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Journal of Virology, November 1998, p. 9329-9336, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Level of ICAM-1 Surface Expression on Virus
Producer Cells Influences both the Amount of Virion-Bound Host
ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity
Jean-Sébastien
Paquette,
Jean-François
Fortin,
Luc
Blanchard, and
Michel J.
Tremblay*
Centre de Recherche en Infectiologie, Centre
Hospitalier Universitaire de Québec, Pavillon CHUL, and
Département de Biologie Médicale, Faculté de
Médecine, Université Laval, Ste-Foy, Québec, Canada
G1V 4G2
Received 5 February 1998/Accepted 4 August 1998
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ABSTRACT |
Using virions harvested from 293T cells stably expressing either
low or high levels of surface ICAM-1, we determined that the number of
virus-embedded host ICAM-1 proteins is positively influenced by the
expression level of ICAM-1 on virus producer cells. Moreover, the
increase in virion-bound host cell membrane ICAM-1 led to a concomitant
enhancement of virus infectivity when a T-cell-tropic strain of human
immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also
seen when primary human cells were infected with virions pseudotyped
with the envelope protein from a macrophage-tropic HIV-1 isolate, thus
ruling out any envelope-specific effect. We also observed that target
cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to
infection with HIV-1 particles bearing on their surfaces large numbers
of host-derived ICAM-1 proteins. Given that cellular activation of
leukocytes is known to modify the conformational state of LFA-1 and
induce ICAM-1 surface expression, it is tempting to speculate that
activation of virus-infected cells will lead to the production of HIV-1
particles bearing more host ICAM-1 on their surfaces and that such
progeny virions will preferentially infect and replicate more
efficiently in activated cells which are prevalent in lymphoid organs.
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TEXT |
Viruses are obligate intracellular
parasites, and they are thus absolutely dependent on the host cell for
essential functions such as the generation of metabolic machinery and
protein synthesis. Such a strong dependence on the host cellular
machinery is probably linked with the limited genetic resources of
viruses. These infectious agents have overcome their genetic
limitations by showing a strong adaptation to their host. For example,
human immunodeficiency virus type 1 (HIV-1) uses the surface CD4
glycoprotein and chemokine receptors to infect its target cells
(1, 19, 21, 22, 24, 25, 27, 43). In addition, due to their
high rate of mutations, viruses can adapt themselves to their host by
natural selection in an attempt to optimize their life cycle to assure their survival. Enveloped viruses such as HIV-1 acquire their lipid
membranes and their own envelope proteins during the process known as
budding. A characteristic of the propagation of HIV-1 within its target
is the incorporation of several host-encoded proteins during the
extrusion of the virus particles from the infected cells. Indeed, the
outer surface of HIV-1 has been demonstrated to be composed of numerous
host cell membrane constituents including major histocompatibility
complex class II (MHC-II) determinants (HLA-DR, -DP, and -DQ),
2-microglobulin, CD43, CD44, CD55, CD59, CD63, CD71, and
adhesion receptors such as ICAM-1 and LFA-1 (2, 3, 12, 13, 16, 26,
33, 38, 40, 47-49, 59, 62). The incorporation of these
host-derived molecules seems to be a selective process since not all
cell surface molecules are found embedded within HIV-1. For example,
the transmembrane protein tyrosine phosphatase CD45 is not acquired by
newly formed HIV-1 progeny virions (49) despite the fact
that it represents the most abundant molecule at the surfaces of
leukocytes (65).
Accumulating evidence indicates that virion-bound host proteins are
functional and that they seem to confer protection against the harsh
environment surrounding HIV-1 (64). More specifically, the
neutralizing capacity of sera from HIV-1-infected individuals was
enhanced by the addition of anti-LFA-1 antibodies, thus suggesting an
important role for this host-encoded glycoprotein in the process of
infection (36). In addition, the physical presence of host cell membrane MHC-II, HLA-DR1, and ICAM-1 on HIV-1 has been shown to
lead to an enhancement of virus infectivity that is due to the
interactions between virion-bound host molecules and their physiological counterreceptors found on the surfaces of target cells
(10, 11, 30, 55).
The basis of the present work is founded on several published
observations. First, host-derived ICAM-1 is incorporated into nascent
HIV-1 progeny viruses and the presence of cell membrane host molecules
is also detected in plasma-derived HIV-1 isolates (12, 13,
32, 47, 58). Second, HIV-1 infectivity is significantly increased
by the acquisition of host-encoded ICAM-1 (30, 55). Third,
target cells are more susceptible to infection with ICAM-1-bearing HIV-1 progeny viruses if they express on their surfaces the natural ligand of ICAM-1, LFA-1, in its activated form (31). Fourth, ICAM-1 is normally expressed in small amounts on peripheral blood leukocytes but is strongly induced by cytokines such as tumor necrosis
factor alpha (TNF-
), gamma interferon (IFN-
), and interleukin 1 (IL-1) (15). Fifth, increased levels of TNF-, IFN-,
and IL-1 have been observed during the course of HIV-1 infection
(51). The major aim of the present study was to assess if
the number of virus-acquired host cell membrane ICAM-1 proteins is
influenced by the level of ICAM-1 that is expressed on the surfaces of
virus producer cells. If so, we were next interested in determining whether virus infectivity was modified by the relative numbers of
host-encoded ICAM-1 proteins acquired by newly formed HIV-1 particles.
Finally, we have also studied the functional relevance of differences
in the levels of virus-embedded host ICAM-1 when infection is carried
out with target cells expressing LFA-1 in either the low- or
high-affinity conformational state.
Main characteristics of cells used in this study.
Peripheral
blood mononuclear cells (PBMCs) from healthy donors were isolated by
Ficoll-Hypaque density gradient centrifugation and were cultured in
complete culture medium in the presence of 3 µg of
phytohemagglutinin-protein (Sigma, St. Louis, Mo.) per ml and 30 U of
recombinant human IL-2 (rhIL-2) per ml for 3 days at 37°C under a 5%
CO2 atmosphere prior to viral infection. The Jurkat-tat cell line is a derivative of Jurkat E6.1 cells
that stably expresses the HIV-1 Tat protein, and PM1 is a clonal
derivative of HUT 78 (14, 45). 293T cells are human
embryonic kidney cells which are negative for ICAM-1 expression, while
Jurkat-tat and PM1 cells express high levels of LFA-1 on
their surfaces (data not shown). Three distinct populations of 293T
cells that are either negative for ICAM-1 expression (parental cells
termed Null-ICAM-1) or express either low (Lo-ICAM-1) or high
(Hi-ICAM-1) levels of surface ICAM-1 were used in this study. These
cell lines were obtained by transfecting 293T cells with
pCMV-Hygro to obtain Null-ICAM-1 and by cotransfecting 293T
cells with pCD1.8, a eukaryotic expression vector containing the entire
human ICAM-1 cDNA (63), and pCMV-Hygro (10:1
ratio) to derive ICAM-1-expressing 293T cells. All transfections were
performed by a modified version of the calcium phosphate
(CaPO4) transfection protocol as described previously (11, 30, 31). Transfected 293T cells were maintained under selective pressure with hygromycin B (400 µg/ml; Calbiochem) for 3 weeks and were next sorted by fluorescence-activated cell sorter analysis with the use of the anti-ICAM-1 RR1/1.1.1 antibody. The Lo-ICAM-1 population was sorted by gating in the
low-mean-fluorescence-intensity region, whereas cells with the highest
mean fluorescence intensity were kept for the Hi-ICAM-1 population.
Production of virus stocks.
Viral particles differing only by
the absence or the presence of host-derived ICAM-1 proteins on their
surfaces were produced by CaPO4 transfection of pHXB-Luc
(17) or, for pseudotyped virions, of
pNL4-3-Luc-E
R+ plus pcDNA-1/Ada-M
env, in Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 293T cells as
described previously (30). Briefly, a typical transfection
experiment was carried out with 10 µg of pHXB-Luc, while for the
production of virions pseudotyped with the Ada-M Env protein, we used 2 µg of pNL4-3-Luc-ER+ and 10 µg of
pcDNA-1/Ada-M. Null-ICAM-1 virus preparations were produced with
parental 293T cells that do not express ICAM-1, whereas Lo- and
Hi-ICAM-1 progeny virions were produced from cells expressing low and
high levels of surface ICAM-1, respectively. All virus preparations
underwent only one freeze-thaw cycle before the initiation of infection
studies. Virus stocks were normalized for virion content by a
commercial assay for p24 (Organon Teknika, Durham, N.C.). The
standardization on p24 content 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 (30).
Antibodies and purified proteins.
Anti-ICAM-1 (anti-CD54)
antibodies RR1/1.1.1 and R6.5 were provided by Robert Rothlein
(Boehringer Ingelheim, Ridgefield, Conn.) (56).
LFA-1-activating antibody NKI-L16 (anti-CD11a) was obtained from Carl
C. Figdor (University Hospital Nijmegen, Nijmegen, The Netherlands)
(42). The ICAM-1 fusion protein (ICAM-1-immunoglobulin G2b
[IgG2b]) is composed of the extracellular part of ICAM-1 fused to the
hinge region and constant H chain domains 2 and 3 of a mouse IgG2b and
was purified by an immunoaffinity column. This fusion protein was
supplied by Eric Lundgren (University of Umeå, Umeå, Sweden) and has
been described before (37). F105, a human monoclonal
antibody directed against a conformational epitope on HIV-1 gp120
mapping to the CD4 binding site, was obtained from Marshall Posner
through the AIDS Repository Reagent Program (54). Sheep
anti-HIV-1 gp120 was supplied by the AIDS Repository Reagent Program
and was purified with a mAbTrap protein G affinity column according to
the manufacturer's instructions (Pharmacia LKB Biotechnology AB,
Uppsala, Sweden). Purified recombinant gp120 (rgp120; strain HIV-1IIIB) was obtained from Genentech, Inc. (South San
Francisco, Calif.). Sheep anti-HIV-1 gp120 and R6.5 were biotinylated
with N-hydroxysuccinimide-long-chain biotin according to
the supplier's instructions (Pierce, Rockford, Ill.). Fluorescein
isothiocyanate-conjugated goat anti-mouse IgG was purchased from
Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.).
Virus infection and HIV-1 entry assay.
Similar amounts of each
recombinant luciferase-encoding virus stock (5 ng of p24 for
Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 virions) were incubated with
target cells (105) for 90 min at 37°C. In some
experiments, target cells were either left untreated or treated with
the LFA-1-activating antibody NKI-L16 at 1 µg/ml for 30 min at
37°C. The cells were next washed with phosphate-buffered saline
(PBS), resuspended in 100 µl of complete culture medium supplemented
with 30 U of rhIL-2 per ml for PBMCs, and transferred to a 96-well
flat-bottom tissue culture plate (Microtest III, Falcon; Becton
Dickinson, Lincoln Park, N.J.). Cells were kept for 24 (Jurkat-tat and PM1 cells) or 72 h (PBMCs) at 37°C.
After this final incubation, cells were lysed as described previously
(11, 30). Luciferase activity was monitored with a
microplate luminometer (MLX; Dynex Technologies, Chantilly, Va.). In
some experiments, cells were treated with herbimycin A (1 µg/ml) for
60 min at 37°C prior to infection. In this set of experiments,
cellular viability was estimated by the MTS assay as described
previously (9). Internalization of Null-ICAM-1, Lo-ICAM-1,
and Hi-ICAM-1 virions was monitored by a recently described method
(46). Briefly, for each sample, 5 × 106
Jurkat-tat cells were washed with PBS and resuspended into 1 ml of an HIV-1 suspension containing 100 ng of p24 in culture medium
supplemented with 10% fetal bovine serum (FBS). Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 virus preparations were incubated with target
cells for 2.5 h at 37°C. Cells were washed two times with ice-cold PBS and then resuspended in 1 ml of ice-cold Dulbecco modified
Eagle medium (DMEM) (without FBS) containing pronase (Boehringer
Mannheim, Laval, Quebec, Canada) at 0.1 mg per ml for 5 min at 4°C.
Cells were washed immediately with 2 ml of ice-cold DMEM containing
10% FBS and three times with ice-cold PBS to eliminate pronase. Cells
were resuspended in 1 ml of complete RPMI medium to which was added 200 µl of disruption buffer (2.5% Triton X-100 in PBS). Cells were
agitated for 10 min at room temperature and then stored at 
20°C
until assayed for p24 content.
Quantitative determination of virion-bound host ICAM-1 and
gp120.
The presence of virion-bound ICAM-1 and viral gp120
proteins in our virus preparations was assessed by in-house enzymatic assays. Cell-free Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 virus stocks
were pelleted under ultracentrifugation conditions that are sufficient
to sediment whole viruses (Heraeus model Contifuge 28RS; 12,000 rpm for
90 min at 4°C) (11). Next, the virus pellets were gently
resuspended in PBS in 1/20 of the initial volume and were stored at
20°C until assayed. The virus-embedded host ICAM-1 level was
assessed with R6.5 as the coating antibody and biotinylated RR1/1.1.1
as the secondary antibody, while the gp120 level was quantitated with
F105 (first antibody) and biotinylated sheep anti-HIV-1 gp120 (second
antibody). Proteins were visualized with a streptavidin-horseradish
peroxidase conjugate (streptavidin-HRP40; Research Diagnostics Inc.,
Flanders, N.J.). Appropriate dilutions of rgp120 and ICAM-1-IgG2b were
used to produce standard curves.
The level of ICAM-1 surface expression on virus producer cells
quantitatively modulates the amount of host-encoded ICAM-1 acquired by
HIV-1.
In an attempt to gain more knowledge on cellular factors
affecting the process of incorporation of host cell membrane proteins by progeny HIV-1 particles, we investigated whether the expression level of ICAM-1 can affect the number of virion-embedded host-derived ICAM-1 proteins. To attain this goal, we generated two different 293T
cell populations bearing different levels of ICAM-1 by means of stable
transfection and cell sorting. This specific cell line was selected
because it is recognized as being highly transfectable (53).
In addition, a significant advantage of this cell line over lymphoid
cells is that virus preparations produced by transfected 293T cells are
relatively free of cellular constituents (see below), while purified
virus stocks harvested from acutely HIV-1-infected lymphoid cell lines
have been reported to be heavily contaminated with microvesicles that
cosediment with sucrose gradient-purified virions (6, 35).
The parental 293T cells (Null-ICAM-1) are negative for ICAM-1
expression, whereas the mean fluorescence intensities (indicative of
the number of ICAM-1 proteins per cell) measured for Lo- and Hi-ICAM-1
293T cells were found to be 12.8 and 44.1, respectively (data not
shown).
Progeny viruses were initially produced by transiently transfecting
Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 293T cells with pHXB-Luc. We
evaluated the relative levels of viral p24 and gp120 proteins, as well
as the quantities of virus-embedded host ICAM-1 in such virus
preparations. We determined by enzymatic assays that the molar ratios
of viral p24 to virus-embedded host ICAM-1 for Lo-ICAM-1 and Hi-ICAM-1
virus stocks were 1:0.001 and 1:0.016, respectively (Table 1). It
should be noted that these molar ratios were calculated on the basis of
known molecular masses: 24.0 kDa for viral p24 and 90.0 kDa for ICAM-1.
Assuming 3,000 p24 molecules per virion (5), this places the
numbers of ICAM-1 molecules per Lo-ICAM-1 and Hi-ICAM-1 virion at about
3 and 49, respectively (Table 1). Thus,
the number of virus-embedded host ICAM-1 proteins in virions produced
by Hi-ICAM-1 cells is 16-fold greater than that in progeny viruses
harvested from Lo-ICAM-1 293T cells. We can conclude that the relation
between the expression level of ICAM-1 on the surfaces of virus
producer cells and the amount of virion-bound ICAM-1 is not linear
considering that there is a fourfold increase in ICAM-1 surface
expression in Hi-ICAM-1 cells compared to Lo-ICAM-1 cells (mean
fluorescence intensity of 44.1 for Hi-ICAM-1 293T cells versus 12.8 for
Lo-ICAM-1 293T cells). Interestingly, the molar ratios of viral gp120
to p24 were comparable for Null-ICAM-1, Lo-ICAM-1, and Hi-ICAM-1 virus stocks (data not shown). This last finding suggests that the
incorporation of different levels of host-encoded ICAM-1 does not
affect the external envelope spike density on HIV-1 particles. Next,
the presence of cellular contaminants composed of microvesicles loaded with cellular ICAM-1 proteins was assessed with ultracentrifuged supernatants from mock-transfected Lo-ICAM-1 and Hi-ICAM-1 293T cells.
Enzymatic analyses of such samples revealed that pellets from
mock-transfected Lo-ICAM-1 and Hi-ICAM-1 293T cells contained 0.3 and
4.8 ng of ICAM-1, respectively, per ml. This indicates that cellular
contaminants represent only 5 to 6% of the total host-derived ICAM-1
proteins, which are detected in similar quantities in ultracentrifuged
supernatants from Lo-ICAM-1 and Hi-ICAM-1 cells transiently transfected
with pHXB-Luc (0.3 versus 5.5 ng/ml for Lo-ICAM-1 and 4.8 versus 79.8 ng/ml for Hi-ICAM-1).
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TABLE 1.
Characterization of virus preparations produced by
transient transfection of Null-, Lo-, and Hi-ICAM-1 293T cells
with pHXB-Luc
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Virus infectivity is enhanced by increasing the amount of
virion-bound host ICAM-1.
The next logical step was to determine
if the observed quantitative changes in the numbers of virus-acquired
host ICAM-1 proteins could affect HIV-1 infectivity. First, we
inoculated a highly susceptible CD4+ T-lymphoid-cell line
(Jurkat-tat) with identical amounts of each virus
preparation standardized in terms of p24. It is clear from the results
illustrated in Fig. 1 that virus
infectivity is increased in a manner that is dependent on the quantity
of virion-bound host ICAM-1 proteins. Indeed, Lo-ICAM-1 progeny virions
are more infectious than HIV-1 particles devoid of host-encoded ICAM-1 (2.3- to 3.2-fold increase compared to Null-ICAM-1 virions), but the
former are less infectious than Hi-ICAM-1 virus particles (4.3- to
5.9-fold increase compared to Null-ICAM-1 virions). However, the
observed enhancement of virus infectivity does not seem to correlate in
a perfectly linear way with the corresponding level of host cell
membrane ICAM-1 proteins physically present within mature HXB-Luc viral
entities. A clear example is provided by the fact that only a 2-fold
increase is seen when virus infectivities of Hi-ICAM-1 and Lo-ICAM-1
virus stocks are compared, whereas Hi-ICAM-1 virions incorporate
16-fold more host ICAM-1 proteins than Lo-ICAM-1 progeny viruses (49 ICAM-1 molecules for Hi-ICAM-1 versus 3 for Lo-ICAM-1). The positive
correlation between virion-bound host ICAM-1 and HIV-1 infectivity is
not an isolated phenomenon since this observation was made with Null-,
Lo-, and Hi-ICAM-1 virus preparations originating from two independent
transfections (stocks 1 and 2). The observation that Null-, Lo-, and
Hi-ICAM-1-purified virus preparations contain comparable levels of
gp120 per virion (data not shown) demonstrates that the increase of
HIV-1 infectivity is not due to variations in the gp120 content but
rather to the number of virion-embedded host cell membrane ICAM-1
proteins (Table 1). To more closely parallel physiological conditions,
similar experiments were conducted with mitogen-stimulated PBMCs
isolated from three healthy donors as the targets. The infection of
primary mononuclear cells with Null-, Lo-, and Hi-ICAM-1 virus stocks led to an effect on virus infectivity that was still positively modulated by the amount of virion-bound host ICAM-1 (Fig.
2). More specifically, Lo-ICAM-1 virions
were 3.1- to 4.6-fold more infectious than Null-ICAM-1 HIV-1 particles,
while Hi-ICAM-1 viruses were 6.8- to 14.8-fold more infectious than
isogenic progeny viruses devoid of host-encoded ICAM-1 (Null-ICAM-1).
The exact mechanism(s) responsible for such a wide variation in the
enhancement of virus infectivity for the Hi-ICAM-1 HIV-1 particles
(6.8- to 14.8-fold) is undefined, but it might be linked with
differences between donors with regard to the level of surface
expression of LFA-1, the natural counterreceptor of ICAM-1.
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FIG. 1.
The number of virion-bound host ICAM-1 proteins
positively affects HIV-1 infectivity. Jurkat-tat cells were
infected with standardized amounts of virus stocks composed of Null-,
Lo-, and Hi-ICAM-1 HIV-1 particles produced by two independent
transfection experiments (5 ng of p24). Cells were incubated for 90 min
at 37°C and then washed with PBS. Thereafter, the cells were further
incubated for 24 h at 37°C. Finally, cells were lysed and
luciferase activity, expressed in RLU was monitored as described in
Materials and Methods. Results are the means ± standard errors of
the means for quadruplicate samples and are representative of three
independent experiments. Negative controls consisted of uninfected
Jurkat-tat cells. Luciferase activity values for
mock-infected cells were 0.35 ± 0.03 RLU for stock 1 and
0.37 ± 0.04 RLU for stock 2.
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FIG. 2.
The positive effect on HIV-1 infectivity mediated by the
amount of virion-bound host ICAM-1 is also seen in primary human cells.
Similar amounts of Null-, Lo-, and Hi-ICAM-1 virus preparations were
used to inoculate freshly isolated PBMCs from three different healthy
donors (5 ng of p24). Virions and target cells were incubated together
for 90 min at 37°C and washed with PBS, and incubation at 37°C was
pursued for an additional 72 h. Next, cells were lysed and
luciferase activity was monitored as described in Materials and
Methods. Results are the means ± standard errors of the means for
quadruplicate samples and are representative of three independent
experiments. Negative controls consisted of mock-infected PBMCs.
Luciferase activity values for mock-infected cells were 0.44 ± 0.07 RLU for donor A, 0.41 ± 0.02 RLU for donor B, and 0.43 ± 0.03 RLU for donor C.
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The enhancement of virus infectivity conferred by host-derived
ICAM-1 is not due to signal transduction through LFA-1 but rather is
linked with an increase in the process of virus entry.
A previous
study has reported that LFA-1 possesses signaling properties, as it
leads to activation of phospholipase C-1 (41). In order
to verify whether virion-bound ICAM-1 could generate signaling in the
target cells that could upregulate virus expression, target cells were
pretreated with herbimycin A, a potent tyrosine kinase inhibitor which
has been shown to abrogate LFA-1-mediated signal transduction events
(41). Virus infectivity was increased by the presence of
host-derived ICAM-1 despite treatment with herbimycin A (data not
shown). These data suggest that the positive effect of host-encoded
ICAM-1 on virus infectivity is not the result of a postinfection
LFA-1-mediated intracellular signaling event. A virus entry assay was
next performed to determine if virus-acquired host ICAM-1 could modify
the early steps in the virus infection cycle. Proteolytic enzymes
(pronase) were used to eliminate noninternalized virus particles
attached to the cell surface. Indeed, treatment of cells with pronase
has been recently shown to remove cell surface CD4, thus eliminating
any virions attached to its primary receptor (46). This
virus entry assay showed that the percentage of progeny viruses
entering susceptible cells was correlated with the number of
virion-bound host ICAM-1 proteins. The percentages of virus entry for
Null-, Lo-, and Hi-ICAM-1 virions were 30.2, 37.5, and 46.8%,
respectively (the percentages were determined by dividing the p24
content after pronase digestion by the total p24 content in the absence
of pronase treatment). It can thus be concluded that virus-acquired
host cell membrane ICAM-1 proteins are positively modulating the early
stages of the virus infection process.
Expression of a high-affinity LFA-1 form strongly enhanced the
susceptibility of target cells to infection by Hi-ICAM-1 virions.
It has been previously reported that the susceptibility to infection
with ICAM-1-bearing HIV-1 particles is increased upon surface
expression of LFA-1 in a conformational state of high affinity for
ICAM-1 (31). It was thus of interest to determine whether
this cellular factor could differentially modulate the process of
infection with Lo- and Hi-ICAM-1 virions. The conformational change of
LFA-1 was accomplished by treating target cells with anti-LFA-1
monoclonal antibody NKI-L16 (42). In Jurkat-tat
cells, treatment with NKI-L16 has no noticeable effect on the process of infection with Null-ICAM-1 progeny viruses, while the susceptibility of target cells to infection with Lo-ICAM-1 HIV-1 particles was slightly enhanced (1.8-fold increase) by modifying the conformational state of LFA-1 (70.5 versus 39.1 relative light units [RLU]) (Table 2). In sharp contrast to this
observation, the expression of an activated form of LFA-1 was found to
dramatically affect cellular susceptibility to infection with Hi-ICAM-1
virions. Indeed, a 7.3-fold increase in the level of infection with
Hi-ICAM-1 virions was seen by changing the conformational state of
LFA-1 on the surfaces of Jurkat-tat cells (535.1 RLU with
NKI-L16 versus 73.3 RLU without NKI-L16). Interestingly, target cells
bearing LFA-1 in the conformational state of high affinity for ICAM-1
were found to be 26-fold more susceptible to infection by Hi-ICAM-1
progeny viruses than to infection by Null-ICAM-1 HIV-1 particles (535.1 RLU with Hi-ICAM-1 versus 20.8 RLU with Null-ICAM-1). These data were
confirmed with another cell line since the susceptibility of PM1 cells
to infection by Null-ICAM-1 particles was unaffected by treating PM1
cells with NKI-L16, while a similar NKI-L16 treatment resulted in
slight and strong increases in cellular susceptibility to infection
with Lo- and Hi-ICAM-1 virions, respectively. To determine whether the
same phenomenon can also occur in human primary cells, we infected
PBMCs from a healthy donor with standardized amounts of Null-, Lo-, and
Hi-ICAM-1 virus stocks. In agreement with our previous results obtained
with T-lymphoid-cell lines, cellular susceptibility to infection by
Null-ICAM-1 virions was unaffected by the conformational state of LFA-1
while treatment with NKI-L16 resulted in a 1.9-fold increase in
susceptibility to infection with Lo-ICAM-1 HIV-1 (87.6 versus 47.3 RLU). Again, cellular susceptibility to infection with Hi-ICAM-1
progeny virions was markedly affected by modifying the conformational
state of LFA-1 since treatment of PBMCs with the LFA-1-activating
antibody led to a 6.9-fold increase (994.6 versus 143.8 RLU). The
enhancement of cellular susceptibility to virus infection is even more
substantial, reaching a 54-fold increase when the levels of infection
of PBMCs treated with NKI-L16 produced by Hi- and Null-ICAM-1 virus
preparations were compared (994.6 RLU for Hi-ICAM-1 versus 18.5 RLU for
Null-ICAM-1).
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TABLE 2.
Lymphoid and primary cells expressing an activated form
of surface LFA-1 are much more susceptible to infection by Hi-ICAM-1
progeny viruses
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Infectivity of a primary macrophage-tropic HIV-1 isolate is
enhanced by increasing the amount of virion-bound host ICAM-1.
All
the previous experiments were carried out with a T-cell line-adapted
laboratory strain of HIV-1 (HXB-Luc, which is derived from HXB-2D). We
next sought to determine whether the enhancement of virus infectivity
due to virus-incorporated host ICAM-1 can be influenced by the nature
of the viral envelope glycoprotein. This possibility was evaluated by
infecting human PBMCs with Null-, Lo-, and Hi-ICAM-1 progeny virions
pseudotyped with the envelope from Ada-M, a primary macrophage-tropic
strain of HIV-1 (34). An increase in virus infectivity,
which correlates with the level of ICAM-1 on the surfaces of the virus
producer cells, was observed for Lo-ICAM-1 and Hi-ICAM-1 virus
preparations compared to Null-ICAM-1 virions (Fig.
3). Again, expression of the
high-affinity LFA-1 form further increased the susceptibility of
PBMCs to infection with Lo-ICAM-1 and Hi-ICAM-1 progeny virions
bearing the Ada-M envelope but not susceptibility to infection with
Null-ICAM-1 viruses.
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FIG. 3.
The increase in virus infectivity mediated by the amount
of virion-bound host ICAM-1 is also seen in PBMCs infected with a
primary macrophage-tropic HIV-1 isolate. PBMCs were either left
untreated or treated with NKI-L16 for 30 min at 37°C prior to
infection with similar numbers of Null-, Lo-, and Hi-ICAM-1 virions
pseudotyped with the envelope protein from Ada-M (5 ng of p24). Viruses
were incubated with PBMCs for 90 min at 37°C, and cells were washed
with PBS. The cells were kept in culture for 72 h before
luciferase activity was monitored. Results are the means ± standard errors of the means for quadruplicate samples and are
representative of three independent experiments. Negative controls
consisted of mock-infected PBMCs. Luciferase activity values for
mock-infected cells were 0.38 ± 0.11 RLU for untreated cells and
0.27 ± 0.02 RLU for cells treated with NKI-L16.
|
|
In summary, the expression level of ICAM-1 on the surfaces of virus
producer cells was found to influence the relative number
of
host-encoded ICAM-1 proteins that are acquired by budding HIV-1
particles (Table
1). Indeed, we noticed that the level of ICAM-1
on the
surfaces of virus producer cells directly influenced the
number of
virion-bound ICAM-1 proteins in a nonlinear manner.
The functional
significance of this observation was next investigated
by infecting
Jurkat-
tat, PM1, and primary human cells with Null-,
Lo-,
and Hi-ICAM-1 HXB-Luc particles. We observed that the presence
of
increasing numbers of virus-embedded host ICAM-1 proteins led
to a
concomitant enhancement of virus infectivity (Fig.
1 and
2). The
increase in virus infectivity for Lo-ICAM-1 and Hi-ICAM-1
viruses
compared to that for the Null-ICAM-1 virus was not due
to any
LFA-1-mediated signaling events. Based on our previous
observation
(
30) and on results from the present virus entry
assay (see
the data above), it can be stated that the marked increase
in virus
infectivity is due to interactions between virus-incorporated
host
ICAM-1 and LFA-1 proteins expressed on the surfaces of target
cells.
LFA-1 is the physiological counterreceptor for ICAM-1 and
is a member
of the integrin family that is primarily expressed
on lymphocytes,
granulocytes, monocytes, and macrophages, with
elevated levels on
memory T cells (
60). The affinity constant
determined for
the binding of multimeric ICAM-1 to LFA-1 is similar
to the strong
binding constant for the gp120-CD4 interaction
(
Kd = 4 nM) (
44,
67). Therefore, it
is not surprising to discover
that virion-bound host ICAM-1 enhances
HIV-1 infectivity through
its positive action on the process of virus
entry, i.e., by favoring
attachment and/or entry. It has been shown
that the whole process
leading to HIV-1 entry is sequential
(
61). Indeed, a series
of events has to take place in order
to achieve virus internalization.
The additional interaction between
virus-embedded ICAM-1 and cell
surface LFA-1 may result in the
stabilization of the viral entity
on the surface of the target cell.
The final result will be an
increase in the efficiency of the whole
virus entry process.
LFA-1 can be found in two distinct states of affinity for ICAM-1, the
low- and high-affinity conformational states of LFA-1.
This
conformational modification can be induced by several agents,
including
phorbol esters, chemoattractants, and antibodies specific
for surface
receptors such as the T-cell receptor-CD3 complex,
CD2, and MHC II
(
23). Treatment with the anti-LFA-1 NKI-L16
monoclonal
antibody has also been reported to induce a similar
conformational
change of LFA-1 because this antibody binds to
a peripheral domain and
opens a link site for ICAM-1 (
42). We
then assessed if this
property of LFA-1 can result in the modulation
of the susceptibility of
target cells to infection with Lo- and
Hi-ICAM-1 HXB-Luc particles by
treating target cells with NKI-L16.
Cellular susceptibility to
infection by Hi-ICAM-1 HXB-Luc virions
was found to be markedly
accentuated when target cells carried
LFA-1 on their surfaces in the
conformational state of high affinity
for ICAM-1 (Table
2).
The external envelope glycoprotein of HIV-1 (gp120) is known to play an
essential role in the viral replicative cycle, including
a role in
tropism (T cell- or macrophage-tropic), replication
kinetics, and
syncytium induction (
18,
28). Since the first
part of our
experiments was performed with T-cell-tropic (X4 virus)
HXB-2D, and
considering the crucial role played by the viral gp120
in the
pathogenesis of HIV-1 infection, we considered that the
inclusion in
our study of a viral strain with a macrophage-tropic
(R5) phenotype was
of critical importance. Similar observations
were made when Null-, Lo-,
and Hi-ICAM-1 virus preparations pseudotyped
with the envelope protein
from the R5 Ada-M isolate were used
(Fig.
3). The physiological
relevance of this finding is great
based on the concept that
macrophage-tropic strains are preferentially
transmitted between
individuals and predominate during the asymptomatic
phase of HIV-1
infection, which generally lasts several years
(
4).
It can be argued that these data were collected by using progeny HIV-1
particles produced by transient transfection of human
embryonic kidney
cells, a cell type that does not serve as a natural
reservoir for
HIV-1. However, our technical approach is validated
by the fact that
the quantitative determination of virus-acquired
host proteins cannot
be accomplished for virus preparations produced
by lymphoid cells
because these are known to be heavily contaminated
with cellular
proteins (
6,
35). In this regard, we estimated
that the
molar ratios of HIV-1 p24 to host cell membrane ICAM-1
proteins
detected within purified Lo- and Hi-ICAM-1 HXB-Luc particles
were
1:0.001 and 1:0.016, respectively (Table
1). These figures
are much
lower than those previously observed by Capobianchi et
al., who have
calculated a molar ratio for viral p24 proteins
to virion-bound host
ICAM-1 proteins of 1:0.14 (
13). Such a
wide difference might
be attributed to their use of a T-lymphoid-cell
line to produce sucrose
gradient-purified virus preparations.
A recent report has shown that
all uninfected T-cell lines tested
(H9, CEM-SS, DAUDI, and MOLT-3)
secreted large amounts of microvesicles
that banded at the same density
as that for HIV-1 when fractionated
by sucrose gradient centrifugation
(
6). The same study has
demonstrated that mitogen-stimulated
human PBMCs also produce
microvesicles loaded with cellular proteins.
It should be noted
that we have established that Lo- and Hi-ICAM-1 293T
cells secrete
relatively minimal quantities of such microvesicles,
therefore
indicating that these cells represent an appropriate tool to
quantitatively
evaluate virion-associated cellular proteins.
The precise mechanism(s) responsible for the incorporation of foreign
constituents by nascent viral entities is still undefined.
However,
results from the present study allow us to speculate
about the
factor(s) responsible for the insertion of at least
host-encoded ICAM-1
molecules into newly formed HIV-1 particles.
It can be postulated that
the incorporation of host cell membrane
ICAM-1 molecules into budding
virions is probably not due to a
direct physical interaction between
ICAM-1 and a viral protein
present within the mature particle. This
assumption is based on
the fact that Null-, Lo-, and Hi-ICAM-1 virus
stocks originated
from the same genetic source (HXB-Luc or
pNL4-3-Luc-E
R
+) and the only difference
between virus preparations was the producer
cells (Null-, Lo-, or
Hi-ICAM-1 293T cells). A possible scenario
to explain the acquisition
of host proteins by HIV-1 is that nascent
virions emerge from specific
sites at the cellular membrane that
would be loaded with patches
composed of host cell components
such as ICAM-1. This postulate is
based primarily on previous
studies that have showed that the budding
of HIV-1 is restricted
to a certain localized region of the cell
membrane (
20,
39).
The fact that copolarization of ICAM-1
and budding viral entities
were observed at the site of cell-to-cell
contact during HIV-1-induced
syncytium formation supports this idea
(
26). This would explain
why ICAM-1 is incorporated in a
nonlinear manner into the virus
envelope.
It is well known that secondary lymphoid organs such as lymph nodes
constitute a natural viral reservoir in HIV-1-infected
patients.
Interestingly, a fairly high proportion of immune cells
residing within
the lymph nodes are in an activated state since
antigen-specific immune
responses primarily take place in these
peripheral lymphoid tissues
(
52). This is exemplified by the
nodal migration of
activated CD4
+ T lymphocytes during a natural antigenic
response (
57). It
is well established that the replication
of HIV-1 is tightly linked
to the proliferative state of the cells and
that cellular activation
is thus necessary for a productive HIV-1
infection of CD4
+ T lymphocytes. Indeed, HIV-1 particles
have the ability to bind
to and fuse with both quiescent and activated
CD4
+ T cells (
29,
69). However, in nondividing T
lymphocytes,
HIV-1 is unable to complete a full replicative life cycle
(
7,
8,
68,
70). The intense cellular activation observed in
the lymphoid organs will also lead to the upregulation of ICAM-1
expression and consequently, as shown in this study, to a higher
number
of host-derived ICAM-1 proteins acquired by budding HIV-1
particles.
The observation that activated germinal-center B cells
secrete TNF-
,
a proinflammatory cytokine known to induce ICAM-1
expression
(
66), supports this concept. In addition, cellular
activation will also lead to surface expression of the activated
conformational state of LFA-1. This conformational modification
of
LFA-1 can alter the host cell-virus interaction, as shown by
our
previous work (
31) and the present data indicating that
cellular susceptibility to infection with Hi-ICAM-1 virions is
markedly
enhanced by the expression on target cells of the activated
form of
LFA-1. The activated cellular populations living within
the lymph nodes
represent thus an ideal cellular environment to
facilitate the initial
process of virus infection and to achieve
more productive virus
replication events. Results from clinical
studies confirm that lymphoid
organs constitute preferential anatomical
sites for HIV-1 replication,
as 5 to 10 times more virus-infected
cells were found in the lymphoid
organs (lymph nodes, adenoids,
and tonsils) than in the peripheral
blood of HIV-1-infected individuals
(
50).
There are now many reasons to believe that host cell membrane
constituents acquired by HIV-1 play a critical role in the virus
life
cycle (
64). The data we have presented here provide
supplementary
evidence of the functionality of cellular proteins
embedded within
mature HIV-1 particles. On the basis of our results, we
propose
a model in which cellular activation will lead to the
production
of virions bearing high numbers of host-encoded ICAM-1
proteins.
The activated cellular state will also result in the
expression
of LFA-1 in a conformational state of high affinity for
ICAM-1
on the surfaces of target cells. The combined action of these
two factors will ultimately increase the efficiency of the process
of
virus infection. Hence, by using the activated cells of the
immune
system, HIV-1 is able to increase its propagation rate
in strategic
tissues such as the secondary lymphoid organs.
| |
ACKNOWLEDGMENTS |
We thank M. Dufour for excellent technical assistance in flow
cytometric studies. We are grateful to D. Baltimore for pHXB-Luc, M. Emmerman for pCMV-Hygro, N. Landau for
pNL4-3-Luc-ER+ and pcDNA-1/Ada-M
env, T. Springer for pCD1.8, W. C. Greene for 293T
cells, R. Rothlein for RR1/1.1.1 and R6.5 antibodies, and C. C. Fidgor for NKI-L16 antibodies. We are indebted to the NIH AIDS Research
and Reference Reagent Program for kindly providing the following items:
PM1 and Jurkat-tat cells and the anti-gp120 F105 antibody.
This work was supported by a grant to M.J.T. from the Medical Research
Council of Canada (MRC) (grant MT-14438). M.J.T. is the recipient of a
scholarship award from the Fonds de la Recherche en Santé du
Québec. J.-S.P. and J.-F.F. hold Ph.D. fellowships from the
Natural Sciences and Engineering Research Council of Canada and the
MRC, respectively.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
d'ImmunoRétrovirologie Humaine, Centre de Recherche en
Infectiologie, RC709, Centre Hospitalier Universitaire de Québec,
Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec, Canada G1V
4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail:
Michel.J.Tremblay{at}crchul.ulaval.ca.
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Journal of Virology, November 1998, p. 9329-9336, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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