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Journal of Virology, September 2002, p. 8776-8786, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8776-8786.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Direct and Quantitative Single-Cell Analysis of Human Immunodeficiency Virus Type 1 Reactivation from Latency

Olaf Kutsch,1 Etty N. Benveniste,1 George M. Shaw,2,3 and David N. Levy2*

Departments of Cell Biology,1 Medicine, The University of Alabama at Birmingham,2 The Howard Hughes Medical Institute, Birmingham, Alabama3

Received 21 December 2001/ Accepted 21 May 2002


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ABSTRACT
 
The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.


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INTRODUCTION
 
The regulation of retrovirus expression within the infected host is controlled at many levels by both viral and host factors. For complex retroviruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2, several viral elements contribute cis and trans functions that regulate virus expression within host cells (23). The infected host cell, on the other hand, provides the transcription and translation machinery essential for the expression of viral proteins and viral replication. Following integration of the viral cDNA into the cellular genome, HIV-1 expression leads to the production of infectious virus, frequently resulting in the death of the host cell. In some instances viral expression can be down-modulated, leaving the provirus in a latent state characterized by low or absent viral mRNA and protein production (11, 48). This latent state may persist within the host cell for the natural life span of the cell or until external factors induce the virus to resume expression. A substantial reservoir of latently infected cells has recently been shown to be established early in HIV infection in vivo within macrophages and memory T cells (3, 9, 14, 16, 18, 26, 27, 37, 41, 52). This reservoir of latently infected cells is thought to be a contributing factor to the failure of highly active antiretroviral therapy to eradicate HIV-1 from the host (15, 16, 19). Thus, a better understanding of the underlying molecular mechanisms of HIV-1 latency and reactivation is needed in order to develop targeted therapies that could control or eradicate latently infected cells.

To date, it has been impossible to expand chronically infected primary cells; thus the most appropriate in vitro cell models for viral latency have been HIV-1-infected transformed cell lines such as ACH-2, J1.1, U1, and OM-10.1 (10, 22, 28, 29, 44). These cell lines contain one or two copies of integrated virus and constitutively display low levels of HIV-1 gene expression. Studies of these cells have revealed important roles for the site of viral integration (54), for cellular (33-35, 43, 49, 56) and viral proteins (30, 38, 39, 42, 47), and for histone acetylation and DNA methylation (4, 5, 51, 53) in the establishment and maintenance of latency. Nevertheless, the state of latency in these cells, on a population basis or at the single-cell level, can only be determined by indirect and time-consuming procedures (i.e., p24 enzyme-linked immunosorbent assay [ELISA], reverse transcriptase assay, and intracellular staining for viral proteins). As such, research on HIV-1 latency would benefit from a relevant model that is amenable to rapid and efficient analysis and through which useful pharmacological compounds capable of controlling HIV-1 reactivation may be efficiently screened.

To this end, we describe a reporter system to study HIV-1 latency and reactivation that combines the benefits of a latently infected immortal cell line with the convenience of using enhanced green fluorescent protein (EGFP) as a marker for HIV-1 expression. To establish this system, two recombinant HIV-1 viruses based on the dual-tropic 89.6 strain and the T-cell-tropic NL4-3 strain were engineered to express EGFP, while preserving all viral nucleotide sequences and potential cis elements. Following infection, three clonal, latently infected cell lines, representing both T-cell (Jurkat) and monocyte/macrophage (THP-1) lineages were developed. In the resulting cell lines, named JNLGFP, J89GFP, and THP89GFP, EGFP fluorescence is tightly linked to HIV-1 protein production and can be used as a quantitative marker for HIV-1 expression on a single-cell basis by fluorescence microscopy or flow cytometry and can be used on a population basis by fluorometry.

We find that different stimuli which are known to promote viral expression (tumor necrosis factor alpha [TNF-{alpha}], interleukin-1ß [IL-1ß], gamma interferon [IFN-{gamma}], phorbol 12-myristate 13-acetate [PMA], and trichostatin A [TSA]) differ in both the percentage of cells which demonstrate viral reactivation and the extent of reactivation within individual cells. Differences between the T-cell and macrophage cell lines were seen as well, highlighting the apparent complexity of the processes involved in HIV-1 reactivation.

The ability of this system to quantify HIV-1 reactivation and subsequent replication on a single-cell level can be simultaneously combined with additional analyses (e.g., cell cycle analysis, apoptosis detection, and antibody staining techniques). Using this method we observe that virus activation and cellular activation by proinflammatory cytokines can be uncoupled. In THP89GFP cells IFN-{gamma} induces cellular activation in the entire cell population while stimulating virus expression on a small subset of these cells. Likewise, low doses of TNF-{alpha} could induce expression of cellular activation markers in the complete population of cells while activating virus expression in only a subset.

Following TNF-{alpha}-induced virus reactivation in THP89GFP cells, a recapitulation of the latency induction process is observed over time, in which the population of cells shows a synchronous and progressive down-modulation of HIV-1 expression, reconstituting a fully latent and reactivatable state.


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MATERIALS AND METHODS
 
Cell culture and reagents. Jurkat-derived and THP-1-derived cell lines were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 10% heat-inactivated fetal bovine serum. THP89GFP cells grow semiadherent; therefore all experiments using THP89GFP cells were performed in ultralow-attachment plates (Costar, Acton, Mass.). 293T cells were maintained in Dulbecco's modified Eagle medium supplemented as for RPMI 1640. Cytokines (TNF-{alpha}, IL-1ß, IL-2, IL-6, IFN-{gamma}, and lymphotoxin alpha [LT-{alpha}]) were obtained from R & D Systems (Minneapolis, Minn.). PMA and TSA were purchased from Sigma (St. Louis, Mo.). HIV-1 p24 Gag protein ELISA kits were purchased from Coulter Diagnostics (Miami, Fla.).

Construction of HIV-1 89ENG and HIV-1 NLENG1. NL4-3 hemigenomic plasmids p83-5 and p83-10 were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program from Ronald Desrosiers (2, 20). The 89.6 hemigenomic plasmids were a kind gift from Ronald Collman, University of Pennsylvania School of Medicine, Philadelphia, Pa. (2, 20). In brief, recombinant hemigenomic HIV-1 plasmids 3'89ENG and 3'NLENG1 were similarly constructed by using primer extension and sequence overlap extension (Deep Vent polymerase; New England Biolabs) to link the EGFP coding sequence to the HIV-1 genome and to introduce desired restriction sites and Kozak sequences into the DNA fragments to be ligated. The cloning was designed such that the EGFP open reading frame was placed directly between the env and nef genes within the HIV-1 sequence (Fig. 1).



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  		FIG. 1. Schematic diagram of HIV-1 89ENG and HIV-1 NLENG1. (A and B) Wild-type HIV-1 (A) and recombinant EGFP viruses (B), showing placement of the EGFP gene within the HIV-1 genome. (C and D) DNA sequences of the 5' and 3' junctions between HIV-1 and the EGFP gene. *, translational stop codon.

For NLENG1, three PCR products were generated; these products represented (i) the NL4-3 genome from the unique BamHI site within env to the junction between the end of env and the start of the EGFP gene, (ii) the EGFP gene, and (iii) NL4-3 from the Kozak sequence within the junction with the EGFP gene to the unique BbrPI site within the 3' LTR of NL4-3. Products i and ii were then linked by using PCR sequence overlap extension, and the resulting product was cut with BamHI and NcoI (within the Kozak sequence). Product iii was cut with NcoI and BbrPI, and the BamHI-BbrPI fragment was removed from p83-10. p83-10 plus the cut PCR products were ligated to create 3'NLENG1.

For 89ENG, three PCR products were generated similarly to those for the NLENG1 construct except that the 5' and 3' restriction sites within the 89.6 3' hemigenomic plasmid were BsaBI and NheI sites, respectively. Also, a Kozak site was introduced upstream of the EGFP gene. These three products were cut and cloned into the 3' 89.6 hemigenomic plasmid to generate 3'89ENG.

Generation of virus stocks. 3' and 5' HIV-1 hemigenomic plasmids were linearized at the shared EcoRI site in each plasmid. The DNAs were extracted with phenol-chloroform, precipitated with isopropanol, and resuspended in water. 293T cells were transfected with the two plasmids by using CaPO4 (Stratagene), and 48 h later the supernatants were used to infect CEMx174 cells. At near-peak virus production, as measured visually by EGFP fluorescence and cell death, the medium was changed, and after 24 h this medium was collected, titered, and stored in aliquots at -80°C until use.

Flow-cytometric analysis. Flow-cytometric analysis was performed with a FACStar Plus and CellQuest software (BD Biosciences). For analysis of surface antigen expression, cells were washed with phosphate-buffered saline (PBS) and then preincubated with 50 µl of PBS containing 0.01% azide and 10% rabbit serum to block nonspecific binding. The directly conjugated antibodies were added, incubated at 4°C for 30 min, and washed in 4 ml of PBS prior to flow-cytometric analysis.

Photomicroscopy. Cells were photographed in culture through a Nikon TE300 inverted microscope and Hoffman optics (Modulation Contrast, Inc.) at x100 by using a SenSys:1401E B&W cooled charge-coupled device camera (Photometrics, Inc.). To detect EGFP fluorescence the Piston green fluorescent protein filter set was used (Chroma, Inc.).

Fluorometric analysis of HIV-1 reactivation. Cells were analyzed for cumulative EGFP fluorescence in flat-bottom 96-well tissue culture plates (Costar) in 200 µl of PBS by using a BIO-TEK FL600 fluorometer. Excitation was set at 435 nm, and emission was set at 530 nm. Ideal excitation for EGFP, as given by the manufacturer (Clontech, Palo Alto, Calif.) is at 488 nm, and ideal emission is at 508 nm.


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RESULTS
 
Selection of latent and reactivatable HIV-1-infected clonal cell lines. Jurkat (T-lymphocytic) and THP-1 (promonocytic) cells were infected with the EGFP-containing recombinant viruses NLENG1 (T cell-tropic) and 89ENG (dual-tropic) (Fig. 1). Four days following infection, EGFP-positive cells were cloned by using the automatic cell deposition unit of the FACStar Plus, and the surviving chronically infected clones were monitored for EGFP expression. Clones which lost fluorescence in the majority of cells were selected for further characterization. From this set, clones in which EGFP expression could be reactivated by stimulation with TNF-{alpha}, a powerful activator of the HIV-1 LTR (22, 32), were expanded. Finally, three cell lines representing each combination of virus and natural cellular target, J89GFP and THP89GFP (Fig. 2) and JNLGFP (data not shown), were chosen for further study because of low constitutive EGFP fluorescence and strong up-regulation of EGFP expression following TNF-{alpha} stimulation.



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  		FIG. 2. Induction of EGFP expression in J89GFP and THP89GFP cells by TNF-{alpha} stimulation. JNLGFP, J89GFP, and THP89GFP cells (106/ml) were stimulated with TNF-{alpha} (10 ng/ml), and, 48 h later, EGFP expression was measured by flow cytometry. For the histogram analysis, the parental Jurkat E6-1 and THP-1 cells (gray lines) were used as negative controls, and expression in these cells was compared to EGFP expression in unstimulated (black lines) and TNF-{alpha} (10 ng/ml)-stimulated JNLGFP, J89GFP, and THP89GFP cells (red lines). (D and E) Induction of EGFP expression by TNF-{alpha} as visualized by light microscopy (left) and fluorescence microscopy (right). Black arrows (D) indicate syncytium formation between cells in J89GFP cell cultures following TNF-{alpha} stimulation. Results are representative of five independent experiments.

Correlation of EGFP fluorescence, p24 Gag protein expression, and secretion of infectious viral particles in JNLGFP, J89GFP, and THP89GFP cells. To examine whether EGFP fluorescence in JNLGFP, J89GFP, and THP89GFP cells can be used as a quantitative marker for HIV-1 expression, we stimulated the cells with various concentrations of TNF-{alpha} and after 48 h analyzed EGFP expression, p24 secretion, and infectious-virus production. Cells were further found to express HIV-1 Vpu at levels similar to those seen in CEMx174 cells infected with wild-type HIV-1 89.6. Nef expression in THP89GFP and J89GFP cells was below the detection range of Western blotting (data not shown). In an uninduced state, a small number of cells in each cell line (2 to 5%) exhibited some degree of spontaneous EGFP fluorescence (Fig. 2A and B). In J89GFP and THP89GFP cells, reactivation of EGFP fluorescence over background can be detected at 0.1 ng of TNF-{alpha}/ml (Fig. 3), indicating stimulation of low levels of early HIV-1 gene transcription and translation, which does not lead to the production of measurable secretion of p24 Gag (a late gene product) or infectious virus (Fig. 3). Stimulation with 1 ng of TNF-{alpha}/ml leads to a further increase in expression of EGFP and to measurable production of p24 protein and infectious virus. Higher concentrations of TNF-{alpha} (10 and 100 ng/ml) generated a coordinated enhancement of EGFP expression, p24 secretion, and production of infectious viral particles. A similar correlation of EGFP and p24 expression upon reactivation of latent HIV-1 infection was seen in JNLGFP cells, although this clone produced no infectious viral particles (data not shown). Using fluorescence and visible-light microscopy we also observed that, in J89GFP cells, TNF-{alpha}-mediated HIV-1 reactivation was accompanied by the formation of syncytia (Fig. 2D, lower left), probably resulting from the interaction of the newly expressed HIV-1 envelope protein with cellular CD4 receptors on neighboring cells. The earliest detectable increase in EGFP fluorescence in both cell types was seen at 6 h after stimulation. The proportion of EGFP-positive cells reached its maximum (92 to 98%) after 24 to 48 h, while the mean fluorescence intensity of the population increased until 4 days, suggesting a continuing accumulation of viral proteins over this period of time (Fig. 4). Transduction of cells with murine retrovirus constructs containing the HIV-1 tat gene reactivated virus expression in each cell line, while control viruses lacking the tat gene failed to do so (data not shown). This result indicates that latency in these cells is the result of low LTR activity and not simply the result of suboptimal late-gene expression.



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  		FIG. 3. Correlation of EGFP expression, HIV-1 Gag p24 protein production and secretion of infectious viral particles in J89GFP and THP89GFP cells. J89GFP (A) and THP89GFP (B) cells (106 cells/ml) were stimulated for 48 h with TNF-{alpha} at various concentrations (0 to 100 ng/ml). After 48 h, supernatants were collected and used to determine HIV-1 p24 Gag protein (p24) concentrations by ELISA (squares) and the numbers of infectious viral particles (I.U.) secreted by the cells (circles) were assessed by limiting-dilution endpoint analysis. EGFP expression was analyzed by flow cytometry and is expressed as MCF intensity (bars). Results represent the means ± standard deviations of three independent experiments.



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  		FIG. 4. Kinetics of HIV-1 reactivation following TNF-{alpha} treatment in J89GFP and THP89GFP cells. J89GFP (A) and THP89GFP (B) cells (106 cells/ml) were stimulated with TNF-{alpha} (10 ng/ml) and then subjected to flow-cytometric analysis at the indicated times (3 to 120 h). EGFP expression was determined either as the percentage of EGFP-positive cells in the culture or as MCF intensity of the total population. Untreated control samples were taken after 48 h (open circle, percent EGFP-positive cells; open triangles, MCF intensity). Dead cells were excluded by propidium iodide staining. Results represent the means ± standard deviations of three independent experiments.

Synchronous reversion to latency in THP89GFP cells. We next monitored EGFP expression in THP89EGFP cells over a extended period of time after TNF-{alpha} stimulation in order to examine whether varying the dose of TNF-{alpha} resulted in prolonged or transient reactivation of virus expression. For this analysis we limited the range of TNF-{alpha} to 0.03 to 3 ng/ml, as higher levels led to substantial cell death after 4 days. At each dose of TNF-{alpha} tested, the maximum percentage of cells that were EGFP positive was achieved after 24 to 48 h. While the fluorescence intensity peaked during this time as well in the cultures receiving 0.03 to 0.3 ng/ml, peak fluorescence intensity was observed after 4 days in the cultures receiving the highest doses of TNF-{alpha} (1 and 3 ng/ml), consistent with the data from Fig. 4. Over the course of the following days and weeks, the expression of virus as measured by EGFP fluorescence from these cells steadily declined (Fig. 5B), while the percentage of cells which continued to produce at least some virus declined in a much more gradual manner (Fig. 5A). Interestingly, rather than individual cells spontaneously ceasing virus expression, a continual and synchronous decline in virus production from the population of cells was observed (Fig. 5C to F). Upon reexposure to TNF-{alpha}, virus expression, as measured by EGFP fluorescence (Fig. 5A to C; day 28), and infectious-virus production (data not shown) were once again reactivated in these cells, indicating that the process of induction of latency was recapitulated after the first reactivation.



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  		FIG. 5. Synchronous recapitulation of latency induction following TNF-{alpha} stimulation. THP89GFP cells (106 cells/ml) were stimulated with various concentrations of TNF-{alpha} (0.01 to 3.0 ng/ml) (restimulation was with 3.0 ng of TNF-{alpha}/ml on day 24 [down arrows]), and EGFP expression, depicted as the percentage of EGFP-positive cells (A) or the EGFP MCF intensity (B), was monitored by flow cytometry over time. (C to H) Analysis of the synchronous shift of the entire cell population after TNF-{alpha} stimulation from latency to fully active HIV-1 expression and the reversal to a latent state of HIV-1 infection, depicted as dot plots showing EGFP expression and propidium iodide (PI) uptake in THP89GFP cells stimulated with 3 ng of TNF-{alpha}/ml on day 0 (C) and 4 (D), 8 (E), 14 (F), and 24 days (G) after stimulation with 3.0 ng of TNF-{alpha}/ml and after restimulation with 3.0 ng of TNF-{alpha}/ml on day 24.

Reactivation of latent HIV-1 by various cytokines and chemical agents. The replication of HIV-1 in vivo is influenced by the local cellular environment, including cytokines that regulate the immune response. Several of these are known to activate or inhibit HIV-1 replication under various circumstances, and some, such as IL-2, have been explored as part of anti-HIV-1 treatment regimens (17). We tested a panel of cytokines (TNF-{alpha}, LT-{alpha}, IL-1ß, IFN-{gamma}, IL-2, and IL-6) and chemical compounds (PMA and TSA) for their ability to reactivate HIV-1 expression within these cell lines (Table 1). TNF-{alpha}, LT-{alpha}, IL-1ß, and IFN-{gamma} were able to reactivate latent HIV-1 infection in THP89GFP, but only TNF-{alpha} and LT-{alpha} reactivated HIV-1 infection in J89GFP cells, while IFN-{gamma} and IL-1ß failed to do so. Interestingly, after 48 h, only a subpopulation of THP89GFP cells reactivated virus expression in response to IFN-{gamma} and IL-1ß (Table 1). PMA, a potent activator of HIV-1 LTR expression through protein kinase C and ultimately NF-{kappa}B activation (34), produced a strong increase in EGFP expression in J89GFP cells. Its effect on HIV-1 replication in THP89GFP cells was less pronounced, as HIV-1 expression was reactivated in only 57% of the viable cells. TSA, an inhibitor of histone deacetylases, said to reactivate HIV-1 infection (6, 53), also had a strong effect on HIV-1 reactivation in J89GFP cells. The effect of TSA on HIV-1 replication in THP89GFP cells again was less profound (34% reactivation). Although TNF-{alpha}, PMA, and TSA all produced virus reactivation in almost all J89GFP cells, the degree of reactivation within individual cells, as measured by mean channel fluorescence (MCF) intensity, was clearly lower following PMA and TSA exposure than following TNF-{alpha} stimulation at 48 h (Table 1) and all other time points tested (data not shown). Macrophage inhibitory protein 1{alpha}, MCP-1, SDF-1{alpha}, and IP-10 failed to reactivate HIV-1 expression in these cell lines (data not shown).


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  		TABLE 1. Reactivation of latent HIV-1 infection in THP89GFP and J89GFP cells by cytokines and chemical agentsa

Simultaneous analysis of cell activation, CD4 down-modulation and virus reactivation following TNF-{alpha} exposure. The importance of latency to the life cycle of HIV in vivo arises in part from the linkage between the status of immune system activation and the level of virus replication within those cells. It is commonly accepted that the latent state generally exists within the resting pool of CD4-positive T cells and monocytes/macrophages, and, importantly, the activation of those latently infected cells is considered the likely impulse that reactivates virus replication. Using a salient feature of this system, the ability to coordinately monitor virus expression and other cellular events, we simultaneously examined virus expression and the activation state of THP89GFP cells following TNF-{alpha} stimulation (Fig. 6). As an indication of cellular activation, we stained the cells for intercellular adhesion molecule 1 (ICAM-1) expression, which increases in cells of the monocyte/macrophage lineage following TNF-{alpha} exposure (1). While HIV-1 reactivation was achieved in only a subset (67%) of cells exposed to 0.1 ng of TNF-{alpha}/ml, 22% of cells showed full expression of ICAM-1 while remaining negative for HIV-1 expression (Fig. 6B), indicating that in this population cellular activation did not result in viral reactivation. This partial-reactivation pattern could be overcome with increasing levels of TNF-{alpha} (Fig. 6C and D).



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  		FIG. 6. Correlation of HIV-1 reactivation with ICAM-1 and CD4 expression by using two-color flow cytometry. THP89GFP cells (106 cells/ml) were stimulated with various concentrations of TNF-{alpha} (0.1 to 10 ng/ml). Twenty-four hours after stimulation, cells were stained for the expression of ICAM-1 (A to D), as a marker of cell activation, and CD4 (E to H). Levels of EGFP, ICAM-1, and CD4 expression were then quantified by flow-cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. Results are representative of four independent experiments.

Down-modulation of cell surface CD4 following HIV-1 infection is a consequence of coexpression of CD4 and the viral Vpu, Nef, and gp120 envelope proteins within the infected cells (13). CD4 expression on THP89GFP cells is identical to that on the parental THP-1 cells prior to viral reactivation (data not shown), as would be expected in the absence of HIV-1 expression in these cells. Also as expected, TNF-{alpha}-mediated virus reactivation coincided in a concentration-dependent manner with a marked decrease in CD4 surface staining (Fig. 6F to H). At the highest TNF-{alpha} concentration, a substantial reduction of CD4 surface expression was evident in the majority of cells, even at the early time point (24 h) shown (Fig. 6H).

Major histocompatibility complex class II (MHC-II) expression, like that of ICAM-1, is up-regulated on cells of the monocyte/macrophage lineage in response to some proinflammatory signals, such as IFN-{gamma} (Fig. 7B; shown at the optimal concentration for MHC-II induction). Though IFN-{gamma} strongly up-regulated MHC-II expression in these cells, it is a weak inducer of HIV-1 reactivation at any concentration (Fig. 7B and data not shown). This is in stark contrast to TNF-{alpha}, which, while it fails to induce expression of MHC-II (Fig. 7C), at concentrations above 0.1 ng/ml is a powerful inducer of both ICAM-1 and virus expression in the whole cell population. Thus, as also seen in Fig. 6B, in which a low concentration of TNF-{alpha} is used, in THP89GFP cells cellular activation and virus reactivation can be dissociated.



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  		FIG. 7. Differential regulation of MHC-II and HIV-1 expression following stimulation with TNF-{alpha} or IFN-{gamma}. THP89GFP cells (106 cells/ml) were stimulated with IFN-{gamma} (300 U/ml) (B and E) or TNF-{alpha} (1 ng/ml) (C and F). Forty-eight hours after exposure, cells were stained for the expression MHC-II (A to C) or ICAM-1 (D to F). Levels of EGFP, ICAM-1, and MHC-II expression were then quantified by flow-cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. Results are representative of two independent experiments.

Plate-based fluorometric analysis of viral reactivation. We next investigated whether viral reactivation could be conveniently monitored by a 96-well-plate-based fluorometric assay. This type of analysis would be easily scalable for the analysis of many samples in a short period of time using relatively small numbers of cells and reagents. We compared fluorometric analysis using a 96-well format to flow cytometry for sensitivity of EGFP detection in J89GFP cells. Cells were stimulated with various concentrations of TNF-{alpha} for 24 h, and EGFP expression was then measured as cumulative fluorescence with the fluorometer or as MCF intensity by flow cytometry. Flow cytometry here revealed a 25-fold increase in MCF intensity in J89GFP cells stimulated with 100 ng of TNF-{alpha}/ml (Fig. 8A), compared to that in untreated control cells. A 26-fold increase in cumulative fluorescence for cells from the same experiment was measured with the fluorometer, indicating a very close correlation between the two methods. Similar results were obtained for THP89GFP cells (Fig. 8B).



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  		FIG. 8. Comparison of the abilities of flow cytometry and 96-well-plate-based fluorometric analysis to detect changes in the replication state of HIV-1 infection in J89GFP and THP89GFP cells. J89GFP (A) and THP89GFP (B) cells (106 cells/ml) were stimulated with different concentrations of TNF-{alpha} (0 to 100 ng/ml) for 24 h, washed twice with PBS, and analyzed for expression of EGFP by flow cytometry (gray) or 96-well-based fluorometric analysis (black). EGFP fluorescence was determined as MCF intensity by flow cytometry and as cumulative fluorescence (CF) by fluorometric analysis. Results represent the means ± standard deviations of three independent experiments.


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DISCUSSION
 
The need for in vitro models of latency has been partially filled over the past years by HIV-1-infected transformed cell lines such as ACH-2, J1.1, U1, and OM-10.1 (9, 21, 27, 28). Virus expression in these cell lines can be induced by cellular (7, 10) or viral factors (12, 30, 38, 39) and chemical agents (29, 36) or inhibited by pharmacological agents (31, 55). Establishment of latency in these cell lines has been linked to mutations in viral genes such as tat (24) and the TAR region (25), the site of viral integration (54), and to certain cellular (34, 35) and viral proteins (30, 38, 39, 47). A particularly important insight into the regulation of HIV-1 expression comes from the observation that histone acetylation and DNA methylation patterns within and downstream of the viral promoter/enhancer elements can be critical to the suppression of HIV-1 expression or its release (4, 5, 50, 51, 53). HIV-1 Tat participates in these processes in part by recruiting p300 and CREB-binding protein, a protein with histone acetyltransferase activity, to the viral promoter (6, 21, 40). How these DNA acetylation and methylation patterns are established in the first place and how they may be influenced by cellular events are areas of intensive ongoing research.

Studies on the mechanisms governing HIV-1 latency would benefit from an in vitro system where the level and timing of HIV-1 expression can be quantified easily and directly at the single-cell level. The reporter cell lines described here have several features that make them especially useful in this context. The incorporation of the EGFP gene into the HIV-1 genome resulted in control of cellular fluorescence that is strictly coordinated with the expression of viral proteins, permitting immediate and quantitative measurement of the extent of viral expression in cells by using flow cytometry, fluorescence microscopy, or plate-based fluorometry. Flow-cytometric analysis allows both population and single-cell quantification of viral reactivation without any fixation, staining, or other manipulation of the cells that might affect the results or the ability to further manipulate and analyze the relevant cell populations. Multicolor flow-cytometric analysis permits a variety of cellular and viral events to be correlated.

Two cell lines were constructed by using Jurkat cells, which have been widely used as models for T-cell studies. The third cell line is based on THP-1 cells, which constitute the most widely employed transformed cell line for studies of monocytes/macrophages. The THP-1-based latent cell line (THP89GFP) and one of the Jurkat-based cell lines (J89GFP) were infected with 89ENG, whose parental virus, 89.6, is considered a near-primary dual CXCR4- and CCR5-tropic molecular clone (20). The other Jurkat cell line was infected with a recombinant virus (NLENG1) whose parental virus, CXCR40-tropic NL4-3, is the best-characterized HIV-1 strain. We have observed identical patterns in response to different stimuli (TNF-{alpha}, LT-{alpha}, PMA, and TSA) in the two Jurkat-based cell lines infected with the divergent viruses (Table 1 and data not shown). On the other hand, differences in the responses to activating agents between the Jurkat- and THP-1-based cell lines are apparent (Table 1). THP89GFP cells, but not J89GFP cells, responded to IFN-{gamma} and IL-1ß by reactivating HIV-1 expression (Table 1), despite the fact that J89ENG cells responded to IFN-{gamma} and IL-1ß treatment by increasing surface ICAM-1 expression (data not shown). Differences between J89GFP and THP89GFP cells in the responses to PMA and to deacetylase inhibitor TSA were also apparent.

The importance of postintegration viral latency to the natural history of HIV-1 infection in vivo has perhaps been underestimated until fairly recently. The failure of highly active antiretroviral therapy to allow complete removal of virus from the body is due, at least in part, to the reemergence of viral replication from the pool of latently infected cells (15). In the past several years, latent HIV-1 has been identified in resting memory (14, 18, 19, 37) and naive (45) T cells, from which virus may be rescued long after infection of these cells.

The prevailing model of postintegration HIV-1 T-cell latency in vivo requires infection of an activated cell just before the cell enters a quiescent state (46). The reservoir of virus identified within the memory T-cell compartment is thought to be generated by infection of antigen-activated T cells (18, 19), while naive latently infected cells may arise through infection of CD4-positive thymic precursors (8). In either case, it is believed that in vivo viral latency is the direct result of an intracellular environment lacking the necessary factors for efficient transcription of the viral promoter. Following induction of the latent state, maintenance of latency may be assisted through cellular processes directed at the viral promoter region, such as chromatin modifications including histone deacetylation or DNA methylation (4, 5, 50, 51, 53). Because of the extremely long half-lives of these latent viral reservoirs, therapeutic reactivation is thought to be essential to achieve eradication of HIV-1 infection from patients. Administration of IL-2 as part of the treatment schedule has thus far failed to achieve clinical efficacy, emphasizing the difficulty in reaching the latent compartments with the proper signals for virus reactivation.

Unlike latently infected cells in the body, which can be identified ex vivo within the resting population of T lymphocytes, the cell lines described here are constantly proliferating, performing DNA synthesis and mitosis, and expressing genes at high levels. The finding by Brooks et al. (8) that reactivation of HIV-1 gene expression within thymocytes can be achieved in the absence of cellular DNA synthesis indicates that events more closely linked to cell activation than to increased cell proliferation are key to HIV-1 reactivation. NF-{kappa}B is a crucial factor for promoting transcriptional initiation and elongation from the HIV-1 LTR, and its activity may be induced in the absence of cellular proliferation by agents such as PMA and TNF-{alpha} (8, 37).

By exploiting the single-cell analysis capabilities of this system, we found that at least under certain circumstances cellular activation may be induced in the absence of virus reexpression. Using ICAM-1 as a cell activation marker, we observed that HIV-1 reactivation and cell activation can be uncoupled when low concentrations of TNF-{alpha} are applied (Fig. 6B). Low doses of TNF-{alpha} (0.1 ng/ml) apparently resulted in cellular activation of nearly the complete population of cells, as indicated by the uniform up-regulation of ICAM-1 expression, but HIV-1 was reactivated in only a subset of these cells. Also, IFN-{gamma} stimulation, which led to a strong activation of the THP89GFP cells, as indicated by increases in MHC-II and ICAM-1 expression, did not result in substantial reactivation of the latent HIV-1 infection. Each of these patterns persisted for at least 96 h, the longest interval tested, indicating that the uncoupling of cellular and viral activation was not the result of a simple kinetic difference between the two. Therapies which seek to activate HIV-1 expression for the purpose of making these cells vulnerable to antiviral therapy or immune responses may need to consider that not all agents which promote cellular activation may in fact reactivate virus from all cells.

We have also found evidence for stochastic events in creating and maintaining the latent state in cells. The subcloning of unstimulated cells from each of the cell lines described here occasionally generates a cell line that is a constitutive producer of virus. Other subclones differ from the parental cell lines in exhibiting no spontaneously fluorescent cells or detectable p24 Gag protein, and these are uniformly unresponsive to TNF-{alpha} induction of virus expression (data not shown). Thus, while an important role for the site of viral integration has been found for many latently infected cells (54), clearly the outcome of HIV-1 infection is governed by complex and mutable processes. By observing recently infected cells sorted for EGFP fluorescence, we observed loss of EGFP in some cells in as little as 2 days, indicating that down-modulation of HIV-1 gene expression can occur rapidly following infection (data not shown). The defined latent reservoirs in vivo are composed of quiescent nonproliferating cells, but it may be possible for HIV-1 to enter latency in some cells which have yet to transition back to a nonproliferating state. Whether such cells exist in vivo is unknown, but since existing methodologies do not permit their detection, the relevant experiments have not been performed.

Another interesting and useful aspect of the system described herein is the ability of HIV-1 to return to a latent state following reactivation (Fig. 5). Interestingly, following reentrance into the latent state, the virus could be fully reactivated once again, indicating that the original latent state has been reconstituted. As such, this system not only allows for the detailed study of HIV-1 reactivation, but also enables the controlled study of processes involved in the achievement of latency. It is most likely, we believe, that both this recapitulation of latency induction and the stochastic events described above are controlled by cellular modifications at the viral promoter, including histone acetylation and deacetylation events and DNA methylation patterns. Examination of these patterns in clonally related cell lines which exhibit divergent HIV-1 expression properties should be illuminating, and these patterns are the subject of current investigation in our laboratories.


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ACKNOWLEDGMENTS
 
O.K. and E.N.B. were supported in part by NIH grant NH55795 and amfAR research grant 02797-RG. D.N.L. and G.M.S. were supported in part by NIH grants R37 AI35467 and U01 AI41530. D.N.L. was additionally supported by Elizabeth Glaser Pediatric AIDS Foundation Scholar Award PF-77379.

We thank Gautam Bijour for assistance with the fluorometer, Marion Spell for expert operation of the flow cytometer/cell sorter, and Shaun Sparacio for assistance in running E. N. Benveniste's laboratory.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of Alabama at Birmingham, 848 Kaul Bldg., 720 S. 20th St., Birmingham, AL 35294-0024. Phone: (205) 934-0169. Fax: (205) 934-1580. E-mail: levy{at}uab.edu. Back


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Journal of Virology, September 2002, p. 8776-8786, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8776-8786.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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