Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series

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1 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 demédecine, Université Laval, Ste-Foy, Québec, Canada 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. J. Leukoc. Biol. 68: ; Key Words: monocytes/macrophages AIDS/HIV cellular differentiation cell surface molecules transcription factors 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 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 13]. It should be stated that, in contrast to primary monocyte-derived macrophages (MDM), 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) 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. Michel. J.Tremblay@crchul.ulaval.ca Received December 7, 1999; revised June 2, 2000; accepted July 10, Journal of Leukocyte Biology Volume 68, December

2 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 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 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 macrophagelike phenotype, the Mono Mac 1 cell line also presents phenotypic and physiological properties such as cytokine secretion [tumor necrosis factor (TNF- ), interleukin-1 (IL-1 ), 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 g/ml), L-glutamine (2 mm), 1 MEM nonessential amino acids and sodium pyruvic acid (1 mm). Cultures were maintained at 37 C in a humidified atmosphere containing 5% CO 2. 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 ( 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 well s bottom surface. Flow cytometric analysis Untreated (monocyte-like) and LPS-treated (macrophage-like) Mono Mac 1 cells ( ) were incubated for 30 min on ice with saturating concentrations (1 g/10 6 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 L of phosphatebuffered 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/ampbased 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 macrophagetropic 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 ( ) 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 L. After 72 h, 100 L of cell-free supernatant from each well were removed and complemented with 25 L of5 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 L from the lysate was then mixed with 100 L of luciferase assay buffer [210 mm tricine, 1.07 mm (MgCO 3 ) 4 Mg(OH) 2 5H 2 O, 2.67 mm MgSO 4, 0.1 mm EDTA, 270 M CoA, 470 M luciferine, 530 M 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 ( ) in 24-well dishes (final volume of 500 L) with fully infectious progeny viruses (NL4-3, ADA, 89.6/30 ng of p24). Cells were incubated for days, during which aliquots of 100 L 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 100 with an inverted microscope. Genois et al. Mono Mac 1 is susceptible to infection with M-tropic HIV-1 855

3 Viral entry assay Undifferentiated Mono Mac 1 cells ( cells/ml, 250 L/well) were exposed to similar amounts of HIV-1 NL4-3 (X4) or HIV-1 JR-FL (R5) (100 ng of p24) in complete culture medium for 2.5 h at 37 C. Cells were washed twice with 250 L of ice-cold PBS and were next resuspended in 250 L 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 L 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 g of DNA, 200 M of each four deoxynucleotide triphosphates along with MgCl 2 (1 ), Taq buffer (1 ), and 2.5 U of Taq DNA polymerase (Perkin- Elmer Cetus). This reaction mixture was covered with 40 L 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/ was used to amplify a 100-bp fragment specific for the human -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 ( ) were washed with ice-cold PBS and resuspended in 400 L 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 L 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 L 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 L 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 g poly (di-dc), 10 g nuclease-free bovine serum albumin fraction V] containing 1 ng of 32 P-5 -end-labeled double-stranded (dsdna) oligonucleotide. Double-stranded DNA (100 ng) was labeled with [ - 32 P]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 L 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 -CAAGG- GACTTTCCGCTGGGGACTTTCCAGGG-3, respectively. The DNA-protein complexes were migrated on a 4% (w/v) polyacrylamide gel containing 0.5 Tris- 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. 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. Singleround 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 856 Journal of Leukocyte Biology Volume 68, December

4 Fig. 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). known to be susceptible to infection with T cell line-adapted X4 strains of HIV-1 [35]. 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 LPStreated Mono Mac 1 cells (macrophage-like) with fully infectious viruses displaying a tropism for either CD4 T lymphocytes Genois et al. Mono Mac 1 is susceptible to infection with M-tropic HIV-1 857

5 strain of HIV-1 (Fig. 3B, C). 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. 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 Fig. 2. Single-round infection assays with recombinant luciferase-encoding virions. (A) Untreated (open bars) and LPS-treated (filled bars) Mono Mac 1 cells ( ) 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 ( ) 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. (HIV-1 NL4-3 ), monocyte/macrophages (HIV-1 ADA ), or both of them (HIV ). 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 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-1 BaL 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 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 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 858 Journal of Leukocyte Biology Volume 68, December

6 Fig. 3. Kinetics of virus infection with fully infectious HIV-1 particles. Untreated (open circles) and LPS-treated Mono Mac 1 cells (filled circles; ) were infected with fully infectious strains of HIV-1 (NL4-3/Ttropic, 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. TABLE 1. Viral strain Internalization of X4 and R5 Strains of HIV-1 in Undifferentiated Mono Mac 1 Cells Total p24 levels in the absence of pronase (pg/ml) a Intracellular p24 levels in the presence of pronase (pg/ml) Percentage of virus entry (intracellular/total p24 levels) NL4-3 (X4) JR-FL (R5) a Results shown are the mean number SD for triplicate samples. 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 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. 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 Genois et al. Mono Mac 1 is susceptible to infection with M-tropic HIV-1 859

7 with some strains of HIV-1 [45 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 Fig. 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 ( ) 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. infection of LPS-treated Mono Mac 1 cells with NL4-3, a CXCR4-dependent TCLA strain of HIV-1 (data not shown). DISCUSSION Fig. 4. Semiquantitative PCR analysis of viral DNA levels. (A) Untreated (lanes 2 and 4) and LPS-treated Mono Mac 1 cells (lanes 3 and 5) were either left uninfected (lanes 2 and 3) or were infected with HIV-1 BaL (lanes 4 and 5) and cultured for 6 h before DNA extraction. Two sets of primers were included in each tube: an HIV-1 specific primer pair (M667/M661) and an oligonucleotide primer pair specific for human -globin, which serves as an internal control. Ratios between amplified signal with the HIV-1 specific set of primers and the -globin set were calculated for each experimental condition using an -imager spot density calculator. (B) Sensitivity of the current semiquantitive PCR was evaluated through the use of serial plasmid dilutions with the NL4-3 viral clone and the specific primer pair M667/M661. Again, PCR products were calculated for each template dilution using an -imager spot density calculator. 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 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 860 Journal of Leukocyte Biology Volume 68, December

8 Fig. 6. Comparison of NF- B binding activity in untreated and LPS-treated Mono Mac 1 cells. Nuclear extracts from untreated (NT) and LPS-treated Mono Mac 1 cells ( ) were extracted at 1, 2, and 3 days after LPS treatment. Probes corresponding to the consensus NF- B binding domain (A) and HIV-1 enhancer sequence (B) were used to determine specific DNA-protein interactions by EMSA. Specific DNA-protein complexes were identified using 100- fold competitions of cold specific DNA oligonucleotides. 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-1 BaL 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-1 ) [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 downregulation 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 Genois et al. Mono Mac 1 is susceptible to infection with M-tropic HIV-1 861

9 Fig. 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 100 with an inverted microscope. Arrows indicate syncytia. 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 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 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 862 Journal of Leukocyte Biology Volume 68, December

10 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 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. ACKNOWLEDGMENTS 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. 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