and
B7.2 molecules (data not shown). RAJI, RAJI-CD4, and LuSIV cell lines
were maintained in complete culture medium made of RPMI 1640 supplemented with 10% FBS (Invitrogen, San Diego, CA), glutamine (2 mM),
penicillin G (100 U/ml), and streptomycin (100 ␮g/ml). 293T cells are
human embryonic kidney cells that express the simian virus 40 large T Ag
and were kindly provided by Dr. W. C. Greene (J. Gladstone Institutes, San
Francisco, CA). These cells were cultured in DMEM supplemented with
10% FBS, L-glutamine (2 mM), penicillin G (100 U/ml), and streptomycin
(100 ␮g/ml). Flow cytometric analysis revealed that 293T cells are negative for CD28 expression (data not shown). Primary human PBMCs from
healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation. To obtain monocyte-derived macrophages (MDM), PBMCs were
incubated for 1 h at 37°C in 48-well flat-bottom tissue culture plates (Microtest III, Falcon; BD Biosciences, Lincoln Park, NJ) (3 ⫻ 106 cells/ml,
500 ␮l/well). Cells were next washed twice with PBS to remove unadhered
cells and kept in culture for 3 days in RPMI 1640 supplemented with 20%
FBS in the presence of IFN-␥ (500 U/ml). Human tonsillar tissues were
prepared as described previously (29).
Plasmids and preparation of virus stocks
The pHXB-Luc vector leads to the production of single-round infectious
X4 T cell line-adapted viruses, whereas p89.6 is a plasmid that encodes for
the dual-tropic (i.e., R5X4) cytopathic HIV-1 primary isolate 89.6 (30).
The pH␤Apr-1-neo vector codes for a wild-type version of human CD28
and has been described previously (31) (a generous gift from Dr. D. Olive,
Institut National de la Santé et de la Recherche Médicale Unité 119, Marseille, France). Viral particles differing only by the absence (i.e., CD28⫺)
or the presence (i.e., CD28⫹) of host-encoded CD28 proteins on their surface were produced by calcium phosphate coprecipitation in 293T cells as
described previously (15, 16). Virus stocks were normalized for virion
content using an in-house double Ab sandwich ELISA specific for the
major viral p24 protein (29). The standardization on p24 contents is based
on our previous observation indicating that virus preparations harvested
from transfected 293T cells contain minimal amounts of p24 that are not
associated with infectious virions (15). In some experiments, clinical
strains of HIV-1 were produced in acutely infected PBMCs from healthy
donors. At the maximal virus production and before extensive cytopathic
effects were seen, cells were centrifuged at 300 ⫻ g for 5 min and the
virus-containing supernatants were clarified at 2000 ⫻ g for 30 min and
filtered through a 0.45-␮m cellulose acetate membrane to remove cellular
debris. Thereafter, the virus-containing supernatants were stored at ⫺80°C
in aliquots. Two X4 T tropic (i.e., 92HT599 and 93UG070), one R5 macrophage tropic (i.e., 92HT026), and one R5X4 dual tropic (i.e., 92RW009)
primary isolates of HIV-1 were used in our studies (obtained through the
AIDS Research and Reference Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).
Abs and purified fusion/recombinant proteins
The mAb 9.3 is specific for human CD28 and inhibits interaction between
CD28 and B71-/B7-2 (32, 33), while BB-1 is a neutralizing mAb directed
against B7-1 (CD80) (34). Both Abs were kindly provided by Dr. J. A.
Ledbetter (Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, NJ). The two mAbs, BU-63 and FUN-1, were shown to be
specific for B7-2 (CD86) (35). BU-63 has been supplied by Dr. D. L.
Hardie (University of Birmingham, Birmingham, U.K.) and FUN-1 is a
kind gift from Dr. Y. Nozawa (Fukushima Medical College, Fukushima,
Japan). CTLA-4Ig is constituted of the extracellular domain of CTLA-4
fused to the Fc fragment of IgG1. Previous experiments have indicated
that this fusion protein demonstrates a strong affinity for B7-1 and B7-2
molecules and blocks the interaction between CD28 and B7-1/B7-2
(36). FITC-conjugated goat anti-mouse Ab was purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA). SIM.2 is a mAb specific
for human CD4 that binds to a different epitope than Leu 3a, whereas
SIM.4 is also an anti-CD4 Ab, but recognizes the same epitope as Leu 3a
(37). Both Abs block the process of HIV-1 infection, although SIM.2
shows a stronger anti-HIV-1 capacity (38).
Cytofluorometric analyses
Expression of CD28 on the surface of transiently transfected 293T cells
was monitored with the anti-CD28 9.3 Ab. Briefly, an aliquot of transfected
293T cells (1 ⫻ 106) was washed with PBS (pH 7.4). Pelleted cells were
incubated for 30 min on ice with a saturating concentration of the primary
9.3 Ab (1 ␮g/106 cells) in a final volume of 100 ␮l of PBS. The cells were
washed twice with 500 ␮l of PBS and incubated for 30 min with a saturating concentration of FITC-conjugated goat anti-mouse Ab (1 ␮g/106
cells) in a final volume of 100 ␮l of PBS. Finally, cells were washed twice
with PBS and resuspended in 300 ␮l of PBS containing 1% (w/v) paraformaldehyde before cytofluometric analysis (EPICS Elite ESP; Coulter
Electronics, Miami, FL). The same technical strategy was used for monitoring expression of B7-1 and B7-2 on the surface of target cells used for
HIV-1 infection. In this case, monoclonal anti-B7-1 (clone BB-1) and antiB7-2 (clone FUN-1) Abs were used. Surface expression of CD28 on CD4positive T lymphocytes from PBMCs originating from a single healthy
donor was monitored by two-color flow cytometry. PBMCs (1 ⫻ 106) were
first incubated for 30 min on ice with a saturating concentration of the 9.3
Ab (1 ␮g/106 cells) in a final volume of 100 ␮l of PBS. The cells were
washed twice with 500 ␮l of PBS and incubated for 30 min with a saturating concentration of PE-conjugated goat anti-mouse Ab (1 ␮g/106 cells)
in a final volume of 100 ␮l of PBS. Cells were then washed twice and
incubated for 30 min with an FITC-conjugated anti-CD4 mAb (i.e.,
SIM.4). Cells were then washed twice and resuspended in 300 ␮l of PBS
containing 1% (w/v) paraformaldehyde before cytofluometric analysis.
Virus capture assay
The physical presence of host-encoded CD28 glycoproteins on the surface
of HIV-1 particles was investigated using streptavidin-coated magnetic
beads in combination with a biotinylated monoclonal anti-CD28 Ab as
described previously (39). In brief, after incubation of streptavidin-coated
magnetic beads with biotinylated Ab (1 h at room temperature), beads were
washed twice and resuspended in PBS ⫹ 0.1% BSA before used for capture assay. Immunomagnetic beads were incubated overnight at 4°C on a
rotating plate with studied virus preparations (2.5–10 ng of p24). Next,
beads were extensively washed and resuspended in PBS ⫹ 0.1% BSA and
viral p24 contents were quantitated by the p24 assay. A complex made of
streptavidin-coated magnetic beads and biotinylated monoclonal antiCD45 Ab (clone UCHL-1) was used as a negative control based on the
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The homodimeric CD28 glycoprotein is not present on the surface of cells of the monocyte/macrophage lineage, but is constitutively expressed on virtually all human CD4⫹ T lymphocytes
and ⬃50% of CD8-expressing T cells (18, 19). The CD28-mediated signal transduction pathway is considered as one of the dominant costimulatory pathways to achieve the complete activation of
the T cell (20). The CD28 cosignal is triggered by the ligation of
CD28 with its physiological B7-1 (CD80) and B7-2 (CD86) counterligands, which are normally expressed on the surface of the
APC. CD28 must be physically located near the cell-to-cell contact
site between the T cell and the APC to be able to bind to its natural
counterreceptors. Given that a unidirectional budding of HIV-1 has
been shown to occur at the site of cell-to-cell contact (21), it can
be proposed that CD28 is also present within the virus envelope.
The possible acquisition of CD28 by HIV-1 might reveal functional implications considering that B7-1 and B7-2 molecules are
expressed on activated monocytes/macrophages as well as on activated T lymphocytes (18, 22–25), two cell types recognized as
reservoirs of HIV-1 in infected individuals. Thus, the primary objective of the current work was to define whether host-derived
CD28 is incorporated into the HIV-1 envelope, and if so, to study
the functional effect(s) on the HIV-1 biology of such a virus-anchored host constituent.
2763
2764
VIRION-ANCHORED HOST CD28 AND HIV-1
observation that CD45 is excluded from the virus envelope (40). A more
physiological assay was used to assess the presence of foreign CD28 proteins on the exterior of viral entities. This biological test is based on incubation of virus preparations with RAJI, a CD4-negative cell line expressing similar high surface levels of B7-1 and B7-2 (27). Briefly, virus
stocks (20 ng of p24) were exposed to 2.5 ⫻ 105 RAJI cells for 1 h at 37°C
in a total volume of 250 ␮l of complete culture medium. Next, cells were
gently washed twice with 500 ␮l PBS and the pellet was resuspended in
150 ␮l PBS before monitoring for p24 content. Appropriate controls consisted of the RAJI/virus mixture incubated in the presence of either CTLA4Ig, an anti-B7-1 (clone BB-1), or an anti-B7-2 (clone FUN-1) Ab.
Virus infection and luciferase assay
FIGURE 1. Flow cytometric analysis of 293T cells transfected with
pHXB-Luc and a vector coding for human CD28. 293T cells were transiently transfected with pHXB-Luc alone (A) or were cotransfected with
pHXB-Luc and pH␤Apr-1-neo (B). Surface expression of CD28 was monitored by labeling cells with a monoclonal anti-CD28 Ab (clone 9.3, dotted
lines) or an isotype-matched irrelevant commercial control Ab (straight
lines) followed by an FITC-conjugated goat anti-mouse IgG Ab.
HIV-1 infection of MDM and tonsils
MDM were infected with the 89.6 strain of HIV-1 as follow. Culture medium was first removed from the 48-well plates and replaced by an equal
amount of each virus preparation (CD28⫺ and CD28⫹) for 1 h at 37°C (20
ng of p24 in a final volume of 200 ␮l). The cells were next washed twice
with PBS and 500 ␮l of fresh medium was added. After 3 days of infection,
the supernatant was harvested and p24 was measured by ELISA. Tonsil
infection was conducted by slowly applying to the top of each tissue block
an equal amount of each virus preparation (i.e., 5 ␮l of virus stock containing 2.5 ng of p24). The next day, the culture medium was replaced and
the infection was allowed to proceed for 14 days. Half of the medium was
removed at days 6, 9, and 14 and replaced by fresh culture medium. The
concentration of p24 in the culture supernatant was measured by the enzymatic assay. Virus preparations used in this set of experiments were
either pretreated or not for 30 min at 37°C with the anti-CD28 mAb (clone
9.3; 1 ␮g/ml final concentration).
Viral entry assay
surface constituent (Fig. 1). Next, the presence of the costimulatory molecule CD28 on the exterior of HIV-1 particles was assessed using a functional binding assay. Given that RAJI cells are
CD4-negative and are expressing similar elevated levels of the
cognate ligands of CD28, i.e., B7-1 and B7-2 (data not shown), we
hypothesized that such cells could capture virions in a CD28-B71/B7-2-dependent mode. To directly assess the role played by
CD28-B7-1/B7-2 interaction in the possible attachment of CD28bearing progeny viruses on the surface of RAJI, experiments were
also conducted in the presence of the CTLA-4Ig fusion protein
because it binds to both B7-1 and B7-2, but with a 20- to 100-fold
higher affinity than CD28 (41). As shown in Fig. 2, CD28⫹ viruses
were found to bind more significantly to RAJI cells as compared
with CD28⫺ virions (4.5-fold increase, 416 vs 93 pg of captured
RAJI-CD4 cells (5 ⫻ 106 cells/ml, 250 ␮l/well) were exposed to similar
amounts of CD28⫺ and CD28⫹ virus preparations (50 ng of p24) in complete culture medium for 2 h at 37°C. Cells were washed twice with 250 ␮l
of ice-cold PBS and were next incubated for 5 min at 4°C with 250 ␮l of
cold RPMI 1640 (without FBS) supplemented with pronase (Boehringer
Mannheim, Laval, Quebec, Canada) at 0.1 mg/ml. Cells were washed immediately with 2 ml of ice-cold RPMI-10 containing 10% FBS and three
times with ice-cold PBS to eliminate pronase. Cells were resuspended in 1
ml of complete culture medium to which was added 200 ␮l of disruption
buffer (0.5% Triton X-100 in PBS). Finally, cells were agitated for 10 min
at room temperature and then stored at ⫺20°C until assayed for p24
content.
Results
The host-derived costimulatory molecule CD28 is efficiently
incorporated within the HIV-1 envelope
We initially used our previously described transient transfection
and expression system (15, 16) to define whether host cell surface
CD28 glycoprotein is efficiently acquired by mature HIV-1 particles. To this end, we transiently cotransfected 293T cells with an
infectious molecular clone of HIV-1 (pHXB-Luc) and a mammalian expression vector coding for the human CD28 glycoprotein
(pH␤Apr-1-neo). Flow cytometric studies indicated that cotransfection with both vectors resulted in expression of a high level of
CD28 in 293T cells that do not constitutively express this cell
FIGURE 2. Capture of isogenic CD28⫺ and CD28⫹ HIV-1 particles
with RAJI cells. Similar amounts of CD28⫺ and CD28⫹ virions, standardized in terms of p24 content, were incubated with RAJI cells for 1 h at 37°C
in the absence or the presence of the CTLA-4Ig fusion protein. Cells were
next washed several times with PBS and the levels of RAJI-associated viral
p24 were estimated by performing an in-house p24 enzymatic assay. Data
shown represent the means ⫾ SD from triplicate samples and these results
are representative of three different experiments.
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Similar amounts of each recombinant luciferase-encoding virus stocks (10
ng of p24 for CD28⫺ and CD28⫹) were used to inoculate RAJI-CD4 cells
(1 ⫻ 105) in a 96-well flat-bottom tissue culture plate (Microtest III, Falcon; BD Biosciences). In some experiments, cells were either left untreated
or were treated with CTLA-4Ig, the anti-B7-1 Ab BB1, or the anti-B7-2 Ab
BU-63 (10 ␮g/ml) for 30 min at 4°C before inoculation with HIV-1. Cells
were also treated when appropriate for 5 min at 37°C with increasing
concentrations of the anti-CD4 SIM.2 Ab (0, 0.2, 1, 2.5, 5, and 10 ␮g/ml)
before virus infection (5 ng of p24 for CD28⫺ or CD28⫹ virions). Virus
preparations used for the infection of LuSIV cells (strain 89.6) were pretreated or not for 30 min at 37°C with the anti-CD28 mAb (clone 9.3) or
an isotype-matched irrelevant control Ab (1 ␮g/ml final concentration)
before infection with HIV-1 (10 ng of p24 and 1 ⫻ 105 cells). Cells were
kept incubated at 37°C for 48 h (RAJI-CD4 and LuSIV). Finally, cells were
lysed and luciferase activity was monitored using a microplate luminometer (MLX; Dynex Technologies, Chantilly, VA). Luciferase activity is
expressed as relative light units.
The Journal of Immunology
2765
viral p24). The observed enhancement of attachment of CD28⫹
viral particles to RAJI cells was abrogated by CTLA-4Ig, thus
indicating that it is conferred by the interaction between virionanchored host CD28 and cell surface B7-1/B7-2.
Infectivity of CD28-bearing virions is markedly enhanced for
target cells expressing high levels of B7-1/B7-2
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We next assessed whether the acquisition of host-derived CD28 by
HIV-1 could modulate virus infectivity by up-regulating the virus
attachment/entry process. This possibility was tested using RAJICD4, a RAJI derivative stably expressing CD4 that has been
shown to be highly susceptible to HIV-1 infection (13). It should
be stressed that flow cytometric analyses revealed that RAJI-CD4
cells, as it is the case for the parental RAJI cell line, express considerable amounts of surface B7-1/B7-2. Virus infection experiments were performed with three distinct virus stocks originating
from independent transfections. As illustrated in Fig. 3A, CD28
increases virus infectivity for RAJI-CD4 cells by up to 12.3- to
17.2-fold. Virus infection studies were next conducted in the presence of CTLA-4Ig to demonstrate without any doubt the crucial
role played by virus-anchored host CD28 in the noticed increase in
virus infectivity. Treatment of the RAJI-CD4 and CD28⫹ HIV-1
particles mixture with CTLA-4Ig was found to totally abrogate the
increase in virus-encoded reporter gene activity conferred by virally embedded host CD28 (Fig. 3B). Similar results were obtained
following infection of another B7-1/B7-2-expressing cell line, i.e.,
LuSIV, with the dual-tropic strain 89.6. In this experimental setting, an 8-fold increase of HIV-1 infectivity was conferred by the
presence of host CD28 in the viral envelope (Fig. 3C). Again, the
enhancement of virus infectivity was abrogated by a pretreatment
with a blocker of the interaction between CD28 and B7-1/B7-2.
Interestingly, an isotype-matched irrelevant Ab (control Ab) had
no effect on infectivity of CD28-bearing virions.
It has been shown that B7-1 and B7-2 bind to overlapping but
not identical sites on CD28 (42– 45). Thus, we investigated the
involvement of either CD28/B7-1 or CD28/B7-2 interaction in the
observed enhancement of virus infectivity. As expected, infection
of RAJI-CD4 cells with virions devoid of host-encoded CD28
(CD28⫺) was unaffected by treatment with either BB-1 (anti-B7-1)
or BU-63 (anti-B7-2) (Fig. 4A). Infectivity of progeny viruses
bearing host CD28 in their envelope was more markedly affected
by the addition of the monoclonal anti-B7-1 Ab than by treatment
with the monoclonal anti-B7-2 Ab. The combined action of the
two Abs resulted in an almost complete inhibition of the increase
in virus infectivity conferred by the virion-anchored host CD28
glycoprotein. We next studied in further detail the ability of BB-1
and BU-63 to abrogate the increase in virus infectivity of CD28⫹
HIV-1 particles for RAJI-CD4 cells. As expected, treatment of the
mixture composed of CD28⫺ virions and RAJI cells (CD4-negative) with either BB-1 or BU-63 had no noticeable effect on viral
binding to RAJI cells (Fig. 4B). However, the overall attachment
process of CD28⫹ viruses to RAJI cells was more markedly diminished by BB-1 as compared with BU-63.
We also investigated whether the increase in the virus attachment of CD28-bearing virions to susceptible target cells could lead
to a concomitant augmentation of virus internalization. For this
purpose, we performed a virus entry assay based on the use of a
potent mixture of proteolytic enzymes (pronase) that eliminate
noninternalized virus particles attached to the cell surface (46, 47).
Results from Table I indicate that attachment of CD28⫹ HIV-1
particles to RAJI-CD4 cells is quantitatively more important as
compared with the binding efficiency of isogenic progeny viruses
devoid of host-encoded CD28 (3166 vs 536 pg/ml, i.e., a 6-fold
FIGURE 3. Infectivity of CD28⫺ and CD28⫹ HIV-1 particles when
using RAJI-CD4 and LuSIV cells as targets. A, Recombinant luciferaseencoding virus stocks (HXB-Luc) originating from three independent
transfection protocols were used to inoculate RAJI-CD4 cells (10 ng of
p24). B, Viral infection with isogenic CD28⫺ and CD28⫹ virions (10 ng of
p24) was performed with RAJI-CD4 cells either in the presence or the
absence of the CTLA-4Ig fusion protein. C, Isogenic CD28⫺ and CD28⫹
dual-tropic 89.6 virions were used to inoculate LuSIV cells (10 ng of p24).
Virus preparations were treated either with an anti-CD28 mAb (i.e., clone
9.3) or an isotype-matched irrelevant control Ab. Cells were incubated at
37°C for 48 h before evaluation of luciferase activity. Results shown are
the mean ⫾ SD of triplicate samples and these results are representative of
three different experiments.
increase). The removal of noninternalized virus particles attached
to the cell surface by pronase demonstrate that intracellular viral
p24 represented 12.3 and 33.7% of the total input for CD28⫺ and
2766
VIRION-ANCHORED HOST CD28 AND HIV-1
The degree of virion-anchored host CD28 and HIV-1 infectivity
are both influenced by the level of CD28 expression on the
surface of virus producer cells
The phenomenon of CD28 incorporation has physiological
significance for the HIV-1 life cycle
CD28⫹ virions, respectively. In these experiments, treatment of
the mixture composed of CD28⫹ progeny viruses and RAJI-CD4
cells with CTLA-4Ig decreased the percentage of virus entry to
14.9%, a value comparable to the one seen when RAJI-CD4 cells
are inoculated with CD28⫺ HIV-1 particles (i.e., 12.3%).
To explore the relative importance of the alternative infection
pathway (i.e., CD28 and B7-1/B7-2) by comparison with the major
pathway (i.e., gp120 and CD4), RAJI-CD4 cells were treated with
a known blocker of the gp120-CD4 interaction. As shown in Fig.
5A, the anti-CD4 SIM.2 Ab was found to mediate a dose-dependent inhibition of infection by both CD28⫺ and CD28⫹ viruses.
However, CD28⫹ progeny viruses were found to be more resistant
to neutralization by the anti-CD4 Ab than were CD28⫺ viruses
(Fig. 5B). Such results demonstrate the importance of the additional secondary CD28-B7-1/B7-2 interactions when the primary
CD4-gp120 association is compromised.
To directly demonstrate the relevance of CD28 incorporation in
HIV-1 biology, isogenic CD28⫺ and CD28⫹ dual-tropic virus
stocks were used to infect more natural B7-1/B7-2-expressing target cells. For this purpose, MDM were derived from fresh PBMCs
and grown for 3 days in the presence of IFN-␥ to induce expression of the costimulatory B7-1 and B7-2 molecules. FACS analyses revealed that this treatment led to a noticeable up-regulation
of B7-1 and B7-2 on the surface of MDM (data not shown). Such
IFN-␥-treated MDM were next inoculated with the dual-tropic
HIV-1 strain 89.6 bearing or not on its surface the CD28 glycoprotein. As depicted in Fig. 7A, CD28-bearing virions were found
to be more infectious for MDM than isogenic viruses devoid of
foreign CD28 (compare 7300 and 3400 pg/ml of p24). Pretreatment of our viral stocks with the anti-CD28 mAb 9.3 diminished
the process of infection with CD28⫹ virions, while it had no effect
on the replicative capacity of CD28⫺ HIV-1 particles.
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FIGURE 4. Infectivity and capture of CD28⫺ and CD28⫹ virions in the
presence of monoclonal anti-B7-1 and anti-B7-2 Abs. A, RAJI-CD4 cells
were infected with isogenic CD28⫺ and CD28⫹ viruses in the absence or
the presence of monoclonal anti-B7-1 (clone BB-1) and/or anti-B7-2 (clone
BU-63) Abs. Cells were incubated at 37°C for 48 h before evaluation of
virus-encoded luciferase activity. B, RAJI cells (CD4-negative) were exposed to CD28⫺ and CD28⫹ progeny viruses for 1 h at 37°C in the absence
or the presence of BB-1 or BU-63. Cells were next washed several times
with PBS and the levels of RAJI-associated viral p24 were monitored by
performing a p24 assay. Data shown represent the means ⫾ SD of three
determinations and these results are representative of three different
experiments.
The biological significance of our findings was investigated by first
defining whether the incorporation rate of CD28 and virus infectivity were affected by the amounts of surface CD28 on 293T cells
and second by comparing the levels of expression of CD28 between 293T cells and natural cellular reservoirs of HIV-1, i.e.,
CD4-expressing T lymphocytes. To test whether differences in surface expression levels of CD28 can quantitatively affect the
amounts of virion-bound foreign CD28, different virus stocks were
made by transfecting 293T cells with increasing amounts of the
CD28-encoding vector. Results from flow cytometric analyses indicate that there is a correlation between the introduced CD28
expression plasmid and the levels of CD28 expressed on the surface of 293T cells (Fig. 6A). The next step was to evaluate the
degree of virally embedded host CD28 in progeny virions produced by 293T cells that express varying levels of surface CD28.
The presence of these proteins of host origin on the exterior of the
virion makes them accessible to reagents such as Abs specific for
the protein of interest. This method has been used with success to
immunoprecipitate the viral entity from a variety of preparations.
We previously developed a virus capture assay based on magnetic
beads coated with an Ab directed against the studied cellular protein(s) (48). A more sensitive version of this virus capture test was
used in the present work that relies on the use of streptavidincoated magnetic beads in combination with a biotinylated monoclonal anti-CD28 Ab (39). An isotype-matched biotinylated antiCD45 Ab (clone UCHL-1) was used as a control to estimate the
nonspecific binding of viruses to magnetic beads. As shown in Fig.
6B, the virus recovery rates were directly influenced by the levels
of CD28 expressed on the surface of 293T cells. Interestingly, the
observed quantitative changes in the amounts of host-encoded
CD28 acquired by HIV-1 seem to correlate in an almost linear way
with virus infectivity (Fig. 6C). Because CD4-expressing T lymphocyte represents the major CD28-positive cell type that is infected by HIV-1 in vivo, we next examined surface levels of CD28
on such primary human cells. We noticed that human CD4⫹ T
cells express a significant amount of surface CD28 that is comparable to the amount of CD28 found on the surface of 293T cells
transiently transfected with 2–10 ␮g of the CD28-encoding plasmid (compare Fig. 6, A and D). This suggests that viral entities will
emerge from target cells in vivo expressing levels of surface CD28
protein sufficient to modulate HIV-1 infectivity.
The Journal of Immunology
2767
Table I. Internalization of isogenic CD28⫺ and CD28⫹ virions in RAJI-CD4 cells
Virus Stocks
(treatment)
CD28⫺ (without
CTLA-4Ig)
CD28⫺ (with
CTLA-4Ig)
CD28⫹ (without
CTLA-4Ig)
CD28⫹ (with
CTLA-4Ig)
a
b
Total p24 Levels in the
Absence of Pronase
(pg/ml)a
Intracellular p24 Levels in
the Presence of Pronase
(pg/ml)
536 ⫾ 96
66 ⫾ 12
Percentage of Virus Entry
(intracellular/total p24 levels)
12.3
NTb
NT
NA
3166 ⫾ 58
1066 ⫾ 57
33.7
93 ⫾ 12
14.9
623 ⫾ 130
Results shown are the mean number ⫹ SD for triplicate samples.
NT, Not tested; NA, not applicable.
89.6 virus stocks in the absence or the presence of the blocking
anti-CD28 Ab. CD28-bearing HIV-1 particles were again more
infectious than their CD28⫺ counterparts (compare 66,000 and
32,000 pg/ml of p24) and pretreatment with 9.3 reduced infectivity
of CD28⫹ only (Fig. 7B). To more clearly establish the physiological significance of the presence of host-derived CD28 when
inserted within the virus envelope, we assessed whether a blocking
anti-CD28 Ab could affect replication of a clinical strain of HIV-1
produced in PBMCs. Treatment of the R5X4 clinical isolate of
HIV-1 92RW009 with 9.3 resulted in a diminution of virus production in IFN-␥-treated MDM (Fig. 8). Replication of this clinical
strain of HIV-1 was unaffected by an isotype-matched irrelevant
Ab (data not shown).
Host-derived CD28 is detected on the surface of clinical isolates
of HIV-1 produced in primary human mononuclear cells
The virus capture assay was used to assess the physical presence of
foreign CD28 on the surface of clinical strains of HIV-1 produced
during normal infection of human PBMCs. Two different T cell
tropic (X4) clinical isolates of HIV-1 expanded on human PBMCs
were more efficiently captured by an anti-CD28 Ab than by an Ab
recognizing CD45 (Fig. 9A). The use of RAJI cells to capture such
progeny viruses showed that the binding of 92HT599 and
93UG070 to these cells is partly mediated through an interaction
between CD28 and B7-1/B7-2 because treatment with CTLA-4Ig
diminishes the amounts of viruses attached to the cell surface (Fig.
9B). Virus capture studies were also performed with macrophage
(R5) and dual-tropic (R5X4) primary isolates of HIV-1 because
CD28-expressing CD4⫹ T lymphocytes are susceptible to infection by such viruses under in vivo situations. These viruses were
expanded in PBMCs from two healthy donors. Precipitated viruses
are presented as the ratio between virions captured with anti-CD28
and viruses captured with anti-CD45 because there were quantitative differences in the virus-associated p24 content for the virus
preparations tested (data not shown). As depicted in Fig. 9C, magnetic beads coated with anti-CD28 efficiently captured T-, M-, and
dual-tropic field isolates of HIV-1 produced by PBMCs from two
healthy donors.
FIGURE 5. Sensitivity of CD28⫺ and CD28⫹ virions to neutralization
by an agent blocking the gp120-CD4 interaction. A, RAJI-CD4 cells were
incubated for 5 min at 37°C with the indicated amounts of the anti-CD4
SIM.2 Ab. Such treated cells were inoculated with CD28⫺ and CD28⫹
luciferase-encoding virus stocks (5 ng of p24). Cells were next incubated
at 37°C for 48 h before evaluation of the luciferase activity. B, Inhibition
of virus infectivity was calculated with the following formula: percentage
of inhibition ⫽ [1 ⫺ (virus treated with SIM.2/untreated virus) ⫻ 100%].
Results shown are the mean ⫾ SD for triplicate samples and are representative of two independent experiments.
Discussion
The binding event represents the first step in the virus replicative
cycle. The attachment of viruses to the surface of the target cell
was previously considered to involve simple recognition to a single-cell surface molecule by virus attachment proteins. Results
from studies aimed at understanding virus-host interactions revealed that virus binding is a more complex phenomenon. For
example, some viruses recognized more than one receptor and
sometimes a secondary binding step has been shown to follow the
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A tissue culture system has been previously developed as a
model for studying HIV-1 pathogenesis (49). This experimental
system consists of histocultures of human lymphoid tissues (i.e.,
tonsils) that preserve their general cytoarchitecture. Human lymphoid tissues cultured ex vivo harbor different cell subsets which
may express B7-1 and/or B7-2 molecules (e.g., activated T lymphocytes, macrophages, and dendritic cells). Histocultures of human tonsils were thus infected with isogenic CD28⫺ and CD28⫹
2768
VIRION-ANCHORED HOST CD28 AND HIV-1
FIGURE 6. Evaluation of CD28 surface expression, virion-anchored
host CD28, and HIV-1 infectivity. A, Flow cytometric analyses were conducted to monitor levels of CD28 surface expression on 293T cells transiently transfected with pHXB-Luc and increasing concentrations of the
human CD28-encoding vector (i.e., pH␤Apr-1-neo). MFV, mean fluorescence value. B, Virus stocks were captured by using a combination of
streptavidin-coated magnetic beads and a monoclonal anti-CD45 (control)
or anti-CD28 Ab. C, Virus preparations were used to inoculate RAJI-CD4
cells as described in Fig. 3. D, Expression of CD28 was also assessed on
primary human CD4-positive T lymphocytes using a two-color flow cytometry protocol as described in Materials and Methods.
initial binding event (50). This is exemplified by the observation
that HIV-1 recognizes both CD4 and galactosyl ceramide as primary cellular receptors and some members of the seven-transmembrane chemokine receptor family served as coreceptors for virus
entry (51). More evidence suggests that other surface molecules
might also be playing an active potentiating role in the whole process of HIV-1 binding. Given that the interaction between gp120
from primary viruses and cellular CD4 is often weak, these secondary interactions would act by stabilizing or strengthening the
initial virus-cell contact and would allow ligation of gp120 to the
appropriate chemokine coreceptor. This type of secondary interaction can be biologically important for a pathogen such as HIV-1
found in fluids with flow where a rapid and firm docking of the
virus to its host cell is essential. Previous work has indicated that
incorporation of foreign HLA-DR and ICAM-1 proteins in HIV-1
contributes to virus infectivity by increasing the binding/entry process (12, 14, 15). In the present study, we provide evidence indicating that the costimulatory molecule CD28, which is constitutively expressed on virtually all human CD4⫹ T lymphocytes, is
incorporated within the HIV-1 envelope and is enhancing the process of infection through a potentiating effect on the early events
of virus replication. The noticed enhancement of virus infectivity
conferred by the acquisition of host CD28 is primarily due to the
interaction between CD28 and B7-1. Our observation is consistent
with a previous work that has reported that although B7-1 and
B7-2 bind CD28 with similar low affinities and CTLA-4 with similar high affinities (52), B7-2 has faster dissociation kinetic than
B7-1 (53). Given that the increase in virus infectivity is resulting
from an augmentation of the overall attachment process, it is thus
logical to observe that infectivity of CD28-bearing HIV-1 particles
is more affected when CD28/B7-1 interaction is blocked than when
CD28/B7-2 interaction is abrogated. However, it is important to
note that two lines of evidence suggest that the sole CD28-B7
interaction is not sufficient per se to allow HIV-1 infection. First,
infection with CD28-bearing virions is completely abolished when
using an anti-CD4 Ab that blocks the gp120-CD4 interaction (Fig.
5, A and B). Second, the CD4-negative parental RAJI cell line is
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FIGURE 7. Infectivity of CD28⫺ and CD28⫹ virions when using MDM
and human lymphoid tissues as targets. Primary human MDM treated with
IFN-␥ (A) and human tonsils (B) were infected with isogenic CD28⫺ and
CD28⫹ dual-tropic 89.6 viruses in the absence or the presence of a blocking anti-CD28 Ab (i.e., clone 9.3). Virus production was estimated by
measuring p24 levels at 3 and 14 days postinfection for MDM and human
tonsils, respectively. Data shown represent the means ⫾ SD of three determinations and these results are representative of three different experiments.
The Journal of Immunology
2769
not susceptible to infection with CD28-bearing HIV-1 particles
despite surface expression of CXCR4 and high levels of B7-1/
B7-2 (data not shown). The interaction between the external viral
envelope and CD4 remains thus essential for infection of B7-expressing cells with HIV-1 carrying foreign CD28 in their envelope.
Results from infectivity studies conducted in MDM are particularly informative since induction of the two costimulatory B7-1
and B7-2 molecules after activation of this cell type is a wellestablished phenomenon. For example, microbial components
such as bacterial LPS (54) and some cytokines have the capacity to
up-regulate expression of these two costimulatory molecules on
MDM. Among the various cytokines involved during the immune
response, IFN-␥ is recognized as the major macrophage-activated
molecule able to increase surface expression of B7-1/B7-2 on cells
of the monocyte/macrophage lineage (55–57). The presence of the
additional CD28/B7 interaction between the virus and its target
could represent an important step in the establishment of the infection as indicated by the observation that IFN-␥-treated MDM
are more susceptible to infection with CD28-bearing virions.
Members of the B7 family are also expressed on a variety of APCs
and CD4-positive T lymphocytes, particularly in human lymphoid
tissue where a high activation state is prevailing (58). Virus infectivity was also positively affected by the acquisition of CD28 when
human lymphoid organs cultured ex vivo were used as targets.
Such data reveal some clinical significance since these tissues are
considered major reservoirs of HIV-1 and sites of intense virus
replication. Interestingly, T lymphocytes have been demonstrated
to express both B7-1 and B7-2 following stimulation (22–25), and
their expression levels are augmented in HIV-1-infected individuals (59 – 61).
As stated above, one obvious mechanism to explain the noticed
increase of HIV-1 infectivity conferred by the presence of hostencoded CD28 is by a direct contribution to the early events of the
viral life cycle, namely binding and entry. The fact that CD28
increases virus entry into RAJI-CD4 cells by ⬎3-fold entirely supports this idea. It should be stated that a similar conclusion has
been drawn to explain the previously described augmentation of
infectivity for progeny viruses bearing host cell membrane CD44,
MHC-I, MHC-II, and ICAM-1 proteins (12–15, 62). Maréchal and
colleagues (46) have previously shown that HIV-1 entry occurs
both through plasma membrane fusion, leading to productive in-
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FIGURE 8. Replication of a clinical isolate of HIV-1 in human MDM
following treatment with a blocking anti-CD28 Ab. The macrophage-tropic
clinical strain of HIV-1 92RW009 that was produced in PBMCs was either
left untreated or was treated with the anti-CD28 9.3 before inoculation of
IFN-␥-treated MDM. Virus-infected target cells were analyzed for p24
concentration in the culture medium at the indicated times. Data shown
represent the means ⫹ SD of three determinations and these results are
representative of three different experiments.
FIGURE 9. Capture of virions with immunomagnetic beads and RAJI
cells. A, Virus stocks from transiently transfected 293T cells (CD28⫺ and
CD28⫹) and clinical isolates of HIV-1 expanded in primary human cells
were incubated with a combination made of streptavidin-coated magnetic
beads and a monoclonal anti-CD28 or anti-CD45 Ab. B, Such virus preparations were also incubated with RAJI cells as described in Fig. 2. C, T-,
M-, and dual-tropic clinical isolates of HIV-1 were produced by infecting
PBMCs from two healthy donors. Such virus stocks were subjected to the
virus capture assay. Results shown are the mean ⫾ SD of triplicate samples
and are representative of three different experiments.
fection, and through endocytosis, usually leading to nonproduction
infection. Their work has also revealed that most of the virus p24
taken up by cells is not due to infectious virus, but to particles that
2770
understanding of the exact contribution of virion-anchored host
molecules to the life cycle of HIV-1 is crucial to provide insights
into the pathogenesis of this retroviral disease.
Acknowledgments
We thank M. Dufour for technical assistance in flow cytometry studies.
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undergo endocytosis and end up in lysosomes. Although the experimental approach used in the present study to assess virus entry
does not permit discrimination between cytosolic p24 and nonspecific vesicular uptake, our observation that the process of HIV-1
infection is modulated by incorporation of host CD28 and blockers
of CD28-B7-1/B7-2 interactions is an indication that cytosolic p24
is also augmented by the presence of virion-anchored host CD28.
Our findings indicate also that the presence of the additional
CD28/B7 interaction can fulfill a certain role in the infection process when the major CD4/gp120 interaction is compromised by a
blocking agent. Our observations might reveal great physiological
relevance considering that the stabilization of the viral particle on
the host-cell membrane by this additional interaction will favor the
ensuing gp120-CD4 association.
The process of HIV-1 attachement to host cells is considered to
be essentially mediated by the association between cellular CD4
and virion-associated gp120. This postulate originates from early
studies suggesting that virus attachment to T cells correlates with
CD4 expression (63, 64) and is gp120-dependent (64, 65). However, the binding of gp120 to cellular CD4 might not be sufficient
per se for an efficient docking of the virus to cell types such as
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the additional interaction mediated by CD28 and B7-1/B7-2 facilitates the binding and entry process of HIV-1. Moreover, follicular
dendritic cells that are CD4-negative have the capacity to trap
large quantities of HIV-1 on their surfaces via dendritic cell-specific ICAM-3 grabbing nonintegrin (68, 69). It is believed that this
process could be involved in the migration of the virus from the
mucous to the lymphoid organs. It is conceivable that other viruscell interactions mediated by adhesion molecules could participate
in this process. The interaction between CD28 and B7 family
members represents a good candidate because follicular dendritic
cells express large amounts of B7 molecules (70).
The current work has not explored the possible effect of CD28B7-1/B7-2 signal transduction pathway in HIV-1 replication. This
scenario should not be ignored considering the observation that
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lymphocytes resulting in activation of some specific transcription
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several proteins in RAJI cells (72). Proliferation of B cells was
found to be arrested upon engagement of B7-1 on the surface of B
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transduction events resulting from the interaction between virionbound host CD28 and cell surface B7-1/B7-2 can affect HIV-1
transcriptional activity and/or target cell function(s).
The physiological relevance of the insertion of host-derived
CD28 in the HIV-1 envelope is high considering that CD28 is
constitutively expressed on virtually all CD4⫹ T lymphocytes, a
cellular subpopulation considered as a major reservoir of HIV-1,
and that the level of CD28 found on freshly isolated CD4⫹ T cells
is comparable to the amount of CD28 expressed on transfected
293T cells used in the present study. Moreover, the observation
that treatment of clinical virus isolates with a blocking anti-CD28
Ab leads to a decrease of HIV-1 production in MDM is an indication that the level of foreign CD28 acquired by clinical strains of
HIV-1 is sufficient to play a role in the virus life cycle.
In conclusion, these experiments further reinforce the idea that
the biology of HIV-1 is influenced by the nature of host cell membrane constituents found embedded within virions. Our data confirm the high degree of complexity of interactions occurring between a pathogen such as HIV-1 and its cellular target. A better
VIRION-ANCHORED HOST CD28 AND HIV-1
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