(Journal of Leukocyte Biology. 2000;68:854-864.)
© 2000
by Society for Leukocyte Biology
Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series
Nicolas Genois,
Gilles A Robichaud 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
Correspondence: Michel J. Tremblay, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail:
Michel.J.Tremblay{at}crchul.ulaval.ca
 |
ABSTRACT
|
|---|
Throughout the years, most researchers have used continuous cell lines
as in vitro models to evaluate the immunopathogenesis of
human immunodeficiency virus type-1 (HIV-1) infection. Unfortunately,
the most commonly used monocytoid malignant cells have not been shown
to adequately mimic primary human monocyte-derived macrophages, at
least with respect to HIV-1 infection. The Mono Mac 1 cell line has
been defined as a model system for studying biochemical, immunological,
and genetic functions of human cells of the monocyte/macrophage
lineage. In this study, we have investigated whether Mono Mac 1
represents an in vitro culture system for HIV-1 infection.
Flow cytometric analyses revealed that Mono Mac 1 are positive for the
HIV-1 primary receptor (CD4), as well as for the coreceptors (CXCR4,
CCR5, and CCR3). Infectivity experiments conducted with recombinant
luciferase-encoding and fully infectious viruses demonstrated that Mono
Mac 1 can support a highly productive infection with both macrophage-
and dual-tropic isolates of HIV-1. Furthermore, differentiation of such
cells led to a marked increase in virus production. Data from
semiquantitative polymerase chain reaction analysis and mobility shift
assays indicated that enhanced virus production in differentiated Mono
Mac 1 cells was most likely related to an increase in nuclear
translocation of NF-
B. Mono Mac 1 can thus be considered as a human
monocytoid cell line representing a proper in vitro system
for studying the complex interactions between HIV-1 and cells of the
monocyte/macrophage lineage.
Key Words: monocytes/macrophages AIDS/HIV cellular differentiation cell surface molecules transcription factors
 |
INTRODUCTION
|
|---|
Different cell types are recognized as potential targets for the
human immunodeficiency virus type-1 (HIV-1). The major cellular
reservoirs during HIV-1 infection are cells of the mononuclear
phagocyte series (i.e., monocyte/macrophages) and CD4-expressing cells
of the T-helper lymphocyte subset [1
, 2
].
Tissue macrophages are considered as crucial target cells for virus
infectivity and propagation because, in contrast to viral replication
in CD4+ T lymphocytes, these cells are relatively
refractory to the HIV-1-mediated cytopathic effects. It has been
reported that during primary infection, most viral strains bear a
tropism for macrophages (M-tropic) or both T lymphocytes and
macrophages (dual-tropic) [3
]. However, during the
course of the disease, viral tropism is altered, giving rise to a
particular attraction toward CD4+ T lymphocytes ultimately
receiving the appropriate appellation as T cell tropic (T-tropic) viral
strains [4
]. This change of attraction usually
correlates with a switch in coreceptor usage by the virus, mainly from
CCR5 (R5) for M-tropic to CXCR4 (X4) for T-tropic HIV-1 isolates
[5
6
7
8
9
].
Primary human monocyte/macrophages have been extensively used to define
the role played by cells of the mononuclear phagocyte lineage in the
context of HIV-1 infection. Unfortunately, major problems have been
reported in in vitro studies when using such cells as a
target for HIV-1 infection. For example, marked differences in the
ability to support a productive HIV-1 infection were seen depending on
several variables such as the purity of the isolated population, the
maturation state of the cell (length of time in culture), and the
degree of macrophage adherence [10
]. The donor source
represents another confounding factor influencing the magnitude of
HIV-1 replication. Promonocytic cell lines and primary
monocyte/macrophages display some similarities with respect to
expression of surface markers (e.g., CD14, MHC-II) and functional
capacities (e.g., antigen presentation, accessory cell functions). This
has prompted several investigators to use human myeloid leukemic cell
lines as models to examine the ability of cells of the
monocyte/macrophage lineage to support productive infection with
monocytotropic strains of HIV-1. The most frequently used immortalized
monocytoid cell lines to study HIV-1 infection in vitro
remain the leukemia cell lines U937, THP-1, and HL-60
[11
12
13
]. It should be stated that, in contrast to
primary monocyte-derived macrophages (MDM), these monocytic cell lines
once differentiated to a macrophage phenotype, by means of agents such
as phorbol 12-myristate 13-acetate (PMA) or bacterial
lipopolysaccharide (LPS), display a low permissiveness to infection by
M-tropic isolates of HIV-1 [14
15
16
17
]. Therefore,
differentiated monocytoid cell lines show a very different pattern of
susceptibility to infection by various M-tropic HIV-1 strains and do
not reflect their corresponding primary cell types in studies focused
on viral host cell tropism [14
, 15
,
18
19
20
21
].
The established human tumor cell line Mono Mac 1 has recently been
defined as a good model system to study hematological and immunological
properties of cells from a monocytoid origin because this cell line
displays morphological, histochemical, phenotypic, as well as
functional properties of mature monocytes (e.g., phagocytosis, Fc
receptor, and specific surface marker expression) [22
,
23
]. Furthermore, after LPS treatment, which has been
reported to induce a macrophage-like phenotype, the Mono Mac 1 cell
line also presents phenotypic and physiological properties such as
cytokine secretion [tumor necrosis factor
(TNF-
),
interleukin-1ß (IL-1ß), granulocyte colony-stimulating factor
(G-CSF), and granulocyte-macrophage CSF (GM-CSF)] and specific antigen
presentation (CD14), which resemble mature MDMs [22
,
23
]. Given that U937, THP-1, and HL-60 are considered
either as cells at early stages of monocytic differentiation (U937 and
THP-1) or are clearly myeloid (HL-60) and are poorly susceptible to
infection with M-tropic viral strains, we tested the susceptibility of
the human cell line Mono Mac 1 to HIV-1 infection. In this study, we
provide evidence that Mono Mac 1 represents an appropriate model system
that parallels primary MDM properties in terms of necessary surface
receptor/coreceptors mediating HIV-1 infection by both M- and
dual-tropic strains of HIV-1, infection kinetics, and cellular
differentiation events. Therefore, Mono Mac 1 can be considered as a
representative biological system for in vitro studies of
HIV-1 infection in cells of the monocyte/macrophage lineage.
 |
METHODS
|
|---|
Mono Mac 1 and induction of cellular differentiation
Mono Mac 1 was initially derived from the Mono Mac parental cell
line, which was itself established from the peripheral blood of a
patient diagnosed with an acute peripheral monoblastic leukemia
[22
]. According to morphological, cytochemical, and
immunological criteria, this cell line was assigned to the monocytic
lineage [23
]. The Mono Mac 1 cell line (available
through the German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) was cultured in complete culture medium made of
RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS; GIBCO-BRL, Grand Island, NY), penicillin-streptomycin (100
µg/mL), L-glutamine (2 mM), 1x MEM nonessential amino
acids and sodium pyruvic acid (1 mM). Cultures were maintained at
37°C in a humidified atmosphere containing 5% CO2.
Exponentially growing cells were used throughout our experiments. Mono
Mac 1 cells were induced to differentiate into a more mature phenotype
(i.e., macrophage-like) by treatment with bacterial LPS or phorbol
ester PMA. In brief, cells were adjusted with fresh medium and cultured
in 24-well plates (1 x 106 cells/well in 1 mL). Cells
were then incubated for 72 h in the presence of 10 ng/mL of LPS
(from Escherichia coli serotype 0111:b4, Sigma, St. Louis,
MO) or 20 ng/mL of PMA (Sigma). Differentiation characteristics were
determined by morphological changes such as cell clustering, spreading,
and cellular adhesion to the wells bottom surface.
Flow cytometric analysis
Untreated (monocyte-like) and LPS-treated (macrophage-like) Mono
Mac 1 cells (5 x 105) were incubated for 30 min on
ice with saturating concentrations (1 µg/106 cells) of
monoclonal antibodies directed against CD4 (clone SIM.4), CCR5 (clone
2D7), CXCR4 (clone 12G5), CCR3 (clone 7B11), and CD14 (clone CRIS-6).
Cells were next incubated for 30 min on ice with a saturating
concentration of a secondary antibody constituted of
R-phycoerythrin-conjugated goat anti-mouse immunoglobulin G (IgG) from
Caltag Laboratories (San Francisco, CA). Finally, cells were
resuspended in 500 µL of phosphate-buffered saline (PBS) containing
1% (w/v) paraformaldehyde before flow cytometry analysis (EPICS XL,
Coulter, Miami, FL). Controls consisted of commercial isotype-matched
irrelevant murine monoclonal antibodies (Sigma). Anti-CD4 (SIM.4) and
anti-CCR3 (7B11) monoclonal antibodies were kindly supplied by the AIDS
Research and Reference Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases. Anti-CXCR4 (12G5) and
anti-CCR5 (2D7) monoclonal antibody were purchased from PharMingen,
while anti-CD14 (CRIS-6) was obtained from Biosource International.
Plasmids and preparation of virus stocks
The NL4-3 luciferase backbone
(pNL4-3-Luc-E-R+) and pcDNA-I/Amp-based
expression vectors coding for HIV-1 ADA (R5), JR-FL (R5), BaL (R5),
HXB2 (X4), or amphotropic murine leukemia virus (A-MLV) full-length
envelope proteins were generously provided by Nathaniel R. Landau (The
Salk Institute for Biological Studies, La Jolla, CA)
[9
]. The infectious molecular clones pNL4-3 (X4)
[24
] and p89.6 (R5X4) [25
] were also used
in this study. pNL4-3 was provided by the AIDS Repository Program,
whereas pcDNA-I/89.6 was obtained from Ronald Collman (Pulmonary and
Critical Care Division, University of Pennsylvania, Philadelphia, PA).
The ADA macrophage-tropic virions [26
] were prepared by
infecting primary human MDM. The ADA strain of HIV-1 was kindly
provided by Howard E. Gendelman, as cell-free supernatant from infected
MDM, through the AIDS Repository Program. The pHCMV-G expressing the
broad-host-range vesicular stomatitis virus envelope glycoprotein G
(VSV-G) from the human cytomegalovirus (HCMV) promoter has been
described previously [27
]. Fully infectious viral
entities (NL4-3, ADA, 89.6) and luciferase reporter viruses pseudotyped
with various envelope proteins (ADA, JR-FL, BaL, HXB2, A-MLV, VSV-G)
were generated by calcium phosphate cotransfection in 293T cells as
described previously [28
, 29
]. Negative
controls for experiments conducted with luciferase-encoding virions
consisted of envelope-free viruses. Virus-containing supernatants were
harvested 40 h posttransfection and frozen at -85°C until used.
Virus preparations were quantified using a commercial assay for the
viral major core protein p24 (Organon Teknika, Durham, NC).
Viral infection assay
Untreated (monocyte-like) and LPS-treated
(macrophage-like) Mono Mac 1 cells (1 x 105) were
seeded in 96-well dishes in complete culture medium and infected with
luciferase-encoding pseudotyped virions (10 ng of p24) in a total
volume of 200 µL. After 72 h, 100 µL of cell-free supernatant
from each well were removed and complemented with 25 µL of 5x
Promega cell culture lysis buffer [125 mM triphosphate (pH 7.8), 10 mM
dithiothreitol (DTT), 5% Triton X-100, 50% glycerol] for a 30-min
incubation period at room temperature. An aliquot of 20 µL from the
lysate was then mixed with 100 µL of luciferase assay buffer [210 mM
tricine, 1.07 mM (MgCO3)4
Mg(OH)2 5H2O, 2.67 mM MgSO4, 0.1
mM EDTA, 270 µM CoA, 470 µM luciferine, 530 µM ATP, 33.3 mM
DTT]. Finally, luciferase activity was monitored using a microplate
luminometer (MLX; Dynex Technologies, Chantilly, VA). Kinetics of viral
infection was assessed by infecting untreated (monocyte-like) and
LPS-treated (macrophage-like) Mono Mac 1 cells (3 x
105) in 24-well dishes (final volume of 500 µL) with
fully infectious progeny viruses (NL4-3, ADA, 89.6/30 ng of
p24). Cells were incubated for 2532 days, during which aliquots of
100 µL of cell-free supernatant were retrieved to evaluate reverse
transcriptase activity as described previously [30
].
Virally infected Mono Mac 1 cells were observed in light microscopy for
the presence of HIV-1-mediated syncytium formation and were
photographed at a magnification of x100 with an inverted microscope.
Viral entry assay
Undifferentiated Mono Mac 1 cells (1 x 106
cells/mL, 250 µL/well) were exposed to similar amounts of
HIV-1NL4-3 (X4) or HIV-1JR-FL (R5) (100 ng of
p24) in complete culture medium for 2.5 h at 37°C. Cells were
washed twice with 250 µL of ice-cold PBS and were next resuspended in
250 µL of cold RPMI 1640 (without FBS) containing pronase (Boehringer
Mannheim, Laval, Quebec) at 0.1 mg/mL for 5 min at 4°C. 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
µL of disruption buffer (0.5% Triton X-100 in PBST). Cells were
agitated for 10 min at room temperature and then stored at -20°C
until assayed for p24 content.
Semiquantitative PCR analysis
After LPS treatment and infection with HIV-1, total cellular DNA
was isolated using a DNA extraction kit (Quiagen). The PCR reaction
mixture was made of 1.5 µg of DNA, 200 µM of each four
deoxynucleotide triphosphates along with MgCl2 (1x), Taq
buffer (1x), and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus).
This reaction mixture was covered with 40 µL of mineral oil and then
subjected to denaturation (10 min at 94°C) followed by 27 cycles of 1
min at 94°C, 2 min at 56°C, 3 min at 72°C, and finally 10 min at
72°C for complete polymerization. Primer pairs used for these
experiments included M667/M661, which can detect full-length or nearly
completely synthesized HIV-1 DNA (200-bp fragment) [31
].
In parallel, the primer set 14-33/123-104 was used to amplify a 100-bp
fragment specific for the human ß-globin gene.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared according to the previously
described microscale preparation protocol [32
]. Briefly,
untreated and LPS-treated Mono Mac 1 cells (5 x 106)
were washed with ice-cold PBS and resuspended in 400 µL of cold
buffer A [10 mM
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic
acid (HEPES; pH7.9), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and
0.5 mM phenylmethylsulfonyl fluoride(PMSF)]. The cells were allowed to
swell on ice for 15 min, after which 25 µL of a 10% solution of
Nonidet P-40 is added and the tube is vigorously vortexed for 30 s. The homogenate was centrifuged for 10 s at 12,000 g.
The supernatant fraction was discarded and the pellet was resuspended
in 50 µL of cold buffer C [20 mM HEPES-KOH (pH 7.9), 0.4 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF] and incubated at 4°C
for 15 min on a shaking platform. Cellular debris were removed by
centrifugation at 12,000 g for 20 min at 4°C and the
supernatant fractions were stored at -70°C until used. Ten
micrograms of nuclear extracts were used to perform EMSA as determined
by the bicinchoninic acid assay (BCA) with a commercial protein assay
reagent (Pierce, Rockford, IL). Nuclear extracts were incubated for 20
min at room temperature in 15 µL of the binding buffer [100 mM HEPES
(pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol,
5 mM EDTA, 250 mM NaCl, 2 µg poly (dI-dC), 10 µg nuclease-free
bovine serum albumin fraction V] containing 1 ng of
32P-5-end-labeled double-stranded (dsDNA)
oligonucleotide. Double-stranded DNA (100 ng) was labeled with
[
-32P]ATP and T4 polynucleotide kinase in a kinase
buffer (New England Biolabs, Beverly, MA). This mixture was incubated
for 30 min at 37°C and the reaction was stopped with 5 µL of 0.2 M
EDTA. The labeled oligonucleotide was extracted with phenol/chloroform
and passed through a G-50 spin column. The dsDNA oligonucleotide, which
was used as a probe, contained the consensus NF-
B and HIV-1
enhancer-binding site corresponding to the sequences
5-ATGTGAGGGGACTTTCCCAGGC-3 and
5-CAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3, respectively. The DNA-protein
complexes were migrated on a 4% (w/v) polyacrylamide gel containing
0.5xTris-Borate-EDTA. The gels were then subsequently dried and
autoradiographed. Specific DNA-protein complexes were identified
through the use of 100-fold competitions of cold specific DNA
oligonucleotides.
 |
RESULTS
|
|---|
HIV-1 primary receptor and coreceptor expression on Mono Mac1
To define susceptibility of Mono Mac 1 to HIV-1 infection, we
studied by flow cytometry the phenotype of this cell line. First, the
monocytic surface marker CD14 was found to be expressed at low levels
on untreated Mono Mac 1 cells (data not shown). Given that CD14
expression is recognized as a good phenotypic marker for monocyte
differentiation, Mono Mac 1 cells were then treated with potent
inducers of maturation (i.e., LPS and PMA). Treatment of Mono Mac-1
cells with either LPS or PMA was found to slightly up-regulate the CD14
staining (data not shown).
Next, in order to evaluate the functional capacity of Mono Mac 1 to act
as a cellular target for HIV-1 infection, we measured by flow cytometry
the levels of HIV-1 primary receptor and coreceptors at the cell
surface. The cell-surface markers CD4, CCR5, CXCR4, and CCR3 were
expressed on 58, 100, 73, and 40%, respectively, of untreated Mono Mac
1 cells (Fig. 1
, left panels). It is interesting to note that, as it is the case
for Mono Mac 1 cells, primary human MDM have also been reported to be
positive for CD4 and to express detectable levels of CXCR4 on their
membranes [33
]. Treatment of Mono Mac 1 cells with 10
ng/mL of LPS for 72 h induces a slight decrease in the levels of
surface expression of CD4, CXCR4, and CCR3 (Fig. 1
, right panels).
However, LPS-treatment of Mono Mac 1 slightly increases the intensity
of CCR5 staining from a specific mean fluorescence intensity of 15.3 in
undifferentiated cells (monocyte-like) to 17.7 for differentiated cells
(macrophage-like). Maturation of Mono Mac 1 cells was confirmed by the
observation that LPS treatment induces an increased adherence to the
plastic surface of the culture vessels (data not shown).
Differentiation of Mono Mac 1 was further distinguished by
morphological changes of the cells because treatment with LPS resulted
in the development of irregular shapes and more extensive projections
of cell membranes (data not shown). These results suggest that
LPS-mediated maturation of Mono Mac 1 cells from an immature
(monocyte-like) to a more mature phenotype (macrophage-like) mimics the
primary human monocyte/macrophage lineage.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Flow cytometric studies of HIV-1 primary cellular receptor and
coreceptors. Mono Mac 1 cells were either left untreated or were
treated for 72 h with LPS (10 ng/mL) before staining with anti-CD4
(clone SIM.4), anti-CCR5 (clone 2D7), anti-CXCR4 (clone 12G5), and
anti-CCR3 (clone 7B11) monoclonal antibodies (dotted lines). Controls
consisted of cells incubated with an isotype-matched irrelevant
monoclonal antibody (solid lines).
|
|
Mono Mac 1 cells are highly permissive to infection with M-tropic
strains of HIV-1
Having established that Mono Mac 1 cells do express on their
surface the necessary HIV-1 primary receptor and coreceptors, we next
defined their permissiveness to virus infection. Single-round infection
assays were carried out using recombinant luciferase encoding virions
pseudotyped with full-length envelope proteins from either HXB2, BaL,
ADA, or JR-FL. These recombinant progeny viruses were used as
prototypes for lymphocytotropic (HXB2) and monocytotropic (BaL, ADA,
JR-FL) viruses, respectively. Untreated and LPS-treated Mono Mac 1
cells were found to be weakly permissive to infection with viruses
bearing T-tropic Env proteins (HXB2; Fig. 2A
). Our data are consistent with the recent demonstration that
primary human MDM are weakly permissive to infection with
CXCR4-dependent T cell line-adapted (TCLA) strains of HIV-1
[21
, 34
]. To demonstrate that the low
permissiveness of Mono Mac 1 to an X4 isolate of HIV-1 is not due to a
lack of infectivity of our virus stock, we used a cell line highly
susceptible to infection with T-tropic strains of HIV-1. Results from
Figure 2B
clearly indicate that our luciferase-encoded virus
preparation pseudotyped with envelope proteins from HXB2 is highly
infectious for WE17/10, an IL-2-dependent human T cell line known to be
susceptible to infection with T cell line-adapted X4 strains of HIV-1
[35
].

View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. Single-round infection assays with recombinant luciferase-encoding
virions. (A) Untreated (open bars) and LPS-treated (filled bars) Mono
Mac 1 cells (1 x 105) were infected with HIV-1-based
luciferase reporter viruses (10 ng of p24) bearing either T- (HXB2) or
M-tropic (BaL, ADA, JR-FL) envelope proteins. (B) WE17/10 cells (1 x 105) were infected with HIV-1-based luciferase reporter
viruses (10 ng of p24) bearing either T- (HXB2) or M-tropic (BaL)
envelope proteins. Infection was allowed to proceed for 72 h
before lysis of the samples. Viral infections were translated in
luciferase activity that was read with a luminometer apparatus. Results
are shown as the mean ± SD of samples carried out in
triplicate. These data are representative of three independent
experiments. Controls consisted of cells infected with Env-deficient
luciferase-encoding viruses.
|
|
Infection of these human monocytic cells with virions pseudotyped with
M-tropic Env proteins showed a different pattern. Indeed, untreated
Mono Mac 1 cells were found to be more susceptible to infection with
HIV-1-based luciferase reporter viruses bearing all tested M-tropic
envelope proteins than T-tropic Env. Moreover, LPS-treatment of Mono
Mac 1 cells resulted in a significant increase in reporter gene
activity after infection with M-tropic strains. Thus, the human
monocytoid cell line Mono Mac 1 can be productively infected with HIV-1
particles bearing commonly studied M-tropic Env proteins (i.e., BaL,
ADA, JR-FL) and virus production in such cells is markedly enhanced
upon cellular differentiation.
Kinetics studies of HIV-1 infection
Our next interest was to assess the capacity of this human
monocytic cell line to support replication of unmodified complete
virions because HIV-1 replication is known to be a complex process that
is regulated by proteins of viral and cellular origin that has been
shown to work in cis and in trans. This goal was
achieved by infecting both untreated (monocyte-like) and LPS-treated
Mono Mac 1 cells (macrophage-like) with fully infectious viruses
displaying a tropism for either CD4+ T lymphocytes
(HIV-1NL4-3), monocyte/macrophages (HIV-1ADA),
or both of them (HIV-189.6). In this set of experiments,
the extent of virus replication was investigated by measuring reverse
transcriptase activity in cell-free culture supernatants. In accord
with our previous infection studies with T-tropic recombinant virions,
a weak virus production was detected at a very late time point in
untreated and LPS-treated Mono Mac 1 cells infected with the
CXCR4-dependent T cell line-adapted (TCLA) strain of HIV-1, NL4-3
(Fig. 3A
). In contrast, a sustained and much stronger virus production was
observed in undifferentiated Mono Mac 1 cells infected with the
M-tropic ADA isolate and the dual-tropic 89.6 strain of HIV-1 (Fig. 3B
3C)
. Again, virus production in cells infected with M-tropic isolates
was enhanced upon treatment of Mono Mac 1 with LPS, which confirms our
previous data with HIV-1-based luciferase reporter viruses.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 3. Kinetics of virus infection with fully infectious HIV-1 particles.
Untreated (open circles) and LPS-treated Mono Mac 1 cells (filled
circles; 3 x 105) were infected with fully infectious
strains of HIV-1 (NL4-3/T-tropic, ADA/M-tropic, and 89.6/dual-tropic;
30 ng of p24). Virus production was monitored by measuring reverse
transcriptase activity in cell-free supernatants at the indicated time
points. Data shown as the mean of triplicate samples and are
representative of two independent experiments.
|
|
Higher susceptibility of Mono Mac 1 to infection with
macrophage-tropic isolates is due to an enhancement of virus entry
We next attempted to define the mechanism(s) responsible for the
lower susceptibility of Mono Mac 1 cells to infection with virions
bearing T-tropic envelope proteins despite CXCR4 expression. Data from
a virus entry assay revealed that T-tropic virions are blocked at an
early stage of infection (Table 1
).
Higher virus production in differentiated Mono Mac 1 cells is due
to increased levels of endogenous nuclear NF-
B
Our next series of experiments was aimed at the identification of
the putative mechanism responsible for the enhancement of virus
expression in LPS-treated Mono Mac 1 cells (macrophage-like). First, we
measured the initial steps in the virus replicative cycle by performing
a semiquantitative PCR assay with a combination of primers (M667/M661)
known to recognize only full-length or nearly completely synthesized
viral DNA [31
]. The macrophage-tropic isolate
HIV-1BaL was used for these studies because our previous
experiments have shown that infection with this specific viral strain
led to the most dramatic difference in susceptibility to virus
infection between untreated and LPS-treated Mono Mac 1 cells (Fig. 2)
.
As depicted in Figure 4A
, the observed increase in virus production depending on the
cellular maturation stage (i.e., undifferentiated vs. differentiated)
could not be attributed to an enhancement in the early events of the
HIV-1 life cycle (i.e., binding, fusion, entry, and reverse
transcription). To demonstrate the sensitivity of our semi-quantitative
PCR analysis to detect differences in viral DNA input, serial plasmid
dilutions of the viral clone NL4-3 were amplified to demonstrate the
detection of various template concentrations (Fig. 4B)
.
The ubiquitous mammalian transcription factor NF-
B has been shown to
act as a major constituent in HIV-1 LTR-driven regulation and to be
essential for efficient HIV-1 expression [36
37
38
].
Reports have also indicated that nuclear NF-
B is constitutively
expressed in cells of the monocyte/macrophage lineage (i.e., monocytic
cell lines, blood monocytes, tissue macrophages)
[38
39
40
41
]. Moreover, constitutive NF-
B can be further
increased with stage of maturation [38
]. As illustrated
in Figures 2
and 3
, replication of M- and dual-tropic HIV-1 strains was
more efficient in LPS-treated than untreated Mono Mac 1 cells. In an
attempt to define whether the observed up-regulation in virus
production could be related to an increase in nuclear translocation of
NF-
B, we conducted virus infection assays with luciferase reporter
viruses bearing either the amphotropic murine leukemia virus (A-MLV)
envelope protein or vesicular stomatitis virus envelope glycoprotein G
(VSV-G). Virions pseudotyped with A-MLV and VSV-G Env proteins will
enter Mono Mac 1 cells in a CD4/CCR5/CXCR4-independent manner. Higher
levels of virus-encoded reporter gene activity was still seen in
differentiated Mono Mac 1 cells compared with untreated cells after
infection with A-MLV and VSV-G pseudotyped recombinant viruses
(Fig. 5
). Data from this set of experiments suggest that the intracellular
milieu prevailing inside treated Mono Mac 1 cells is more favorable for
HIV-1 replication than in untreated cells. The role played by NF-
B
in the increase of virus replication was assessed by performing gel
mobility shift assays in cell lysates before and after LPS treatment.
After treatment of Mono Mac 1 cells with LPS, a noticeable increase in
endogenous nuclear NF-
B levels was detected when using a
radiolabeled probe corresponding to the consensus NF-
B binding
sequence (Fig. 6A
). Given that the crucial role played by NF-
B in up-regulation
of HIV-1 transcriptional activity is due to its association to the
HIV-1 LTR enhancer region [42
43
44
], we next performed
EMSA using a radiolabeled probe containing the complete HIV-1
enhancer-binding sequence. Again, binding of NF-
B to the HIV-1 LTR
enhancer domain was increased upon treatment of Mono Mac 1 cells with
LPS (Fig. 6B) . Altogether, results from these studies suggest that the
increase in virus production that is seen in more mature Mono Mac 1
cells (macrophage-like) compared with Mono Mac 1 cells displaying a
more immature phenotype (monocyte-like) is most likely due to higher
levels of endogenous nuclear NF-
B.

View larger version (33K):
[in this window]
[in a new window]
|
Figure 5. Single-round infection experiments with CD4/CCR5/CXCR4-independent
reporter gene-encoding progeny viruses. Untreated (open bars) and
LPS-treated (filled bars) Mono Mac 1 cells (1 x 105)
were infected with HIV-1-based luciferase reporter viruses (10 ng of
p24) pseudotyped with A-MLV and VSV-G Envs. Samples were lysed after a
72-h incubation period and read for luciferase activity with a
luminometer. Results are shown as the mean ± SD of
samples carried out in triplicate. These data are representative of
three independent experiments. Controls consisted of cells infected
with Env-deficient luciferase-encoding viruses.
|
|
Syncytium formation is observed after HIV-1 infection of Mono Mac 1
In the process of HIV-1 pathogenesis, virus-mediated syncytium
formation has been proposed as being responsible to some extent for
CD4+ T lymphocyte depletion. Cell-to-cell fusion has also
been reported to take place after infection of MDM with some strains of
HIV-1 [45
46
47
]. During the course of infection of Mono
Mac 1 cells with fully infectious complete virions (NL4-3, ADA, 89.6),
visual observations were conducted by light microscopy. Syncytium
formation was detected in LPS-treated Mono Mac 1 cells infected with
either ADA or 89.6 (Fig. 7
). Larger and qualitatively more numerous syncytia were noticed in
LPS-treated cells infected with 89.6 compared with LPS-treated Mono Mac
1 inoculated with ADA. This finding is in correlation with the greater
virus production seen in 89.6-infected cells compared with Mono Mac 1
cells infected with ADA (Fig. 3)
. In reference to NL4-3 virus
production, syncytium formation, as expected, was not detected after
infection of LPS-treated Mono Mac 1 cells with NL4-3, a CXCR4-dependent
TCLA strain of HIV-1 (data not shown).

View larger version (133K):
[in this window]
[in a new window]
|
Figure 7. Virus-mediated syncytium formation by M- and dual-tropic HIV-1 strains.
Undifferentiated (A) and LPS-treated Mono Mac 1 cells (B, C, and D)
were either left uninfected (A, B) or were infected with
fully-infectious ADA (M-tropic; panel C) and 89.6 (dual-tropic; panel
D). Cells were analyzed for HIV-1-mediated syncytium formation and
photographed at a magnification of x100 with an inverted microscope.
Arrows indicate syncytia.
|
|
 |
DISCUSSION
|
|---|
Laboratory and clinical strains of HIV-1 have been divided into
two distinct categories depending on their cellular tropism,
replication kinetics, and propensity to mediate cell-to-cell conjugate
or syncytia formation [48
49
50
]. Viral isolates
displaying a tropism for monocyte/macrophages (slow/low and non
syncytia-inducing/NSI) can infect both MDM and primary CD4+
T lymphocytes but do not usually infect established T cell lines. On
the opposite, HIV-1 strains with a tropism for T cell lines (rapid/high
and syncytia-inducing/SI) can grow in leukemic T cell lines and primary
CD4+ T lymphocytes. Unfortunately, studies with M-tropic
isolates of HIV-1 are greatly complicated by the fact that none of the
most extensively used monocytoid cell lines properly represent primary
human monocyte/macrophages. A clear example of this is provided by the
observation that a highly productive infection with M-tropic HIV-1
isolates cannot be achieved in monocytoid cell lines currently in use.
Because of the crucial role played by cells of the monocyte/macrophage
lineage in the pathogenesis of HIV-1 infection, the discovery of a
monocytoid cell line showing similarities with primary human
monocyte/macrophages, with respect to susceptibility to HIV-1
infection, would represent a useful tool for the study of virus-cell
interactions. Therefore, the central objective of our series of
investigations was to determine whether the human monocytic cell line
Mono Mac 1 represents a suitable in vitro model system for
studying the infection of cells of the monocytic lineage with
monocytotropic HIV-1 strains.
As specified above, previous reports have demonstrated that the most
widely used human cell lines of monocytoid origin do not represent an
ideal system to investigate the effects of HIV-1 infection on cells of
the monocyte/macrophage lineage [14
, 15
,
17
, 20
, 51
, 52
].
For example, Valentin and colleagues discovered that the M-tropic
isolate HIV-1BaL was unable to establish a productive
infection in any of the five human monocytoid cell lines tested (U937,
THP-1, RC2A, Mono Mac 6, and DD) [20
]. It was thus
concluded that these human continuous cell lines do not parallel
primary MDM, at least with respect to susceptibility to infection with
M-tropic HIV-1 variants. With this information in mind, we focused our
attention on Mono Mac 1, a recently established human cell line showing
characteristics of mature blood monocytes. This line has already been
extensively characterized in terms of reactive oxygen intermediates and
lysozyme production, phagocytosis, surface expression of specific
markers (e.g., CD13, CD14, CD15, CD68, MHC class II), and cytokine
production in response to LPS or PMA treatment (e.g., TNF-
, G-CSF,
M-CSF, GM-CSF, IL-1ß) [22
, 23
].
We first proceeded to the immunophenotyping of Mono Mac 1 cells using
flow microfluorimetry. We report here that Mono Mac 1 are expressing
CD14, a marker found on cells committed to myeloid/monocytic lineages,
and are also positive for CD4, the primary cellular receptor for HIV-1.
It is interesting to note that our flow cytometric studies revealed
that Mono Mac 1 cells also express the chemokine coreceptors CXCR4,
CCR5, and CCR3. Differentiation of Mono Mac 1 to a more mature
phenotype (macrophage-like) by treatment with bacterial-derived LPS
resulted in a slight reduction of CD4, CXCR4, and CCR3 surface markers.
In contrast, CCR5 antigen expression increased after LPS treatment,
which is in agreement with a recent report showing that CCR5 mRNA level
is up-regulated in PMA-treated HL-60 cells [21
]. These
observations correlate also with results from other groups who have
reported an up-regulation of CCR5 after differentiation of primary
monocytes from healthy donors [53
, 54
]. It
can be concluded that the Mono Mac 1 cell line is to date one of the
only monocytoid cell lines showing properties similar to primary
monocyte/macrophages in response to cellular differentiating agents.
Using single-round infection assays with recombinant
luciferase-encoding virions pseudotyped with T- and M-tropic Env
proteins, we were able to monitor permissiveness of both untreated and
treated Mono Mac 1 cells to viral infection. The final proof of the
susceptibility of Mono Mac 1 to virus infection was attained using
fully infectious T-, M-, and dual-tropic isolates of HIV-1. Viral
infection experiments revealed that T-tropic strains of HIV-1 poorly
replicate in both untreated and treated Mono Mac 1 cells. These results
do corroborate the study by Verani and co-workers who have reported
that primary human macrophages are highly resistant to infection with
T-tropic (CXCR4-dependent) TCLA strains of HIV-1 [34
].
However, untreated Mono Mac 1 were found to be highly susceptible to
infection by progeny viruses bearing M-tropic or dual-tropic envelope
proteins. Productive infection with M- and dual-tropic strains was
increased upon LPS-treatment of Mono Mac 1 cells. The observation that
LPS treatment leads to an increase in virus replication despite a
concomitant down-regulation of surface CD4 expression is not a surprise
considering that M-tropic isolates have already been shown to be less
dependent on the level of surface CD4 for infection to proceed
[55
]. Two different mechanisms, which are not mutually
exclusive, might be responsible for the observed higher virus
production in the LPS-treated Mono Mac 1 cells. The first scenario is
linked with the noticed enhancement of surface CCR5 expression (Fig. 1) and the second possibility is related with activation and nuclear
translocation of NF-
B, a transcriptional factor known as a potent
activator of HIV-1 transcription [37
]. Measurement of
viral DNA by a semiquantitative PCR assay revealed that the enhancement
in virus production in LPS-treated Mono Mac 1 cells was not due to a
concomitant increase in the early steps in the virus life cycle, namely
the process of virus binding, fusion, entry, and reverse transcription.
The implication of NF-
B was then studied based on the previously
reported activation of NF-
B after LPS treatment of
monocyte/macrophages [56
57
58
59
]. Of interest to note is
the study by Frankenberger and colleagues showing that constitutive
NF-
B in Mono Mac 1 cells can be further increased with LPS treatment
[40
]. Infectivity experiments conducted with
CCR5-independent virions made of the NL4-3 luciferase backbone
pseudotyped with A-MLV and VSV-G Envs allowed us to propose that
NF-
B activation was most likely responsible for the increase in
virus production seen in LPS-treated Mono Mac 1. This idea was
confirmed by gel mobility shift assays, which revealed that higher
amounts of nuclear NF-
B was detected in differentiated cells
(LPS-treated) compared with untreated Mono Mac 1 cells. Given that
NF-
B strongly binds to the HIV-1 enhancer element, a region in the
viral regulatory sequences known to be essential for the induction of
HIV-1 transcriptional activity [42
43
44
], EMSAs were also
carried out using this sequence as a probe. Again, LPS treatment of
Mono Mac 1 cells was leading to an increase in NF-
B binding activity
to the HIV-1 enhancer domain.
We also report here that the NSI isolate ADA (M-tropic) can form
syncytia in LPS-treated Mono Mac 1 cells. Our data are consistent with
previous observations indicating that Env proteins from M-tropic
strains SF162, JR-FL, and BX08, which are also defined as NSI, can
mediate cell-to-cell fusion [55
, 60
,
61
]. Altogether, these findings indicate that the
traditional nomenclature referring to the tropism of HIV-1 isolates as
either NSI or SI [50
] is no longer valid and should
therefore be revised. It seems clear that the definition of HIV-1
tropism (i.e., T- vs. M-tropic) based on infection of established human
cell lines does not adequately reflect the complex nature of virus-host
cell interactions.
In summary, given that untreated Mono Mac 1 cells display properties of
blood monocytes and that LPS treatment leads to the acquisition by Mono
Mac 1 of a more mature phenotype (macrophage-like), this cell line can
be used as a model system to study various aspects of monocytic
functions. Mono Mac 1 is to date one of the few reported monocytoid
cell lines that seems to effectively mimic primary human
monocyte/macrophages in terms of surface antigen expression, cellular
differentiation events and, as indicated by our findings,
permissiveness to HIV-1 infection by M- and dual-tropic strains of
HIV-1. The usefulness of this monocytoid cell line as a tool for
studying several aspects of the life cycle of HIV-1 is clearly provided
by the observation that differentiation of Mono Mac 1 results in an
enhancement of virus replication. This finding is consistent with
previous studies indicating that the permissiveness of primary human
monocyte/macrophages for HIV-1 infection heavily depends on the state
of cell differentiation [62
63
64
]. Taken together, our
data show that Mono Mac 1 represents a proper system to study, in the
context of a human monocytoid malignant cell line, complex interactions
occurring between HIV-1 and cells of the monocyte/macrophage lineage.
 |
ACKNOWLEDGEMENTS
|
|---|
These studies were rendered feasible by the financial support for
M. J. T. from the Medical Research Council of Canada (grants
MT-14438, GR-14500, and AI-15575). We thank M. Dufour for technical
assistance in flow cytometry studies. This work was performed by
N. G. and G. A. R. in partial fulfillment of M.Sc. and
Ph.D. degrees, respectively, at the Faculty of Graduate Studies,
Department of Medical Biology, Faculty of Medicine, Laval University.
M. J. T. is the recipient of a Medical Research Council of
Canada Scientist Award and G. A. R. holds a Ph.D. Fellowship
from the FRSQ-FCAR Programme Santé. Nicolas Genois and Gilles
Robichaud contributed equally to this work.
Received December 7, 1999;
revised June 2, 2000;
accepted July 10, 2000.
 |
REFERENCES
|
|---|
-
Gendelman, H. E., Oreinstein, J. M., Bach, L. M., Weiser, B., Burger, H., Kalter, D. C., Meltzer, M. S. (1989) The macrophage in the persistence and pathogenesis of HIV infection AIDS 3,475-495[Medline]
-
Schnittman, S. M., Psallidopoulos, M. C., Lane, H. C., Thompson, L., Baseler, M., Massari, F., Fox, C. H., Salzman, N. P., Fauci, A. S. (1989) The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4 Science 245,305-308[Abstract/Free Full Text]
-
Zhu, T., Mo, H., Wang, N., Nam, D.S., Cao, Y., Koup, R. A., Ho, D. D. (1993) Genotypic and phenotypic characterization of HIV-1 in patients with primary infection Science 261,1179-1181
-
Schuitemaker, H., Koot, M., Kootstra, N. A., Dercksen, M. W., de Goede, R. E. Y., van Steenwijk, R. P., Lange, J. M., Schattenkerk, J. K., Miedema, F., Tersmette, M. (1992) Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population J. Virol. 66,1354-1360[Abstract/Free Full Text]
-
Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P. E., Murphy, P. M., Berger, E. A. (1996) CC CKR5: a RANTES, MIP-1
, MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1 Science 272,1955-1959[Abstract]
-
Berson, J. F., Long, D., Doranz, B. J., Rucker, J., Jirik, F. R., Doms, R. W. (1996) A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains J. Virol. 70,6288-6295[Abstract]
-
Feng, Y., Broder, C. C., Kennedy, P. E., Berger, E. A. (1996) HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor Science 272,872-877[Abstract]
-
Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G. L., Newman, W., Gerard, N., Gerard, C., Sodroski, J. (1996) The ß-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates Cell 85,1135-1148[Medline]
-
Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., Landau, N. R. (1996) Identification of a major co-receptor for primary isolates of HIV-1 Nature 381,661-666[Medline]
-
Bennett, S., Breit, S. N. (1994) Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV J. Leukoc. Biol. 56,236-240[Abstract]
-
Sundstrom, C., Nilsson, K. (1976) Establishment and characterization of a human histiocytic lymphoma cell line (U-937) Int. J. Cancer 17,565-577[Medline]
-
Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP-1) Int. J. Cancer 26,171-176[Medline]
-
Collins, S. J., Gallo, R. C., Gallagher, R. E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture Nature 270,347-349[Medline]
-
Collman, R. (1992) Human immunodeficiency virus type 1 tropism for human macrophages Pathobiology 60,213-218[Medline]
-
Schuitemaker, H., Kootstra, N. A., Groenink, M., de Goede, R. E. Y., Miedema, F., Tersmette, M. (1992) Differential tropism of clinical HIV-1 isolates for primary monocytes and promonocytic cell lines AIDS Res. Hum. Retroviruses 8,1679-1682[Medline]
-
Schuitemaker, H., Kootstra, N. A., de Goede, R. E. Y., de Wolf, F., Miedema, F., Tersmette, M. (1991) Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture J. Virol. 65,356-363[Abstract/Free Full Text]
-
Kitano, K., Baldwin, G. C., Raines, M. A., Golde, D. W. (1990) Differentiating agents facilitate infection of myeloid leukemia cell lines by monocytotropic HIV-1 strains Blood 76,1980-1988[Abstract/Free Full Text]
-
Ho, D. D., Rota, T. R., Hirsch, M. S. (1986) Infection of monocyte/macrophages by human T lymphotropic virus type III J. Clin. Invest. 77,1712-1715
-
Collman, R., Hassan, N. F., Walker, R., Godfrey, B., Cutilli, J., Hastings, J. C., Friedman, H., Douglas, S. D., Nathanson, N. (1989) Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1) J. Exp. Med. 170,1149-1163[Abstract/Free Full Text]
-
Valentin, A., Nillson, K., Asjö, B. (1994) Tropism for primary monocytes and for monocytoid cell lines are separate features of HIV-1 variants J. Leukoc. Biol. 56,225-229[Abstract]
-
Di Marzio, P., Tse, J., Landau, N. R. (1998) Chemokine receptor regulation and HIV type 1 tropism in monocyte-macrophages AIDS Res. Human Retroviruses 14,129-138[Medline]
-
Steube, K. G., Teepe, D., Meyer, C., Zaborski, M., Drexler, H. (1997) A model system in haematology and immunology: the human monocytic cell line MONO-MAC-1 Leukemia Res 21,327-335[Medline]
-
Ziegler-Heitbrock, H. W., Thiel, E., Futterer, A., Herzog, V., Wirtz, A., Riethmuller, G. (1988) Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes Int. J. Cancer 41,456-461[Medline]
-
Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., Martin, M. A. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman transfected cells with an infectious molecular clone J. Virol. 59,284-291[Abstract/Free Full Text]
-
Collman, R., Balliet, J. W., Gregory, S. A., Friedman, H., Kolson, D. L., Nathanson, N., Srinivasan, A. (1992) An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1 J. Virol. 66,7517-7521[Abstract/Free Full Text]
-
Gendelman, H. E., Orenstein, J. M., Martin, M. A., Ferrua, C., Mitra, R., Phipps, T., Wahl, L. A., Lane, H. C., Fauci, A. S., Burke, D. S., Skillman, D., Meltzer, M. S. (1988) Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor l-treated monocytes J. Exp. Med. 167,1428-1441[Abstract/Free Full Text]
-
Yee, J. K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. C., Friedmann, T. (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes Proc. Natl. Acad. Sci. USA 91,9564-9568[Abstract/Free Full Text]
-
Fortin, J.-F., Cantin, R., Lamontagne, G., Tremblay, M. (1997) Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhances viral infectivity J. Virol. 71,3588-3596[Abstract]
-
Paquette, J.-S., Fortin, J.-F., Blanchard, L., Tremblay, M. J. (1998) Level of ICAM-1 surface expression on virus producer cells influences both the amount of virion-bound host ICAM-1 and human immunodefiency virus type 1 infectivity J. Virol. 72,9329-9336[Abstract/Free Full Text]
-
Hoffman, A. D., Banapour, B., Levy, J. A. (1985) Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions Virology 147,326-335[Medline]
-
Zack, J. A., Arrigo, S. J., Weitsman, S. R., Go, A. S., Haislip, A., Chen, I. S. Y. (1990) HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure Cell 61,213-222[Medline]
-
Meyer, B. E., Malim, M. H. (1994) The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm Genes Dev 8,1538-1547[Abstract/Free Full Text]
-
Doms, R. W., Peiper, S. C. (1997) Unwelcomed guests with master keys: how HIV uses chemokine receptors for cellular entry Virology 235,179-190[Medline]
-
Verani, A., Pesenti, E., Polo, S., Tresoldi, E., Scarlatti, G., Lusso, P., Siccardi, A. G., Vercelli, D. (1998) CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates J. Immunol. 161,2084-2088[Abstract/Free Full Text]
-
Willard-Gallo, K. E., Van de Keere, F., Kettmann, R. (1990) A specific defect in CD3 gamma-chain gene transcription results in loss of T-cell receptor/CD3 expression late after human immunodeficiency virus infection of a CD4+ T-cell line Proc. Natl. Acad. Sci. USA 87,6713-6717[Abstract/Free Full Text]
-
Kawakami, K., Scheidereit, C., Roeder, R. G. (1988) Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-
B) that actives transcription from a human immunodeficiency virus type 1 promoter in vitro Proc. Natl. Acad. Sci. USA 85,4700-4704[Abstract/Free Full Text]
-
Nabel, G. J., Baltimore, D. (1987) An inducible transcription factor activates expression of human immunodeficiency virus in T cells Nature 326,711-713[Medline]
-
Griffin, G. E., Leung, K., Folks, T. M., Kunkel, S., Nabel, G. J. (1989) Activation of HIV gene expression during monocyte differentiation by induction of NF-
B Nature 339,70-73[Medline]
-
Kaufman, P. A., Weinberg, J. B., Greene, W. C. (1992) Nuclear expression of the 50- and 65-kD Rel-related subunits of nuclear factor-kappa B is differentially regulated in human monocytic cells J. Clin. Invest. 90,121-129
-
Frankenberger, M., Pforte, A., Sternsdorf, T., Passlick, B., Baeuerle, P. A., Ziegler-Heitbrock, H. W. (1994) Constitutive nuclear NF-kappa B in cells of the monocyte lineage Biochem. J. 304,87-94
-
Haas, J. G., Baeuerle, P. A., Riethmuller, G., Ziegler-Heitbrock, H. W. (1990) Molecular mechanisms in down-regulation of tumor necrosis factor expression Proc. Natl. Acad. Sci. USA 87,9563-9567[Abstract/Free Full Text]
-
Alcamí, J., de Lera, T. L., Folgueira, L., Pedraza, M.-A., Jacqué, J.-M., Bachelerie, F., Noriega, A. R., Hay, R. T., Harrich, D., Gaynor, R. B., Virelizier, J.-L., Arenzana-Seisdedos, F. (1995) Absolute dependence on
B responsive elements for initiation and tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes EMBO J 14,1552-1560[Medline]
-
Richard, N., Iacampo, S., Cochrane, A. (1994) HIV-1 Rev is capable of shuttling between the nucleus and the cytoplasm Virology 204,123-131[Medline]
-
Mangasarian, A., Trono, D. (1997) The multifaceted role of HIV Nef Res. Virol. 148,30-33[Medline]
-
Moore, J. P., Trkola, A., Dragic, T. (1997) Co-receptors for HIV-1 entry Curr. Opin. Immunol. 9,551-562[Medline]
-
Healey, D., Dianda, L., Moore, J. P., McDougal, J. S., Moore, M. J., Estess, P., Buck, D., Kwong, P. D., Beverley, P. C. L., Sattentau, Q. J. (1990) Novel anti-CD4 monoclonal antibodies separate HIV infection and fusion of CD4+ cells from virus binding J. Exp. Med. 172,1233-1242[Abstract/Free Full Text]
-
Klasse, P. J., McKeating, J. A. (1993) Soluble CD4 and CD4 immunoglobulin-selected HIV-1 variants: a phenotypic characterization AIDS Res. Human Retroviruses 9,595-604[Medline]
-
Tersmette, M., de Goede, R. E. Y., Al, B. J. M., Winkel, I. N., Gruters, R. A., Cuypers, H. T., Huisman, H. G., Miedema, F. (1988) Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex J. Virol. 62,2026-2032[Abstract/Free Full Text]
-
Cheng-Mayer, C., Seto, D., Tateno, M., Levy, J. A. (1988) Biologic features of HIV-1 that correlate with virulence in the host Science 240,80-82[Abstract/Free Full Text]
-
Fenyo, E. M., Morfeldt-Manson, L., Chiodi, F., Lind, B., von Gegerfelt, A., Albert, J., Olausson, E., Asjo, B. (1988) Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates J. Virol. 62,4414-4419[Abstract/Free Full Text]
-
Ushijima, H., Dairaku, M., Honma, H., Yamaguchi, K., Shimizu, H., Tsuchie, H., Abe, K., Yamamoto, A., Hoshino, H., Muller, W. E. (1991) Human immunodeficiency virus infection in cells of myeloid-monocytic lineage Microbiol. Immunol. 35,487-492[Medline]
-
Meylan, P. R. A., Spina, C. A., Richman, D. D., Kornbluth, R. S. (1993) In vitro differentiation of monocytoid THP-1 cells affects their permissiveness for HIV strains: a model system for studying the cellular basis of HIV differential tropism Virology 193,256-267[Medline]
-
Tuttle, D. L., Harrison, J. K., Anders, C., Sleasman, J. W., Goodenow, M. M. (1998) Expression of CCR5 increases during monocyte differentiation and directly mediates macrophage susceptibility to infection by human immunodeficiency virus type 1 J. Virol. 72,4962-4969[Abstract/Free Full Text]
-
Moriuchi, H., Moriuchi, M., Fauci, A. S. (1998) Differentiation of promonocytic U937 subclones into macrophagelike phenotypes regulates a cellular factor(s) which modulates fusion/entry of macrophagetropic human immunodeficiency virus type 1 J. Virol. 72,3394-3400[Abstract/Free Full Text]
-
Kozak, S. L., Platt, E. J., Madani, N., Ferro, F. E. J., Peden, K., Kabat, D. (1997) CD4, CXCR-4, and CCR-5 dependencies for infections by primary patient and laboratory-adapted isolates of human immunodeficiency virus type 1 J. Virol. 71,873-882[Abstract]
-
Carter, A. B., Monick, M. M., Hunninghake, G. W. (1998) Lipopolysaccharide-induced NF-kappaB activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent Am. J. Respir. Cell. Mol. Biol. 18,384-391[Abstract/Free Full Text]
-
Delude, R. L., Fenton, M. J., Savedra, R. J., Perera, P. Y., Vogel, S. N., Thieringer, R., Golenbock, D. T. (1994) CD14-mediated translocation of nuclear factor-kappa B induced by lipopolysaccharide does not require tyrosine kinase activity J. Biol. Chem. 269,22253-22260[Abstract/Free Full Text]
-
Yoza, B. K., Hu, J. Y. Q., McCall, C. E. (1996) Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation, but not NFkappaB nuclear translocation J. Biol. Chem. 271,18306-18309[Abstract/Free Full Text]
-
Pomerantz, R. J., Feinberg, M. B., Trono, D., Baltimore, D. (1990) Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression J. Exp. Med. 172,253-261[Abstract/Free Full Text]
-
Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., Nagashima, K. A., Cayanan, C., Maddon, P. J., Koup, R. A., Moore, J. P., Paxton, W. A. (1996) HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5 Nature 381,667-673[Medline]
-
Verrier, F. C., Charneau, P., Altmeyer, R., Laurent, S., Borman, A. M., Girard, M. (1997) Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate Proc. Natl. Acad. Sci. USA 94,9326-9331[Abstract/Free Full Text]
-
Kalter, D. C., Nakamura, M., Turpin, J. A., Baca, L. M., Hoover, D. L., Dieffenbach, C., Ralph, P., Gendelman, H. E., Meltzer, M. S. (1991) Enhanced HIV replication in macrophage colony-stimulating factor-treated monocytes J. Immunol. 146,298-306[Abstract]
-
Pauza, C. D., Galindo, J., Richman, D. D. (1988) Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication J. Virol. 62,3558-3564[Abstract/Free Full Text]
-
Broder, C. C., Collman, R. G. (1997) Chemokine receptors and HIV J. Leukoc. Biol. 62,20-29[Abstract]
This article has been cited by other articles:

|
 |

|
 |
 
E. Cassol, M. Alfano, P. Biswas, and G. Poli
Monocyte-derived macrophages and myeloid cell lines as targets of HIV-1 replication and persistence
J. Leukoc. Biol.,
November 1, 2006;
80(5):
1018 - 1030.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. Sun, B. Barbeau, S. Sato, G. Boivin, N. Goyette, and M. J. Tremblay
Syncytium Formation and HIV-1 Replication Are Both Accentuated by Purified Influenza and Virus-associated Neuraminidase
J. Biol. Chem.,
March 15, 2002;
277(12):
9825 - 9833.
[Abstract]
[Full Text]
[PDF]
|
 |
|