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Research Article
1 April 2009

Determinants of the Establishment of Human Immunodeficiency Virus Type 1 Latency


Recent research has emphasized the notion that human immunodeficiency virus type 1 (HIV-1) latency is controlled by a restrictive histone code at, or DNA methylation of, the integrated viral promoter (long terminal repeat [LTR]). The present concept of HIV-1 latency has essentially been patterned from the principles of cellular gene regulation. Here we introduce an experimental system that allows for the qualitative and quantitative kinetic study of latency establishment and maintenance at the population level. In this system, we find no evidence that HIV-1 latency establishment is the consequence of downregulation of initial active infection followed by the establishment of a restrictive histone code at the viral LTR. Latent infection was established following integration of the virus in the absence of viral gene expression (silent integration) and was a function of the NF-κB activation level in the host cell at the time of infection. In the absence of a role for epigenetic regulation, we demonstrate that transcriptional interference, a mechanism that has recently been suggested to add to the stabilization of HIV-1 latency, is the primary mechanism to govern latency maintenance. These findings provide direct experimental evidence that the high number of viral integration events (>90%) found in actively expressed genes of CD4+ memory T cells from highly active antiretroviral therapy-suppressed patients represent indeed latent infection events and that transcriptional interference may be the primary mechanism to control HIV-1 latency in vivo. HIV-1 latency may thus not be governed by the principles of cellular gene regulation, and therapeutic strategies to deplete the pool of latently HIV-1-infected cells should be reconsidered.
Highly active antiretroviral therapy can efficiently suppress human immunodeficiency virus type 1 (HIV-1) replication to below the detection limit. However, even after years of effective viral suppression, cessation of therapy results in the immediate rebound of viremia. During treatment, viral infection is thought to be primarily sustained by a long-lived reservoir of latently infected CD4+ memory T cells (13-15). As a result of the long life span of memory T cells that serve as cellular hosts, the latent HIV-1 reservoir is extremely stable. Details of its half-life (t1/2) are still discussed controversially, with measured t1/2 being up to ∼40 months (20, 27, 53). At this rate, the natural decay of a reservoir consisting of only 1 × 106 cells would take as long as ∼70 years. Thus, as natural depletion of the latent reservoir is unlikely to occur during the lifetime of an infected patient, HIV-1 latency is believed to represent the principal obstacle to curative AIDS therapy (13-19, 21).
To understand the molecular basis of HIV-1 latency, early studies were performed in latently HIV-1-infected transformed clonal cell lines such as ACH-2, J1.1, U1, and OM-10.1 (3, 6-8, 17). Studies of these cells and other systems proposed a role for the site of viral integration (62), for cellular proteins (9-11, 16, 21, 22, 25, 46), for viral proteins (1, 39), and for histone acetylation or DNA methylation in regulating HIV-1 latency (3, 4, 38, 52, 60).
The insights gained from these and other studies produced a model of HIV-1 latency that suggests that the host cell, which initially exhibits a minimum level of activation sufficient to promote infection, returns to a quiescent state (30). Since the virus is dependent on the availability of certain cellular key transcription factors for active gene expression, these key transcription factors are no longer available to the virus as the cells return to a quiescent state, and viral gene expression is shut down prior to the onset of viral cytopathicity or immune clearance. Integration into genome sites that are not supportive of viral transcription could favor this process (51). The latent state of the integrated provirus is then stabilized by the establishment of a suppressive histone code, in particular at the viral promoter (23, 60, 61).
Based on this molecular understanding of HIV-1 latency, several attempts to therapeutically deplete the latent HIV-1 reservoir have been previously made. The underlying concept of these strategies has been to activate the integrated but transcriptionally silent viral promoter. This was attempted either by stimulation of the infected cells (interleukin 2 [IL-2] or anti-CD3 monoclonal antibody [MAb] OKT3) (17, 40, 42) or by triggering changes in the histone composition at the viral promoter using histone deacetylase (HDAC) inhibitors (e.g., valproic acid) to favor viral transcription in the absence of cellular activation (17, 40, 42). These protocols have not resulted in a reduction of the size of the latent reservoir, or the clinical significance of the reported reduction has been disputed (54, 55, 56). While some evidence for the role of histone modifications in HIV-1 latency has been presented in vitro, a more recent comprehensive investigation of HIV-1 integration events in patients, by J. Siliciano and coworkers, is somewhat in conflict with the idea that latency is governed by a restrictive histone code. In this study, Han et al. demonstrate that >95% of all infection events found in memory T cells of infected patients are located in actively expressed genes (30) and are thus integrated in a DNA environment that is unlikely to allow for the formation of a stable suppressive histone code at the latent HIV-1 promoter. Indeed, in a subsequent study, in which the orientation-dependent regulation of HIV-1 gene expression by transcriptional interference was analyzed, no evidence for the establishment of a particular restrictive histone code was provided (31). At the same time, a second study demonstrated the importance of transcriptional interference of the host gene with the integrated virus as a mechanism to stabilize the latent viral expression state (43).
We add to these most recent findings on the role of host gene transcriptional interference as a governing factor for HIV-1 latency by demonstrating that histone deacetylation or DNA methylation is not important for the establishment of latent infection events. Our results reveal that the decision whether a latent infection event is established is a function of the availability of NF-κB at the time of infection. Transcriptional silent integration is a prerequisite for the establishment of a latent infection event, which is then maintained by transcriptional interference. Latent infection events that are governed by transcriptional interference are resistant to reactivation by HDAC or DNA methyltransferase (DNMT) inhibitors. We here discuss how these findings are complementary to current ideas on HIV-1 latency and the possible consequences of these findings for therapeutic depletion of the latent HIV-1 reservoir.


Cell culture and reagents.

All T-cell lines (Jurkat, Molt, SupT1, PM1, C8166, and CEM-GFP) and the CD4-positive B-cell line (AA2) used in infectivity assays were obtained from the NIH AIDS Research and Reference Reagent Program. These cells, as well as the latently HIV-1 infected J89GFP cells (41), were maintained in RPMI 1640 supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum. All latently infected cell lines generated in the described experiments were maintained in supplemented RPMI 1640. Fetal bovine serum was obtained from HyClone (Logan, UT) and was tested on a panel of latently infected cells to ensure that it did not spontaneously trigger HIV-1 reactivation (35, 41).
Anti-CD3 MAb (clone UCHT1) and anti-CD28 MAb (clone CD28.2) were purchased from Pharmingen. The phorbol ester 13-phorbol 12-myristate acetate (PMA) or prostratin and the HDAC inhibitors (sodium butyrate [NaBu], trichostatin A [TSA], and valproic acid), as well as the DNMT inhibitor 5-azacytidine, were obtained from Sigma. The enhanced green fluorescent protein (EGFP) reporter virus that was used, HIV-1 NLENG1-IRES, has been described elsewhere (41, 44).
The HIV-1 reverse transcriptase (RT) inhibitor (RTI) lamivudine, the HIV-1 protease inhibitor (PI) indinavir, and the integrase inhibitor 118-D-24 were obtained from the NIH AIDS Research and Reference Reagent Program. The HIV-1 transcription inhibitor Ro24-7429 was a kind gift from Roche Scientific.

Flow cytometry.

Infection levels in the cell cultures were monitored by flow cytometric (FCM) analysis of EGFP expression. FCM analysis was performed on a Guava EasyCyte (Guava Technologies, Inc.) or an LSRII (Becton Dickinson) cytometer. Cell sorting experiments were performed using a FACSAria flow cytometer (Becton Dickinson). Data analysis was performed using either CellQuest (Becton Dickinson) or Guava Express (Guava Technologies, Inc.).

Determination of absolute cell numbers using Sphero beads.

To determine the level of cell proliferation and cell death in the cell cultures, we added a defined number of Sphero blank calibration particles (Pharmingen) to each individual culture, in which cell numbers had been adjusted by manual counting using a Neubauer chamber. Due to differences in size and granularity, Sphero blank calibration particles (diameter, 6 to 6.4 μm) can be easily distinguished from T cells in a forward scatter/side scatter dot plot analysis using flow cytometry. At the time of analysis, we set up the flow cytometer to acquire a defined amount of beads while acquiring all cells in the live gate. The ratio of beads to cells in each culture allows for the determination of absolute cell numbers for each time point analyzed.

Generation of chronically actively HIV-1-infected reporter Jurkat T cells.

The population of chronically actively infected cells used in the reported HIV-1 transcription inhibitor experiments was generated by infecting a recently described HIV-1 reporter cell line (NOMI; Jurkat T cell based) with a primary patient HIV-1 isolate (CUCY) (36). In the NOMI reporter cells, EGFP expression can be used as a direct and quantitative correlate of HIV-1 expression. Two weeks postinfection (p.i.), EGFP-positive cells were enriched by FCM cell sorting and the resulting EGFP-positive cell population was expanded. We observed that in the next 12 weeks EGFP expression was gradually shut down in >30% of the cells. After that time, the percentage of EGFP-positive cells remained constant. At this time point, the cell culture was subjected to a second round of cell sorting to enrich for EGFP-positive cells and the resulting cell population was employed in the described experiments (see Fig. 3).

In vitro generation and analysis of HIV-1 latency in PBMCs.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque centrifugation and cultured in supplemented RPMI 1640 to which phytohemagglutinin L (PHA-L) (2 μg/ml) and Giant cell tumor conditioned medium (Igen International, Inc., Gaithersburg, MD)-conditioned media (10%) were added. At 2 or 7 days following stimulation, fractions of the parental cell culture were infected with HIV-1 NLENG1-IRES (37). On day 1 p.i. a lamivudine-indinavir combination was added to the infection cultures to inhibit de novo infection and to prevent the formation of preintegration latency. In the absence of IL-2, each culture was maintained until day 14 poststimulation and then subjected to fluorescence-activated cell sorting to remove EGFP-positive and therefore actively HIV-1-expressing cells. The EGFP-negative cell fraction, which would hold noninfected and silently HIV-1-infected cells, was then activated with the indicated stimuli, and reactivation of silent HIV-1 infection was measured 24 h poststimulation by determining EGFP fluorescence using fluorescence-activated cell sorting analysis.

Determination of NF-κB activity profiles.

Nuclear extracts were generated using the NE-PER nuclear and cytoplasmic extraction reagent according to the manufacturer's instructions (Pierce; ThermoScientific). Quantification of NF-κB activity in the respective nuclear extracts was performed using a TransAM NF-κB family transcription factor assay kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA).

PCR for viral integration in actively expressed host genes.

The underlying idea of this nested PCR strategy is that, if the virus is latently integrated into an actively expressed host gene, viral sequences should be detectable in host gene mRNA (30). To exclude the possibility that low-level viral gene transcription could be detected, the 5′ primers are selected to bind prior to the HIV-1 LTR transcription start site. By this means, viral integration into an actively expressed host gene can be determined without any knowledge of the host gene sequence. cDNA from latently infected cell clones was generated using the μMACS mRNA isolation kit with an added digestion step with RNase-free DNase and the μMACS cDNA synthesis module from Miltenyi according to the manufacturer's instructions. PCR amplification was then performed as nested PCR. Primers used for the first amplification are 5′-outLTR, 5′-AGGTGTGGCCTGGGCGGGACT-3′, and 3′-out-gag, 5′-CAGCAAGCCGAGTCCTGCGTCG-3′. The primers 5′-inLTR, 5-GGCGAGCCCTCAGATCCTGC-3, and 3′-in-gag, 5′-GTCCCTGTTCGGGCGCCACTGCTA-3′, were then used for second-round amplification using 5 μl of the first-round PCR product as template. Conditions for both PCRs were as follows: 94°C for 5 min; 30 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 7 min. For visualization, the PCR products were then loaded on a 2% agarose gel and separated by gel electrophoresis. Control reactions in which RT was omitted during cDNA generation were performed to ensure that no genomic DNA was amplified.


Experimental model for the quantitative kinetic analysis of HIV-1 latency establishment.

Understanding the mechanisms governing HIV-1 latency establishment may provide new insights into how to therapeutically interfere with HIV-1 latency in vivo. Currently, HIV-1 latency at the molecular level is studied mostly using latently HIV-1-infected clonal T-cell lines in which HIV-1 latency has already been established at the time point of analysis (e.g., U1, ACH-2, J89GFP, and J-LAT) (28, 37, 41). As such, these cells do not permit the study of mechanisms underlying HIV-1 latency establishment but are limited in use to the study of factors that govern latency maintenance or HIV-1 reactivation.
To study HIV-1 latency establishment at the population level, we initially infected Jurkat T cells with an EGFP-expressing reporter virus (41, 44). Routinely, RTIs and PIs were added to the infection cultures on day 1 p.i. to prevent the establishment of preintegration latency (47) and to restrict continuous de novo infection that would perturb the stability of a possibly established latently infected cell population (reactivation by superinfection). RTIs and PIs were then continuously added throughout the entire experimental period. To follow the level of inactive or silent viral integration events over time and to monitor the establishment of a stable latently HIV-1-infected cell population, we removed samples of the infected parental culture at various time points and stimulated these samples with PMA or tumor necrosis factor alpha (TNF-α), two potent HIV-1-(re)activating agents (29). Levels of active infection in the unstimulated parental culture, as determined by FCM quantification of EGFP expression, were then compared with the levels of total infection in the activated samples 24 h poststimulation. The difference between total infection (% EGFP-positive cells in activated sample) and active infection (% EGFP-positive cells in untreated sample) represented the number of silent integration events.
In the representative experiment depicted in Fig. 1A, the mean active infection level in the parental infection cultures on day 3 p.i. was 28% ± 2%, with 43% ± 3% mean total infection (TNF-α activated), indicating that at this time point, 15% of the cell population contained integrated infection events that were not transcriptionally active but could be activated (silent infection). Active infection levels initially rapidly declined, mostly due to the cytopathic effect of the virus, since no difference in the cell proliferation rates of the uninfected and the infected population could be detected (PKH26 stain; data not shown). After day 10, the decay of active infection levels significantly slowed and complete self-eradication of the active infection took >50 days (Fig. 1B). The pool of silently infected cells also initially quickly decreased, stabilized between days 10 and 15, and remained at the 5% level until the end of the experiment on day 64, when it was evident that this cell population consisted of stably latently HIV-1-infected cells. In control experiments, addition of the integrase inhibitor 118-D-24 on day 1 p.i. produced identical results (data not shown), validating the previously described use of RTIs to prevent preintegration latency (47).

Silent integration is a prerequisite for the establishment of latent infection.

It has been suggested that in vivo cells that are close to a hypothetical minimum intracellular activation threshold become latently HIV-1 infected (30). These cells are sufficiently activated to allow for viral integration and initial viral gene expression. If the cellular activation levels then drop below this arbitrary threshold prior to the onset of any virally induced cytopathicity or immune recognition, viral gene expression is shut off, leaving the virus latently integrated. A second possibility is that the virus integrates but never actively expresses its genes. Obviously, the latter possibility would deprive even a functional immune system of any chance to attack and eradicate these infected cells prior to latency formation. To investigate whether either of these two forms of latency establishment would be predominant, we performed experiments in which we separated actively HIV-1-expressing and thus EGFP-positive cells from the EGFP-negative cell population through fluorescence-activated cell sorting on day 2 p.i. (Fig. 2A). The EGFP-negative population contained noninfected cells, as well as silently infected cells. RTIs and PIs were added to the cultures on day 1 p.i. and continuously replenished throughout the entire duration of the experiment. A part of the parental infection culture was not subjected to cell sorting to serve as a reference culture. The parental population and, after the cell sorting, the EGFP-positive and the EGFP-negative fractions were placed into continuous cell culture, and levels of active and silent infection were quantified throughout a 41-day period, while cell proliferation and cell death were monitored for the first 10 days (Fig. 2B).
If latent HIV-1 infection were to develop exclusively from silencing of active infection, then, after a prolonged culture period, the entire pool of latently infected cells identified in the parental infection culture should be contained in the actively infected, EGFP-positive cell population. Conversely, if HIV-1 latency were to develop following silent integration, then, after an extended experimental period, the sorted EGFP-negative and the unsorted parental population should hold the same level of latently HIV-1-infected cells. Obviously, if both pathways contribute to latency establishment, the total number of latent events observed in the parental culture would be equal to the sum of the latent events observed in the two sorted cell populations following normalization for cell death/proliferation.
The results depicted in Fig. 2 demonstrate that HIV-1 latency is almost exclusively a result of silent integration and originates from the EGFP-negative cell population. In four independent experiments, using Jurkat or Molt-4 T-cell-based HIV-1 infection cultures, we physically separated T cells that were EGFP positive from the EGFP-negative cell fraction (Fig. 2A). Purity of these sorts in all cases was greater than 98%. We then followed the establishment of a latently infected cell population in these sorted cell populations as well as in the parental, unsorted population until day 41 p.i. The kinetics of absolute cell numbers were determined in all cultures for the first 10 days (Fig. 2B). These experiments revealed that the initially EGFP-negative cell populations on day 41 p.i. contained the same level of latent infection events as did the unsorted parental cell cultures (Jurkat cells [Fig. 2C] and Molt-4 cells [Fig. 2D]), arguing that the vast majority of the latently infected cells are derived from the EGFP-negative population and are established by silent integration.
Latently HIV-1-infected cells can also be detected in the initially EGFP-positive cell cultures. However, by tracking cell proliferation and HIV-1-induced cell death in this population, we found that the contribution of these cells to the total population (unsorted) on day 10 p.i. is already less than 1% (Fig. 2B). As latency levels in these populations did not exceed 10%, it can be calculated that the total contribution of latent infection events derived from initially active infection events in the nonsorted population is <0.1% (<1% × 10%). As the separation of the EGFP-positive and EGFP-negative populations had been performed on day 2 p.i., the results further show that the decision whether integration occurs in an active or latent state is made early after infection.

Silencing active HIV-1 infection.

While our results indicate that HIV-1 latency establishment is a function of silent integration, we also sought to investigate the possibility that active infection events could be rendered latent by long-term suppression of active HIV-1 transcription. This idea assumes that pharmacological suppression of transcriptional activity at the integrated viral LTR would assist the establishment of a restrictive histone code at the LTR. This would in turn, as previously suggested, stabilize the promoter in a transcriptionally inactive/latent state. For the experiments shown in Fig. 3, we utilized a NOMI reporter cell population in which chronically actively integrated viruses drive EGFP expression of a stably integrated HIV-1 LTR-EGFP reporter plasmid (36). The cells were treated with an optimal dose of the HIV-1 transcription inhibitor Ro24-7429 (10 μg/ml), and EGFP expression, as a direct correlate of LTR activity, was measured at the indicated time points using FCM analysis. Ro24-7429 was readded to the culture every 5 days, prior to any loss of its fully suppressive activity. Five days after compound addition, maximum suppression of HIV-1 activity was obtained, with EGFP expression being at background level in >40% of the cells. While Ro24-7429 was continuously added to the parental culture, we removed cell culture samples every 5 days and monitored whether virus expression would rebound in all cells (Fig. 3A). Incomplete rebound of EGFP expression in some cells would be indicative that continuous suppression of active HIV-1 infection had resulted either in the establishment of a latent infection or eventually in permanent transcriptional silencing. However, when Ro24-7429 suppression was removed after 5, 10, 15, or 20 days, virus expression rebounded. Even following 30 days of continuous suppression of HIV-1 expression, HIV-1 activity completely recovered in the entire population. The single-cell analysis of one representative experiment is depicted in Fig. 3B. The analysis regions were adjusted according to baseline EGFP fluorescence in uninfected NOMI reporter cells (upper left quadrant). Maximum achievable suppression of HIV-1 expression as indicated by EGFP expression on day 30 was 73% (lower left quadrant). Following cessation of Ro24-7429 addition to the culture, HIV-1 expression gradually rebounded in all cells and on day 48 (lower right quadrant) was found identical at the population level and for mean channel intensity to the levels seen in untreated chronically actively infected NOMI cells (upper right quadrant). These data suggest that prolonged suppression of HIV-1 transcription is insufficient to silence an initially active HIV-1 promoter. Consequently, a restrictive histone code or an inhibitory DNA methylation pattern that could have potentially formed during this time period of suppression was insufficient to stabilize the inactive state of these viral promoters.

Influence of the cell activation state on latency establishment.

Prerequisite for silent infection, viral integration in the absence of viral gene expression is likely a relatively low level of intracellular activity of the host cell at the time point of infection. While several transcription factors have been reported to be involved in HIV-1 transcription (e.g., Sp-1, AP-1, and NFAT), the nuclear presence of active NF-κB, in particular the p50/p65 heterodimer, is necessary for efficient transcription driven by the HIV-1 LTR (5, 24, 25). If latency establishment is controlled by the presence, or rather the absence, of NF-κB, then increasing the levels of active NF-κB by external stimulation prior to infection should abrogate latency formation. In exchange, stimulated cells should exhibit higher levels of active infection, with total infection remaining at the same level, provided that stimulation does not alter expression of the viral receptor/coreceptor profile. To test this hypothesis, we initially infected Jurkat T cells and simultaneously stimulated the cells with PMA or prostratin, two phorbol esters, which are known to (re)activate HIV-1 expression by stimulating NF-κB activity, the key transcription factor in activating HIV-1 expression (Fig. 4A). We then subjected the cells to the standard culture protocol to determine the size of the silent infection on day 3 and the size of latent infection on day 37. As predicted, while silent infection could be observed in the control infection, stimulation either with PMA or with prostratin abrogated the establishment of a pool of silently infected cells at the day 3 time point. This translated into a greatly diminished pool of latently HIV-1-infected cells at the day 37 time point, confirming that the size of the silently infected cell population on day 3 is predictive for the size of the latently infected cell pool at a later time point. This also provides additional evidence that silent integration is a prerequisite for latent infection. Similar results, although slightly less pronounced, could be obtained with other stimuli that target the NF-κB pathway, such as TNF-α or CD3/CD28 MAb stimulation (Fig. 4B). Again stimulation decreased the pool of cells holding silently integrated infection events on day 3 p.i., which translated to a decreased pool of latently infected cells on day 37 p.i. Thus, increasing the intracellular activation level with three different stimuli that trigger cell activation by different signaling pathways but converge in the NF-κB pathway diminished the likelihood of the establishment of latent infection events.
Previous reports have shown that various T-cell lines exhibit different levels of basal NF-κB activation in the absence of stimulation (12). We thus hypothesized that T-cell lines that have been reported to exhibit low NF-κB baseline activity, such as Jurkat or SupT1 cells, would be permissive for the establishment of latent HIV-1 infection, while cell lines with high levels of baseline NF-κB expression, such as PM1 or C8166 cells, would be nonpermissive for the establishment of latent infection (Fig. 5A). As predicted by the NF-κB profiles, infection of Jurkat or SupT1 cells resulted in the establishment of a silently infected cell reservoir, whereas PM1 and C8166 cells did not allow for silent integration to occur (Fig. 5B). Other cell lines frequently used in HIV-1 infection experiments that were found nonpermissive for HIV-1 latency establishment at the population level were the CD4-positive AA2 B-cell line and CEM-GFP cells. It is also noteworthy that the NF-κB activation profile of three latently infected Jurkat T-cell lines (CA5, 3F12, and 11B10) (Fig. 5A) did not differ from the NF-κB activation profile of the parental Jurkat cells, suggesting that once latency is established, it can be maintained in the presence of an NF-κB activation status that in the setting of a de novo infection is generally supportive of active infection.
To explain the finding that latent HIV-1 infection, in a cell line that is permissive for latency establishment at the population level (e.g., Jurkat T cells), is established only in a small fraction of the cells, we must postulate that NF-κB activity at the single-cell level is stochastically distributed around a median value. Only a small portion of the cells exhibit NF-κB activity below the threshold that is required for active HIV-1 transcription and thus, in turn, is permissive for silent integration. If this hypothesis is correct, and the percentage of cells exhibiting subthreshold intracellular activation levels does not change over time, the percentage of latently infected cells relative to the corresponding initial infection level should remain constant and should be independent of the initial infection level. As we can demonstrate that HIV-1 infection is not biased within a Jurkat T-cell population and all cells can be infected (we have achieved infection levels of >95% using an NL43-based EGFP reporter virus), we can test this idea, by infecting Jurkat T cells with various amounts of virus and calculating the ratio of established latent infection (>day 35) over active infection on day 3 for the different infection levels. Analysis on day 3 p.i. revealed that relative silent infection levels increase with decreasing infection levels. This is to be expected as superinfection levels, which would camouflage latent infection events, increase with increasing total infection levels (Fig. 6A). However, as proposed, we found that the ratio of latent infection as determined on day 41 to active or total infection as determined on day 3 was indeed independent of the level of the initial infection and found to be around 7% (6.9% ± 0.6% to 7.5% ± 0.4%) of initial infection levels (Fig. 6B). We repeated this experiment several times throughout a 2-year period and found that the relative level of latency established varied very little between experiments (7 to 10% of total infection). These small alterations can be best explained by minor changes in the level of baseline NF-κB activity throughout the cell population caused by differences in cell culture conditions (e.g., cell density directly prior to the experiments). Thus, these data provide additional evidence that the transcriptional fate of a virus to integrate in a latent state is dependent on the individual host cell activation state at the time of infection.

Establishment of silent/latent HIV-1 infection in primary T cells.

To test whether these findings could be translated into a primary T-cell setting, we hypothesized that HIV-1 infection of primary T cells immediately following stimulation would not allow for the establishment of silent/latent HIV-1 infection, while infection at a later time point, after peak cellular activation, would permit the establishment of silent infection at the population level. We stimulated PBMCs with PHA-L (2 ng/ml), and half of the cell population was infected with an EGFP-reporter virus on day 2 poststimulation (6 to 12% infected cells on day 2 p.i.). From day 1 p.i. the infected cell population was then continuously treated with an RTI-PI combination to prevent the formation of preintegration latency and de novo infection. The second half of the cell population was kept in culture and was infected on day 7 poststimulation, after the peak of PHA-L-induced cell activation. On average, infection levels at this time point were ∼50% lower than peak infection levels (3 to 8%). Again, an RTI-PI combination was added 24 h p.i., and all infection cultures were continued until day 14 poststimulation in the continuous presence of an RTI-PI combination. At this time, all remaining actively infected, and therefore EGFP-positive, cells were removed by flow cytometry-based cell sorting. Immediately following the cell sorting, cells were transferred into fresh cell culture medium and stimulated with an anti-CD3/CD28 MAb combination, IL-2, or TNF-α. A fraction of each cell population was left untreated to serve as a negative control. Again, during this 24-h period, all cultures were continuously supplemented with the RTI-PI combination. Figure 7 depicts the results of experiments using PBMCs from four different donors. In the sorted T-cell populations that were infected on day 2 following activation, only minimal levels of activatable HIV-1 infection were detected (squares). In contrast, silent/latent infection events could be detected in T-cell cultures in which the infection was initiated after the peak of cellular activation. The level of reactivatable infection was dependent on the stimulus, with TNF-α activation providing no increase in HIV-1 levels. In contrast, the anti-CD3/CD28 MAb combination and IL-2 activation produced active HIV-1 infection as indicated by an increase in the expression of EGFP cells relative to untreated control cells. Despite high sorting purity (>99.9% EGFP negative), the controls exhibited increased levels of infection events in which spontaneous HIV-1 reactivation was observed, which is likely to be a result of the physical stress imposed upon the cells during the cell sorting procedure.
Although different stimuli were required, these experimental results are in line with the experimental results obtained using T-cell lines and suggest that high cellular activation levels prohibit the formation of silent HIV-1 infection. While useful to address questions concerning the establishment of HIV-1 latency, the system is likely to have some limitations. The finding that IL-2 (100 U/ml) as a single treatment efficiently activates HIV-1 infection suggests that the cells, while not having an activated phenotype (95% of the cultures were CD25 negative; small forward scatter/side scatter pattern), do not have a true memory T-cell phenotype either. Memory T cells have been reported to express only the low-affinity IL-2 receptor, and we are uncertain whether in vivo latent HIV-1 infection in memory T cells would be reactivated by the applied IL-2 concentrations. In addition, while we can state that infection after the peak of intracellular activation allowed for silent integration of HIV-1, we cannot extend the culture past the day 14 time point in the absence of IL-2 supplementation, in order to test whether the observed silent viral integration state would reflect truly latent infection events found in memory T cells in an in vivo setting.

Histone modifications during HIV-1 latency establishment.

Histone modifications, such as the generation of a repressive histone code, are key mechanisms for the control of cellular gene expression. Histone occupancy is also thought to protect silent cellular promoters from the nonspecific activity of DNMTs, and the absence of the appropriate histone composition could result in permanent transcriptional silencing of the unprotected promoter. Since we have demonstrated that silent integration is key to the establishment of latent HIV-1 infection (Fig. 2), one might assume that, similarly to cellular genes, a restrictive histone composition is formed at a silently/latently integrated HIV-1 promoter to (i) stabilize the silent promoter state and (ii) protect the promoter from nonspecific DNA methylation and subsequent permanent transcriptional silencing. Indeed, the formation of a repressive histone code has been associated with HIV-1 latency (23, 60, 61), and HDAC inhibitors have been reported to reactivate latent HIV-1 infection (49, 52, 60). These findings imply that latency establishment would be regulated by HDAC activity, and we postulated that a lack of HDAC activity would abrogate latency formation. We thus treated Jurkat T cells prior to infection with a single dose of the HDAC inhibitor NaBu (100 μM), valproic acid (300 μg/ml), or TSA (100 nM). These compounds were used at the maximum tolerated concentration without biasing the experimental outcome by affecting cell viability. We found that pretreatment of the cells with a single dose of an HDAC inhibitor (data for NaBu shown in Fig. 8) prior to infection increased the level of infection (28% active infection in control cells versus 37% infection in NaBu-treated cells), demonstrating that the compounds exerted activity at the concentration utilized. In the experiments, NaBu not only increased the level of active infection on day 3 but also increased the size of the generated latently infected cell population on day 37. If HDAC activity were essential for the establishment of latent infection events, then, in the presence of NaBu, we would have expected to see a reduction in the size of the latent reservoir that was found established on day 37 p.i. These data thus suggest that the formation of a restrictive histone code is not essential to establish latent HIV-1 infection. The observed increase in infectivity could be attributed to the ability of HDAC inhibitors to open heterochromatin, thereby allowing for integration of retroviruses into DNA areas that usually are not accessible for integration.
This unexpected finding led us to revisit previous findings that HDAC inhibitors reactivate latent HIV-1 infection in the absence of cellular activation in the cell clones generated under our experimental conditions. For this purpose, we generated ∼800 single-cell clones from the infection culture described for Fig. 1 and stimulated each clone with PMA, the HDAC inhibitor NaBu, the DNMT inhibitor 5-azacytidine, or combinations thereof. Of the ∼800 generated clones, 36 responded to PMA treatment with reactivation as indicated by an increase in the percentage of EGFP-positive cells on day 2 poststimulation (see Fig. 10B and C). None of the clones responded with HIV-1 reactivation to treatment with either the HDAC inhibitor NaBu (100 μM) or the DNMT inhibitor 5-azacytidine (1 μM) alone (data not shown).
For a more detailed analysis, we randomly selected seven of the latent clones that would respond to PMA stimulation with efficient HIV-1 reactivation (>80% EGFP-positive cells). For each of the selected cell clones, a dose-response curve for the HIV-1-activating agents TNF-α, PMA, and prostratin and for the HDAC inhibitors NaBu, valproic acid, and TSA was generated. Levels of HIV-1 reactivation as indicated by EGFP expression and cell viability were determined after 48 h using FCM analysis. Reactivation efficiency relative to drug toxicities for the cell-activating agents in comparison to the HDAC inhibitors is shown in Fig. 9. As expected, TNF-α activation efficiently reactivated latent HIV-1 infection at the population level and the onset of cytotoxicity directly correlated with the increase in active HIV-1 infection levels. Similar results were obtained for the HIV-1-activating phorbol esters PMA and prostratin. Throughout all latently infected cell clones, we found that the ability of HDAC inhibitors to reactivate latent HIV-1 infection was clearly inferior to that of cell-stimulating agents. No HIV-1-reactivating effect of HDAC inhibitors on latent HIV-1 infection was observed until the onset of massive compound-caused cell death. In all cell lines, the observed HDAC inhibitor-caused increase in cytotoxicity was disproportionate to the level of achievable reactivation, suggesting that a cell stress response, rather than changes in a restrictive LTR histone composition, may drive or at least be required for HIV-1 reactivation. The absence of an HDAC inhibitor effect on established latent infection in these cell lines suggests that a restrictive histone code is also not essential to maintain latent HIV-1 infection in these cells.

DNA methylation during HIV-1 latency establishment.

We next tested the influence of the DNMT inhibitor 5-azacytidine on HIV-1 latency establishment. Using the same experimental setup as used above for the HDAC inhibitors, we found no evidence that inhibition of DNMT activity would influence HIV-1 latency establishment (Fig. 10A). However, of the 36 latently infected T-cell clones generated, we identified 18 clones in which the addition of 5-azacytidine to the culture prior to stimulation with PMA would increase the level of HIV-1 reactivation, suggesting that a restrictive DNA methylation pattern had formed on the integrated latent viruses (Fig. 10C). We believe that this theoretical conflict, the lack of influence of DNMT inhibitors during silent integration and early latency establishment (up to day 41) and the formation of an inhibitory DNA methylation pattern at a later time point, is an artifact of the utilization of immortalized T-cell lines and needs to be carefully evaluated for its significance. Evidence for the idea that methylation patterns in our system could be an artifact comes from long-term culture experiments on our latently infected cell populations. We repeatedly observed that continuous growth of latently infected cell populations would decrease the amount of latently infected cells in these cultures without any signs that this decrease could have been the result of spontaneous reactivation and subsequent depletion of these cells from the culture. For example, the cell population depicted in Fig. 1 was initially characterized as holding 5% latently infected cells on day 64 p.i. (Fig. 1). In continuous culture, levels of latently infected cells in the population gradually declined to 3% over the next 6 months (data not shown). While we do not provide direct evidence, this is likely attributable to low-level de novo methylation, enabled by continuous cell division. However, cell replication, which allows for de novo methylation, would not be found in memory T cells, which are considered the in vivo reservoir of latent HIV-1 infection.

Role of immediate active HIV-1 transcription in latency establishment.

The inability to provide sufficient amounts of HIV-1 Tat and thus active transcription has been associated with the establishment of HIV-1 latency. We thus tested the question of whether Tat-mediated HIV-1 transcription activity was relevant for latency formation. For this purpose, we infected Jurkat T cells in the presence of an optimal dose of the HIV-1 transcription inhibitor Ro24-7429 (10 μg/ml) (26, 33). As expected, Ro24-7429 delayed the course of active infection but surprisingly did not alter peak infection levels (Fig. 11). Ro24-7429-mediated inhibition of HIV-1 expression also did not alter the size of the pool of latently HIV-1-infected cells in the infection culture, suggesting that lack of immediate active HIV-1 transcription does not favor latency establishment (Fig. 11). These results are consistent with the inability to generate stable latent infection events from previously active infection events (Fig. 3) and again argue against the establishment of a restrictive histone code as a prerequisite for HIV-1 latency.

Integration of latent virus in actively expressed host genes.

In our experiments, we could not find any evidence for a role of a restrictive histone code for either latency establishment or maintenance. This is in line with previous findings by Han et al., who reported that >90% of all viral integration events in memory T cells of infected patients are found in actively expressed genes (30). Given these findings, we investigated whether the latent integration events in the clonal cell lines generated from our experimental system would be found integrated into actively transcribed host genes and thus reflect the in vivo situation. For this purpose, we used a modification of a PCR strategy described by Han et al. that would exclusively amplify viral sequences integrated into actively transcribed host genes but not viral mRNA products generated under the control of the viral LTR. This was achieved by utilizing 5′ primers that bind the LTR sequence prior to the transcription initiation site and 3′ primers that bind to the gag sequence prior to the splice donor site. Using this experimental strategy, we analyzed cellular mRNA from 13 of the latently infected cell clones described for Fig. 10B that had been archived for the presence of integrated viral genomes. All clones responded to PMA stimulation with reactivation levels of >85%, confirming that the cells were latently infected. Figure 12A shows the reactivation profile of five representative clones. In all 13 analyzed cell clones, the HIV-1 LTR sequence was found integrated into cellular mRNA, demonstrating that HIV-1 in our latently infected cell clones, similar to the in vivo situation, is integrated into actively expressed host genes (Fig. 12B). Combined with the inability of these clones to respond to HDAC inhibitor treatment with HIV-1 reactivation, these data provide quantitative support for the idea that transcriptional host gene interference may be the major mechanism to control HIV-1 latency.


Based on our results, we propose a model for HIV-1 latency in which we differentiate between factors governing latency establishment and mechanisms controlling latency maintenance. According to our data, whether infection occurs in a transcriptionally active or latent state is decided upon viral integration and is a strict function of the availability of NF-κB for binding to the viral promoter at the time of infection. Below a certain threshold of NF-κB availability, the virus will integrate in a transcriptionally silent state (silent integration), which is a prerequisite for latency establishment. Once established, latent infection is maintained by transcriptional interference, as latent viruses are generally found integrated into actively expressed genes and we find no evidence for an involvement of histone modifications or DNA methylation in latency establishment or maintenance.
Until recently, HIV-1 latency was regarded as a gene regulation phenomenon that would be governed by the same mechanisms that control cellular gene expression, histone modifications and DNA methylation. However, findings by Han et al., published in 2004 (30), suggested that viral integration events in CD4-positive memory T cells of highly active antiretroviral therapy-suppressed patients were in >90% of all analyzed events found integrated into the exons or introns of genes that are, in general, actively expressed in memory T cells. This finding is not supportive of the idea that latent integration would be governed by a suppressive histone code, which is unlikely to form in the exon/intron region of actively expressed genes. A likely explanation would be that transcriptional interference in which the transcriptional machinery initiates at the promoter of the gene into which the virus is integrated (host gene) reads through the viral genome. The constant presence of the transcriptional machinery initiating at the host gene promoter then prevents transcription factors from binding to the viral promoter and thus stabilizes latent infection. As the initial ex vivo studies were performed using primary CD4-positive memory T cells, the authors could determine only individual viral integration sites and correlate these integration sites with a general gene expression profile for memory T cells. However, in follow-up studies using the latently infected clonal J-LAT cell line or a clonal cell line in which an HIV-1 reporter construct had been integrated in an orientation-dependent manner relative to the host gene, two groups independently demonstrated that latent HIV-1 infection could indeed be a result of transcriptional interference (31, 43).
We here demonstrate that these results on the governing role of transcriptional interference for HIV-1 latency are not limited to selected clonal cell lines but can be transferred to our population-based experimental system, strongly supporting the idea that transcriptional interference is the primary mechanism controlling HIV-1 latency in vivo. We found that all tested integration events that had given rise to latent HIV-1 infections in our population-based assay had occurred in actively expressed host genes.
The idea that transcriptional interference is the key governing mechanism for the maintenance of HIV-1 latency is supported by several of our other experimental results. First, pretreatment of T cells with the HDAC inhibitor valproic acid, NaBu, or TSA did not prevent latency formation. As HDAC inhibitors have been reported to reactivate latent HIV-1 infection by removing a restrictive histone code, the inhibitors should have prevented latency formation if a restrictive histone code were involved, which was not the case in our experiments. As we have demonstrated that the decision whether a virus integrates in a latent state is made early within the first 48 h after infection and that bolus application of the HDAC inhibitors maintains full activity during this time period, we conclude that a histone code is not required to either establish or maintain latent infection.
Second, this is also suggested by the results obtained using the HIV-1 transcription inhibitor Ro24-7429 during the infection phase. As the data demonstrate, the presence of the inhibitor did not alter the number of total viral integration events but relatively efficiently suppressed active viral transcription in the majority of the infected cells. This should have increased the likelihood of the formation of a suppressive histone code at the viral LTR. However, as our data show, it is unlikely that a restrictive histone code was established, as the application of Ro24-7429 did not alter the level of latency formation.
Third, while we were able to fully suppress HIV-1 expression in a population of otherwise chronically actively HIV-1-infected cells over a prolonged period (>20 days), this extended suppression of viral gene expression was insufficient to generate any latent infection events, which would have been suggestive of the formation of a repressive histone code that would exert control over viral gene expression.
Similarly, we could not find evidence that DNA methylation events are important for the establishment of latent infection. The literature is inconclusive on the possible importance of DNA methylation for the maintenance of HIV-1 latency, and no particular methylation pattern of the viral LTR has been associated with HIV-1 latency (3, 4, 34, 38, 45, 48). We also saw evidence for the formation of DNA methylation patterns that influence reactivation (Fig. 10C), as in a substantial selection of latently infected cell clones the addition of a DNMT inhibitor increased the ability to reactivate latent infection following provision of a cellular stimulus. However, when we continuously passaged cell populations that held latently infected cells, we observed a slow but gradual decrease in the size of the latently infected cell population. As there is no evidence that this decline of the level of latently infected cells is associated with spontaneous HIV-1 reactivation and deletion of these cells from the culture by the ensuing cytopathic effect of the then-active virus, we conclude that the decrease in reactivatable infection events over time is caused by DNA methylation resulting in permanent transcriptional silencing of the integrated viral LTR. This is not particularly surprising, as silencing of retroviral vectors in dividing cells is a problem that has been extensively described (59). As it seems that the occurrence of DNA methylation patterns that are reversible by treatment with 5-azacytidine is associated with continuous cell division, it seems unlikely that a similar restrictive DNA methylation pattern would be established in latently HIV-1-infected, nondividing memory T cells in vivo.
The second finding central to our experiments is that we report silent integration to be a prerequisite to the establishment of latent HIV-1 infection events. As we have demonstrated that low intracellular activation levels are a prerequisite for silent integration, this would require that in vivo or ex vivo low-level-activated or even resting CD4+ T cells could be infected with HIV-1. While HIV-1 preferentially infects activated CD4-positive T cells, it has been experimentally demonstrated that resting T cells can be infected and that infection in these cells is mostly latent (2, 58). Our data generated in immortalized T-cell lines and primary T cells would thus be consistent with these earlier findings in primary T cells. Obviously, our data do not conclusively rule out the possibility that latent infection in primary T cells can be generated by a decrease in the availability of crucial transcription factors that would be associated with the transition of the infected cell to a resting state. However, we demonstrate that the generation of latent infection events is independent of the requirement for a reduction of the cellular activation state, which would be a statistically highly unlikely coincidence. Silent integration of the virus into actively expressed genes is thus a model concept that can explain both latency establishment and maintenance.
We here add to recently presented evidence that HIV-1 latency is not governed by the same mechanisms as is cellular gene expression but, following silent integration, is maintained by transcriptional interference. Even if only a minor portion of the latent infection events, and not the majority of these integration events, as suggested by the work of Han et al. (30) and our results, are governed by transcriptional interference, current therapeutic strategies will have to be revisited. Taken together, these findings have wide-ranging consequences for the future design of therapeutic strategies. Targeted activation of latently infected cells still remains a therapeutic option, but the devastating results from the TGN1412 clinical phase I trial, in which the application of an agonistic anti-CD28 antibody had a near-fatal outcome for all six volunteers, would suggest extremely careful evaluation of this path (32, 50, 57). Fundamentally different ideas will be needed to therapeutically target HIV-1 latency.
FIG. 1.
FIG. 1. Quantitative establishment kinetics of a population of latently infected cells. Jurkat T cells were infected with an EGFP reporter virus. De novo infection was inhibited 24 h p.i. by the addition of a combination of RTIs and PIs. Two samples from the parental infection culture were removed 24 h prior to the indicated time points of the kinetic experiment depicted in panel B and either left nonactivated to determine baseline active infection or stimulated with TNF-α to activate silent/latent HIV-1 infection events (total infection). At the time points indicated, the percentage of EGFP-positive cells was determined in the control samples and the corresponding activated samples by using FCM analysis for EGFP expression. The difference in the percentages of EGFP-expressing cells between the two samples represents the cell population that is silently/latently HIV-1 infected. (A) Histogram analysis of active infection levels (dotted line) and total infection levels following TNF-α stimulation (full line) as determined by FCM analysis of EGFP expression on day 3 p.i. (B) Kinetic analysis of active and total infection levels in the infection cell culture over a total period of 64 days. The results represent the means ± standard deviations of three independent experiments.
FIG. 2.
FIG. 2. Silent integration is the major source of latent infection. Jurkat or Molt T cells were infected with an EGFP reporter virus. Superinfection and the establishment of preintegration latency were inhibited by the addition of an RTI-PI combination 24 h p.i. (A) On day 2 p.i. the EGFP-positive, infected cells were physically separated from the noninfected or silently infected cell population (EGFP negative) by using fluorescence-activated cell sorting. The parental culture and the EGFP-positive and EGFP-negative cell populations were placed in continuous culture to which Sphero beads were added to allow for the determination of absolute cell numbers. (B) Kinetics of absolute cell counts in the sorted EGFP+ and EGFP populations. (C and D) Determination of silent infection levels on day 3 and latent infection levels on day 41 in two independent experiments in the unsorted parental population and the sorted EGFP-negative (−) population in Jurkat T cells (C) or Molt-4 T cells (D) infected with the EGFP reporter virus.
FIG. 3.
FIG. 3. Extended suppression of active HIV-1 transcription does not result in the establishment of latent infection events. (A) A population of chronically actively infected Jurkat T cells in which EGFP expression serves as a quantitative marker of HIV-1 transcription was continuously treated with the HIV-1 transcription inhibitor Ro24-7249. At several time points, samples were removed from the parental culture, and the rebound of HIV-1 transcription was monitored over time. EGFP expression as a surrogate marker was determined by FCM analysis and is expressed as mean channel fluorescence (MCF) of the total population. The results represent the means ± standard deviations of three independent experiments. (B) Analysis of EGFP expression at the population level. EGFP expression was determined in the uninfected parental NOMI reporter cell line (negative), in the untreated infected-cell population (HIV), in the infected-cell population treated with Ro24-7249 for 24 days (HIV + Ro), and in the same treated cell population 24 days following addition of the last dose of Ro24-7249 (Ro removed). Percentages of cells in the respective marker regions and the corresponding MCF intensities are presented for all four culture conditions.
FIG. 4.
FIG. 4. Host cell activation prevents latency establishment. Jurkat T cells were infected with an HIV-1 EGFP reporter virus and simultaneously stimulated with either PMA or prostratin (PRO) (A) or TNF-α or an anti-CD3/CD28 MAb combination (B). Levels of active and total infection on day 3 and on day 37 p.i. in control samples and activated samples were determined by FCM analysis. The results represent the means ± standard deviations of three individual experiments. t tests were performed to determine whether the sizes of the established silent or latent reservoirs in control cells and pretreated cells were significantly different. UN, unstimulated.
FIG. 5.
FIG. 5. Latency formation is cell type dependent and correlates with the basal level of NF-κB activity. (A) Nuclear extracts from the indicated cell lines were generated, and relative baseline levels of NF-κB p50, p65, p52, RelB, and c-Rel were determined using an NF-κB family transcription factor assay kit (Active Motif). (B) Jurkat, SupT1, Molt-4, PM1, C8166, AA2, and CEM-GFP cells were infected with an EGFP reporter virus. On day 1 p.i., RTIs and PIs were added. On day 2 p.i., a sample of each culture was stimulated with PMA (1 ng/ml), and levels of EGFP expression in the control culture and the PMA-activated culture were determined by FCM analysis on day 3 p.i. to determine the level of silent infection. The results represent the means ± standard deviations of three individual experiments.
FIG. 6.
FIG. 6. Latent reservoir size is independent of initial infection level. Jurkat T cells were infected with various levels of an EGFP reporter virus, and active and total infection levels were determined as the percentages of EGFP-positive cells in control or PMA-stimulated samples, respectively, on day 3 and day 32 p.i. by using FCM analysis. (A) Ratio of silent/total infection on day 3 p.i. plotted over the percentage of active infection. (B) Ratio of latent/total infection on day 32 p.i. plotted over the percentage of active infection as determined on day 3 p.i. The results represent the means of three independent experiments ± standard deviations.
FIG. 7.
FIG. 7. Silent HIV-1 infection and reactivation in in vitro HIV-1-infected PBMCs. PBMCs from four different donors were infected with an HIV-1 GFP reporter virus on day 2 (squares) or day 7 after PHA-L stimulation (circles). At 24 h p.i., a lamivudine-indinavir combination was added to the infection cultures to inhibit de novo infection and to prevent formation of preintegration latency. On day 14 following stimulation, HIV-1-expressing EGFP+ cells were removed from the cultures by using fluorescence-activated cell sorting. Following the sorting procedure, the EGFP-negative cells were cultured in supplemented RPMI 1640 to determine levels of spontaneous reactivation (control) or stimulated with an anti-CD3/CD28 MAb combination (UCHT1/CD28.2), IL-2 (100 U/ml), or TNF-α (10 ng/ml). Numbers indicate the percentages of EGFP-positive cells as determined 24 h poststimulation.
FIG. 8.
FIG. 8. HDAC inhibitors do not prevent latency establishment. Jurkat cells were pretreated for 16 h with NaBu (100 μM) and infected with an HIV-1 EGFP reporter virus. Levels of silent infection on day 3 p.i. and levels of latent infection on day 37 were determined by FCM analysis by comparing the level of active infection in unstimulated cultures (UN) and the level of total infection following stimulation (PMA; 3 ng/ml). The results are representative of the means of three independent experiments ± standard deviations.
FIG. 9.
FIG. 9. Reactivation of latent HIV-1 infection. Latently HIV-1-infected cell clones were generated from the day 64 state of the experiment described for Fig. 1. Seven of these clones exhibiting low baseline EGFP expression were then stimulated with increasing concentrations of activating agents (TNF-α, PMA, and prostratin) (left panel) or HDAC inhibitors (valproic acid, NaBu, and TSA) (right panel). At 48 h after stimulation or treatment, levels of HIV-1 reactivation as indicated by EGFP expression and cell viability were determined by FCM analysis.
FIG. 10.
FIG. 10. DNMT inhibitors do not prevent latency establishment. (A) Jurkat cells were pretreated for 16 h with 5-azacytidine (5-Aza; 1 μM) and infected with an HIV-1 EGFP reporter virus. Levels of silent infection on day 3 p.i. and levels of latent infection on day 37 were determined by FCM analysis by comparing the level of active infection in unstimulated cultures (UN) and the level of total infection following PMA stimulation. The results are representative of the means of three independent experiments ± standard deviations. (B and C) From the day 64 cell population described for Fig. 1, ∼800 single-cell clones were generated and characterized for the presence of latent infection, by comparing baseline EGFP expression in unstimulated cells (UN) with EGFP expression levels 24 h after stimulation with either PMA alone or a combination of 5-Aza pretreatment (16 h; 1 μM) and PMA stimulation (3 ng/ml). (B) Eighteen identified latently infected clones that respond with maximal HIV-1 reactivation to PMA stimulation. (C) Eighteen identified clones in which 5-Aza pretreatment enhances PMA-mediated HIV-1 reactivation.
FIG. 11.
FIG. 11. Inhibition of HIV-1 transcription has no influence on latency establishment. (A and B) Jurkat cells were left untreated (A) or treated with 10 μM of the HIV-1 transcription inhibitor Ro24-7249 (B) and infected with an EGFP reporter virus. Levels of active and total infection (+TNF) in the respective cell cultures for the day 3, day 8, and day 15 time points were determined by measuring the percentages of EGFP-positive cells by using FCM analysis. (C) Establishment of latent infection in the control cultures and the Ro24-7249-treated cultures at the day 37 time point. The results are representative of the means of three independent experiments ± standard deviations.
FIG. 12.
FIG. 12. Integration of latent infection events in actively expressed host genes. (A) Baseline EGFP/HIV-1 expression (C) and reactivation response to PMA stimulation of five representative latently infected T-cell clones derived from the infection culture described for Fig. 1. (B) PCR for integration into actively expressed host genes. Cellular mRNA was used to generate cDNA from 13 archived latently infected cell clones (Fig. 10B). Nested PCR to detect viral integration into actively expressed cellular host genes was performed with primer pairs that bind 5′ of the HIV-1 LTR transcription start site and upstream of the HIV-1 gag splice donor site, to exclusively amplify integrated viral LTR sequences that have been generated as part of host gene transcription products. (C) As controls, the PCRs were performed with no input material (C; lane 1) or mRNA from a latently infected cell line (clone 5E3 [L]; lane 2). Lanes 3 to 8 use mRNA from 293T cells that were transfected with the plasmid coding for the EGFP reporter virus (50% transfection efficacy) either in the absence (293T; lanes 3 to 5) or in the presence (293T+RT; lanes 6 to 8) of an RTI. For each transfection, the PCR was performed directly from the isolated mRNA (no reverse transcription; lanes 3 and 6), from cDNA generated from mRNA without prior DNase treatment (lanes 4 and 7), and from cDNA generated from mRNA with prior DNase treatment (lanes 5 and 8). Lane MW, molecular mass markers.


This work was supported through NIH grants R01AI077457 and R01AI064012 and a GCE grant from the Bill and Melinda Gates Foundation (O.K.).


Adams, M., L. Sharmeen, J. Kimpton, J. M. Romeo, J. V. Garcia, B. M. Peterlin, M. Groudine, and M. Emerman. 1994. Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts. Proc. Natl. Acad. Sci. USA91:3862-3866.
Agosto, L. M., J. J. Yu, J. Dai, R. Kaletsky, D. Monie, and U. O'Doherty. 2007. HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration. Virology368:60-72.
Bednarik, D. P., J. A. Cook, and P. M. Pitha. 1990. Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency. EMBO J.9:1157-1164.
Bednarik, D. P., J. D. Mosca, and N. B. K. Raj. 1987. Methylation as a modulator of expression of human immunodeficiency virus. J. Virol.61:1253-1257.
Biswas, D. K., C. M. Ahlers, B. J. Dezube, and A. B. Pardee. 1993. Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat. Proc. Natl. Acad. Sci. USA90:11044-11048.
Brooks, D. G., S. G. Kitchen, C. M. Kitchen, D. D. Scripture-Adams, and J. A. Zack. 2001. Generation of HIV latency during thymopoiesis. Nat. Med.7:459-464.
Bushman, F. D., and R. Craigie. 1992. Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites. Proc. Natl. Acad. Sci. USA89:3458-3462.
Butera, S. T. 2000. Therapeutic targeting of human immunodeficiency virus type-1 latency: current clinical realities and future scientific possibilities. Antivir. Res.48:143-176.
Carbone, A., G. Gaidano, A. Gloghini, L. M. Larocca, D. Capello, V. Canzonieri, A. Antinori, U. Tirelli, B. Falini, and R. Dalla-Favera. 1998. Differential expression of BCL-6, CD138/syndecan-1, and Epstein-Barr virus-encoded latent membrane protein-1 identifies distinct histogenetic subsets of acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas. Blood91:747-755.
Carbone, A., A. Gloghini, L. M. Larocca, D. Capello, F. Pierconti, V. Canzonieri, U. Tirelli, R. Dalla-Favera, and G. Gaidano. 2001. Expression profile of MUM1/IRF4, BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas. Blood97:744-751.
Cavert, W., D. W. Notermans, K. Staskus, S. W. Wietgrefe, M. Zupancic, K. Gebhard, K. Henry, Z. Q. Zhang, R. Mills, H. McDade, C. M. Schuwirth, J. Goudsmit, S. A. Danner, and A. T. Haase. 1997. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science276:960-964.
Chen, B. K., M. B. Feinberg, and D. Baltimore. 1997. The κB sites in the human immunodeficiency virus type 1 long terminal repeat enhance virus replication yet are not absolutely required for viral growth. J. Virol.71:5495-5504.
Chun, T. W., L. Carruth, D. Finzi, X. Shen, J. A. DiGiuseppe, H. Taylor, M. Hermankova, K. Chadwick, J. Margolick, T. C. Quinn, Y. H. Kuo, R. Brookmeyer, M. A. Zeiger, P. Barditch-Crovo, and R. F. Siliciano. 1997. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature387:183-188.
Chun, T. W., R. T. Davey, Jr., M. Ostrowski, J. Shawn Justement, D. Engel, J. I. Mullins, and A. S. Fauci. 2000. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat. Med.6:757-761.
Chun, T. W., D. Engel, M. M. Berrey, T. Shea, L. Corey, and A. S. Fauci. 1998. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc. Natl. Acad. Sci. USA95:8869-8873.
Chun, T. W., D. Engel, S. B. Mizell, L. A. Ehler, and A. S. Fauci. 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med.188:83-91.
Chun, T. W., D. Engel, S. B. Mizell, C. W. Hallahan, M. Fischette, S. Park, R. T. Davey, Jr., M. Dybul, J. A. Kovacs, J. A. Metcalf, J. M. Mican, M. M. Berrey, L. Corey, H. C. Lane, and A. S. Fauci. 1999. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nat. Med.5:651-655.
Chun, T. W., and A. S. Fauci. 1999. Latent reservoirs of HIV: obstacles to the eradication of virus. Proc. Natl. Acad. Sci. USA96:10958-10961.
Chun, T. W., D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, and R. F. Siliciano. 1995. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat. Med.1:1284-1290.
Chun, T. W., J. S. Justement, S. Moir, C. W. Hallahan, J. Maenza, J. I. Mullins, A. C. Collier, L. Corey, and A. S. Fauci. 2007. Decay of the HIV reservoir in patients receiving antiretroviral therapy for extended periods: implications for eradication of virus. J. Infect. Dis.195:1762-1764.
Chun, T. W., L. Stuyver, S. B. Mizell, L. A. Ehler, J. A. Mican, M. Baseler, A. L. Lloyd, M. A. Nowak, and A. S. Fauci. 1997. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA94:13193-13197.
Corbeil, J., D. Sheeter, D. Genini, S. Rought, L. Leoni, P. Du, M. Ferguson, D. R. Masys, J. B. Welsh, J. L. Fink, R. Sasik, D. Huang, J. Drenkow, D. D. Richman, and T. Gingeras. 2001. Temporal gene regulation during HIV-1 infection of human CD4+ T cells. Genome Res.11:1198-1204.
Coull, J. J., F. Romerio, J. M. Sun, J. L. Volker, K. M. Galvin, J. R. Davie, Y. Shi, U. Hansen, and D. M. Margolis. 2000. The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1. J. Virol.74:6790-6799.
Doerre, S., P. Sista, S. C. Sun, D. W. Ballard, and W. C. Greene. 1993. The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus. Proc. Natl. Acad. Sci. USA90:1023-1027.
Duh, E. J., W. J. Maury, T. M. Folks, A. S. Fauci, and A. B. Rabson. 1989. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA86:5974-5978.
Dunne, A. L., H. Siregar, J. Mills, and S. M. Crowe. 1994. HIV replication in chronically infected macrophages is not inhibited by the Tat inhibitors Ro-5-3335 and Ro-24-7429. J. Leukoc. Biol.56:369-373.
Finzi, D., J. Blankson, J. D. Siliciano, J. B. Margolick, K. Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori, C. Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, B. Walker, S. Gange, J. Gallant, and R. F. Siliciano. 1999. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat. Med.5:512-517.
Folks, T. M., K. A. Clouse, J. Justement, A. Rabson, E. Duh, J. H. Kehrl, and A. S. Fauci. 1989. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. Natl. Acad. Sci. USA86:2365-2368.
Folks, T. M., J. Justement, A. Kinter, S. Schnittman, J. Orenstein, G. Poli, and A. S. Fauci. 1988. Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol-12-myristate acetate. J. Immunol.140:1117-1122.
Han, Y., K. Lassen, D. Monie, A. R. Sedaghat, S. Shimoji, X. Liu, T. C. Pierson, J. B. Margolick, R. F. Siliciano, and J. D. Siliciano. 2004. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J. Virol.78:6122-6133.
Han, Y., Y. B. Lin, W. An, J. Xu, H. C. Yang, K. O'Connell, D. Dordai, J. D. Boeke, J. D. Siliciano, and R. F. Siliciano. 2008. Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough. Cell Host Microbe4:134-146.
Hansen, S., and R. G. Leslie. 2006. TGN1412: scrutinizing preclinical trials of antibody-based medicines. Nature441:282.
Haubrich, R. H., C. Flexner, M. M. Lederman, M. Hirsch, C. P. Pettinelli, R. Ginsberg, P. Lietman, F. M. Hamzeh, S. A. Spector, D. D. Richman, et al. 1995. A randomized trial of the activity and safety of Ro 24-7429 (Tat antagonist) versus nucleoside for human immunodeficiency virus infection. J. Infect. Dis.172:1246-1252.
Ishida, T., A. Hamano, T. Koiwa, and T. Watanabe. 2006. 5′ long terminal repeat (LTR)-selective methylation of latently infected HIV-1 provirus that is demethylated by reactivation signals. Retrovirology3:69.
Jones, J., J. Rodgers, M. Heil, J. May, L. White, J. A. Maddry, T. M. Fletcher III, G. M. Shaw, J. L. Hartman IV, and O. Kutsch. 2007. High throughput drug screening for human immunodeficiency virus type 1 reactivating compounds. Assay Drug Dev. Technol.5:181-189.
Jones, J., W. Whitford, F. Wagner, and O. Kutsch. 2007. Optimization of HIV-1 infectivity assays. BioTechniques43:589-594.
Jordan, A., D. Bisgrove, and E. Verdin. 2003. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J.22:1868-1877.
Jordan, A., P. Defechereux, and E. Verdin. 2001. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J.20:1726-1738.
Kao, S. Y., A. F. Calman, P. A. Luciw, and B. M. Peterlin. 1987. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature330:489-493.
Kulkosky, J., G. Nunnari, M. Otero, S. Calarota, G. Dornadula, H. Zhang, A. Malin, J. Sullivan, Y. Xu, J. DeSimone, T. Babinchak, J. Stern, W. Cavert, A. Haase, and R. J. Pomerantz. 2002. Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J. Infect. Dis.186:1403-1411.
Kutsch, O., E. N. Benveniste, G. M. Shaw, and D. N. Levy. 2002. Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. J. Virol.76:8776-8786.
Lehrman, G., I. B. Hogue, S. Palmer, C. Jennings, C. A. Spina, A. Wiegand, A. L. Landay, R. W. Coombs, D. D. Richman, J. W. Mellors, J. M. Coffin, R. J. Bosch, and D. M. Margolis. 2005. Depletion of latent HIV-1 infection in vivo: a proof-of-concept study. Lancet366:549-555.
Lenasi, T., X. Contreras, and B. M. Peterlin. 2008. Transcriptional interference antagonizes proviral gene expression to promote HIV latency. Cell Host Microbe4:123-133.
Levy, D. N., G. M. Aldrovandi, O. Kutsch, and G. M. Shaw. 2004. Dynamics of HIV-1 recombination in its natural target cells. Proc. Natl. Acad. Sci. USA101:4204-4209.
Mikovits, J. A., H. A. Young, P. Vertino, J. P. Issa, P. M. Pitha, S. Turcoski-Corrales, D. D. Taub, C. L. Petrow, S. B. Baylin, and F. W. Ruscetti. 1998. Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-γ) promoter and subsequent downregulation of IFN-γ production. Mol. Cell. Biol.18:5166-5177.
Nabel, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature326:711-713.
Pierson, T. C., Y. Zhou, T. L. Kieffer, C. T. Ruff, C. Buck, and R. F. Siliciano. 2002. Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J. Virol.76:8518-8531.
Pion, M., A. Jordan, A. Biancotto, F. Dequiedt, F. Gondois-Rey, S. Rondeau, R. Vigne, J. Hejnar, E. Verdin, and I. Hirsch. 2003. Transcriptional suppression of in vitro-integrated human immunodeficiency virus type 1 does not correlate with proviral DNA methylation. J. Virol.77:4025-4032.
Quivy, V., E. Adam, Y. Collette, D. Demonte, A. Chariot, C. Vanhulle, B. Berkhout, R. Castellano, Y. de Launoit, A. Burny, J. Piette, V. Bours, and C. Van Lint. 2002. Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-κB and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies. J. Virol.76:11091-11103.
Schneider, C. K., U. Kalinke, and J. Lower. 2006. TGN1412—a regulator's perspective. Nat. Biotechnol.24:493-496.
Schroder, A. R., P. Shinn, H. Chen, C. Berry, J. R. Ecker, and F. Bushman. 2002. HIV-1 integration in the human genome favors active genes and local hotspots. Cell110:521-529.
Sheridan, P. L., T. P. Mayall, E. Verdin, and K. A. Jones. 1997. Histone acetyltransferases regulate HIV-1 enhancer activity in vitro. Genes Dev.11:3327-3340.
Siliciano, J. D., J. Kajdas, D. Finzi, T. C. Quinn, K. Chadwick, J. B. Margolick, C. Kovacs, S. J. Gange, and R. F. Siliciano. 2003. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med.9:727-728.
Siliciano, J. D., J. Lai, M. Callender, E. Pitt, H. Zhang, J. B. Margolick, J. E. Gallant, J. Cofrancesco, Jr., R. D. Moore, S. J. Gange, and R. F. Siliciano. 2007. Stability of the latent reservoir for HIV-1 in patients receiving valproic acid. J. Infect. Dis.195:833-836.
Siliciano, J. M., and R. F. Siliciano. 2005. Targeting HIV reservoirs with valproic acid. Hopkins HIV Rep.17:8-9.
Smith, S. M. 2005. Valproic acid and HIV-1 latency: beyond the sound bite. Retrovirology2:56.
Suntharalingam, G., M. R. Perry, S. Ward, S. J. Brett, A. Castello-Cortes, M. D. Brunner, and N. Panoskaltsis. 2006. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N. Engl. J. Med.355:1018-1028.
Swiggard, W. J., C. Baytop, J. J. Yu, J. Dai, C. Li, R. Schretzenmair, T. Theodosopoulos, and U. O'Doherty. 2005. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J. Virol.79:14179-14188.
Swindle, C. S., H. G. Kim, and C. A. Klug. 2004. Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells. J. Biol. Chem.279:34-41.
Van Lint, C., S. Emiliani, M. Ott, and E. Verdin. 1996. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J.15:1112-1120.
Williams, S. A., L. F. Chen, H. Kwon, C. M. Ruiz-Jarabo, E. Verdin, and W. C. Greene. 2006. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J.25:139-149.
Winslow, B. J., R. J. Pomerantz, O. Bagasra, and D. Trono. 1993. HIV-1 latency due to the site of proviral integration. Virology196:849-854.

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Published In

cover image Journal of Virology
Journal of Virology
Volume 83Number 71 April 2009
Pages: 3078 - 3093
PubMed: 19144703


Received: 30 September 2008
Accepted: 7 January 2009
Published online: 1 April 2009


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Alexandra Duverger
Department of Medicine, Division of Infectious Diseases
Jennifer Jones
Department of Medicine, Division of Infectious Diseases
Jori May
Department of Medicine, Division of Infectious Diseases
Frederic Bibollet-Ruche
Frederic A. Wagner
Department of Medicine, Division of Infectious Diseases
Randall Q. Cron
Pediatric Rheumatology, The University of Alabama at Birmingham, Birmingham, Alabama
Olaf Kutsch [email protected]
Department of Medicine, Division of Infectious Diseases

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