HIV-1 transcription and latency: an update

Latently infected transformed cell lines

The study of HIV-1 latency in transformed cell has revealed many insights into the mechanisms of HIV-1 latency, despite the fact that establishment of latency in such systems is often linked to mutations in viral genes or to an effect specific to the site of integration, perhaps not uniformly representative of the quiescent nature of resting CD4+ T cells in patients. Indeed, each latently infected cell line possesses a different HIV-1 provirus (full-length or not) and may contain one or more copie(s) of the integrated provirus in different integration sites or epigenetic environments. Firstly, several models have been developed based on proviruses with minimal HIV-1 features of the LTR/Tat axis from models carrying a full-length provirus. For example, the Jurkat E4 cells and Jurkat bearing an integrated HIV-1 mini-virus are T-lymphoid Jurkat cells containing one single copy of the integrated provirus. However, E4 Jurkat cells possess a fragment of the HIV-1 pNL4-3 (containing tat, rev, env and vpu) and a short-lived green fluorescent reporter protein (d2EGFP) in place of the nef gene [66, 67] The Jurkat cells bearing an integrated HIV-1 mini-virus posses an HIV-1 reporter mini-virus, where luciferase is produced from the HIV-1 5’LTR as an in-frame fusion with p24gag [68].

Secondly, other models have been proposed that are based on full-length proviruses including ACH2, the inducible HIV-rtTA variant, J-Lat T cell lines and U1 promonocytic cell lines. The ACH2 T-cell line [69] and the promonocytic U1 cell line [70, 71] show minimal constitutive expression of HIV-1 genes, but a marked activation of viral gene expression following treatment with cytokines or mitogens. However, these models present mutations in Tat (U1) [72] or in its RNA target TAR (ACH2) [73], which have been demonstrated to be causative of the latent phenotype of the proviruses integrated in these two cell lines. Ben Berkhout's laboratory has developed stable cell lines containing an HIV-rtTA variant (in which the Tat/TAR axis transcription motifs have been inactivated and replaced by the inducible Tet-ON system [74]). The HIV-rtTA provirus is completely doxycycline-dependent for virus production, it contains the original transcription factor binding sites in the HIV-1 5'LTR, and infected cells have been obtained without selection steps avoiding any bias towards activation markers [75]. However, the implications of the Tat/TAR axis in reactivation cannot be analyzed. More recently, J-Lat cell lines were developed with an HIV-1-based vector containing an intact Tat/TAR axis [76]. These cells whose unique provirus carries the coding sequence for the green fluorescent protein (GFP) replacing the nef gene were selected for a lack of GFP expression under basal conditions [76]. J-Lat cells allow for the rapid assessment of HIV-1 transcriptional activity by cytometric detection of GFP epifluorescence. Consequently, the latently infected transformed cell lines are still useful to study and understand the basic mechanisms governing HIV-1 latency. The first models allow principally the study of HIV-1 promoter activity. The second category permits a complete study of the molecular mechanisms of latency with the influence of produced viral proteins on the HIV-1 promoter but also on host genes.

Interestingly, the J. Karn’s lab has recently generated a HIV-1 latently infected microglial cell line named CHME-5 [77]. To this end, the CHME-5 cell line (primary fetal human microglial cells immortalized with the SV40 large T antigen [78]) was infected with vesicular stomatitis virus G (VSVG)-pseudotyped HIVs bearing a fragment of HIV-1pNL4-3, containing tat, rev, env, and vpu, plus nef adjacent to the reporter gene d2EGFP inserted next to env. GFP + cells were next selected by FACS, and further cultured and allowed to enter into a latent state for 4 weeks. Latency of the CHME-5 cell line was characterized by reactivating expression and evaluating nuclear translocation of NF-kappaB [77].

Latently infected primary cells

Many laboratories have developed new ex vivo experimental primary human CD4+ T cell-based model systems to study HIV-1 latency in a more physiological context (reviewed in [79, 80]). Several groups have attempted to mimic in vitro the infection of activated CD4+ T cells, with HIV-1 or HIV-1 derived vectors, followed by the transition of these infected cells to a resting state to establish latency in memory CD4+ T cells. These cells are difficult to generate and maintain because most CD4+ T cells die soon after activation if not continuously cultured in the presence of specific cytokines. This results in few cells that have transitioned to a quiescent state and contain integrated HIV-1 that can be used for further study. Culturing of CD4+ T cells in the presence of cytokines, such as IL-2 or IL-7, increases their survival and allows time (several weeks) for more cells to transition to a memory state with integrated HIV. However, IL-2 and IL-7 have been implicated in the reactivation of latent HIV-1 [81–83]. Therefore, these cytokines must be used at a concentration that can maintain CD4+ T cells in culture without reactivating latent HIV. Alternatively, activated and HIV-infected CD4+ T cells can be maintained in culture and allowed to transition back to a resting state in the absence of cytokines by utilizing strategies, such as transduction with anti-apoptotic proteins or co-culture with feeder cell lines [84–86]. All these strategies lead to more viable cells that have been infected and returned to a resting state, allowing for HIV-1 latency to be studied in a physiologically relevant setting.

The first model of HIV-1 latency in primary lymphocytes was developed by Sahu et al. [84] and uses a replication-competent virus for the infection. The feeder cell line H80 (a brain tumor-derived cell line) promotes cell survival in the absence of any cytokine and allows the production of long-lived, mostly central memory CD4+ T cells. However, despite being phenotypically similar to resting cells in many respects, a significant fraction of this population continues to express the early activation marker, CD69, suggesting that these cells are not completely resting. In J. Karn’s laboratory, Tyagi et al. [86] developed a model of latency in which cells are infected with VSV-G-pseudotyped, HIV-1 vectors that lack env and encode a fluorescent marker in place of nef. Interestingly, they use HIV-1 vectors that express a mutated Tat with the single amino-acid change (H13L) that permits a quicker establishment of the latent state. This model leads to a relatively homogeneous population of central memory CD4+ T cells, which are not completely resting since they continue to express significant levels of the late activation marker, CD25 (CD69 expression is not measured). Marini et al. [87] have developed a primary latency model presenting the advantage of using a replication-competent wild type HIV-1. The majority of cells exhibits a central memory phenotype and is negative for CD25 (CD69 expression is not shown) but is larger and more granular than freshly isolated CD4+ T cells, suggesting that cells may not be completely resting. A disadvantage of this model is the requirement for IL-7, which has been shown to reactivate HIV-1 in latently infected cells [83, 88]. Bosque and Planelles [89, 90] developed a model in which cells are infected with an envelope-deficient HIV-1 virus that is pseudotyped with HIV-1 Env, thus limiting infection to a single round. Isolated CD4+ T cells were activated first with αCD3/αCD28 antibodies in the presence of IL-2 and then were cultured for several days in three different conditions that produced Th1-helper, Th2-helper, and nonpolarized CD4+ T cells [91]. Biochemical analysis indicated that the Th1 and Th2 populations closely resembled both effector memory and central memory CD4+ T cells, in vivo, while the nonpolarized population more closely resembled central memory CD4+ T cells. However, activation markers such as CD25 and CD69 were not assessed before reactivation, giving no indication if these cells were resting or active. Finally, Siliciano and collaborators [85, 92] increase cell survival in their model by transduction of a lentivector to express BLC2 prior to primary cells activation and expansion by αCD3/αCD28 antibodies and IL-2. Subsequent infection with a HIV-1 vector lacking several genes (including gag, vif, vpr, vpu and env) and encoding GFP in place of nef, an establishment of latency allowed the screening of a small library of random drug-like molecules for anti-latency compounds. This model generates effector memory cells expressing high levels of CD45RO and low levels of CCR7. A small fraction of them still expresses CD69 and CD25.

At variance with the infection of activated primary cells it is possible to infect directly resting (not activated) CD4+ T cells [30, 34, 35]. However, this is an inefficient process owing to several blocks imposed by the cellular environment of resting T cells (including lack of dNTPs needed for reverse transcription, a lack of ATP needed for nuclear import of the viral DNA, as well as a restrictive cortical barrier). Swiggard et al. [34] used spinoculation to efficiently deliver large quantities of replication competent HIV-1 virions to freshly isolated resting CD4+ T cells (a mixture of naïve, TCM and TEM cells) in the absence of any activating stimuli. Integration occurred resulting in latently infected cells, albeit at a much lower frequency than activated CD4+ T cells. Alternatively, Saleh et al. [30] have first stimulated freshly isolated resting CD4+ T cells (naïve/TCM/TEM cells) from uninfected donor blood with the CCR7 (a lymphoid organ homing receptor) ligands, CCL19 and CCL21. These chemokines induce neither CD69 nor CD25 expression, but do increase the susceptibility of resting CD4+ T cells to infection by a replication-competent HIV-1 virus. Interestingly, chemokine-induced changes in the actin cytoskeleton that are involved in cell migration appear to be sufficient to allow HIV-1 integration independently of cell activation [32]. Recent results also showed that memory T cells are more sensitive to chemotactic stimulation,which greatly facilitated HIV-1 infection [93]. Hence, there might be a pathway of direct infection of resting T cells also in vivo during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue. More recently, by using novel reporter viruses, the W. Greene’s group [35] described an improved version of the primary CD4+ T-cell model originally developed in the O’Doherty’s laboratory [34]. The Green’s model allows the study of latency with replication-competent proviruses in all subsets of CD4+ T cells. In addition to the GFP reporter virus that measures the number of cells in which the latent HIV-1 provirus is successfully reactivated, the Green’s lab has created a luciferase-expressing virus that measures overall levels of transcriptional reactivation of latent HIV-1. Importantly, they also generated a novel mCherry-luciferase dual reporter HIV-1 virus to assess simultaneously the number of cells containing reactivated latent provirus in response of a specific inducer (mCherry) and the magnitude of the response within the entire population (luciferase) [35].

In addition to primary CD4+ T cell models, the Collins’s group has generated an ex vivo model for establishing latency in progenitor cells derived from human umbilical cord or bone marrow [94]. CD133+ cells were isolated by magnetic separation and infected with pseudotyped HIV-1 ΔEnv encoding a reporter. Three days after infection, actively infected cells expressing the reporter protein were removed and the remaining cells were resuspended in media with an integrase inhibitor to ensure that increases in reporter gene expression would derive solely from the reactivation of the integrated virus. HIV-1 could establish a latent infection in all of the subsets of HPCs that were examined, including an immature population that includes hematopoietic stem cells and multipotent progenitors. These cells could be reactivated by TNFalpha, SAHA, but not HMBA or 5-aza-2'-deoxycytidine. Although this latter model is preliminary, it appears that differences exist in the establishment of latency in HPCs versus memory T cells that should be analyzed in more detail.

After years when the only experimental models for HIV-1 latency were a series of transformed cell lines carrying an integrated latent provirus, we have entered a new phase with a variety of primary cell-based models. Although it is clear that current research on HIV-1 latency requires such models, the choice of the right system remains difficult. A fundamental issue is that we still don’t know how HIV-1 latently infected resting memory T cells are established in vivo. Either route of infection, direct or after reversal to the quiescent state are plausible and can be recapitulated in vitro. Since each route may impact on the chromatin environment of the integrated virus, it is likely that the results of experiments aimed at understanding the mechanisms of latency and reactivation will be affected. Another non-trivial aspect to consider is the length of the experiment. Ex vivo activation of primary cells, their infection and induction of latency is a lengthy process that takes weeks while direct infection is much quicker. In addition to obvious practical reasons, long term cell culture might induce changes in the chromatin environment of the provirus that do not necessarily mimic the hypothetical in vivo situation. On the other hand, techniques such as spinoculation, where a great quantity of virus is forced to infect a resting cell, are not physiological as well.

To conclude, while these primary cell-based models have several attractive features including the ability to rapidly establish latency in memory CD4+ T cells, it will be important to test each model side-by-side with the others. Only by carefully comparing results from the different models to results obtained with cells isolated from blood of cART-treated HIV-infected patients will it be possible to identify meaningful in vitro models for in vivo HIV-1 latency.

Histone acetylation

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) influence transcription by selectively acetylating or deacetylating the ϵ-amino group of lysine residues in histone tails. Generally, chromatin acetylation by HATs promotes chromatin opening and is associated with active euchromatin, whereas deacetylation by HDACs diminishes the accessibility of the nucleosomal DNA to transcription factors, thereby generating repressive heterochromatin [203]. Moreover, histone acetylation marks enable the recruitment of bromodomain-containing proteins, such as chromatin remodeling complexes and transcriptions factors, which in turn regulate gene expression. The repressive nucleosome nuc-1 is specifically remodeled following PMA or TNFalpha treatment of the cells, coinciding with activation of HIV-1 gene expression [195]. This remodeling includes posttranslational modifications of histone tails. In this context, Van Lint and Verdin reported for the first time 16 years ago that treatment of latently HIV-1-infected cell lines with two HDAC inhibitors (HDACIs), trapoxin and trichostatin A (TSA), induces viral transcription and the remodeling of the repressive nucleosome nuc-1 [196]. These results indicated that one or several HDACs were bound to the HIV-1 promoter under latency conditions and that deacetylation of the HIV-1 promoter chromatin by these enzymes played a role in the establishment and maintenance of HIV-1 latency. This observation also indicated that treatment of HIV-infected patients with HDACIs might be of therapeutic value by forcing the reactivation of HIV-1 expression and the elimination of the latent HIV-1 reservoir (“flushing hypothesis”) [80, 196, 204].

Later experiments revealed that transcriptional repressors recruit HDACs to the HIV-1 promoter (reviewed in [3], Figure 2). Following viral reactivation, acetylation of histones and recruitment of HAT at the HIV-1 promoter have been described by the group of Giacca [205]. To date, numerous studies have demonstrated that HDAC1, HDAC2 and HDAC3 are recruited to the HIV-1 LTR5’ and play an important role in viral latency [206–210]. In microglial cells, Marban et al. showed that the corepressor CTIP2 acts as a recruitment platform for HDAC1 and HDAC2 on the HIV-1 promoter, leading to a heterochromatin environment [207, 211].

Recently, the group of Poli has shown that essential amino acid restriction results in the transcriptional derepression of silenced transgenes including the HIV-1 provirus in T-lymphocytic cells [212]. This derepression correlates with a significant downregulation of HDAC4. These results indicate that HDAC4 behaves as a critical regulator of exogenous transgene expression [212], sensitive to amino acid starvation and suggest that HDAC4 pharmacological inhibition may be necessary to revert HIV-1 transcriptional silencing and lead to reactivation of latent HIV-1. In this context, recently, Xing et al. have provided strong evidence that HDAC6-selective inhibitor M344 is a potent antagonist of HIV-1 latency, acting by increasing the acetylation of histones H3 and H4 in the nuc-1 region of the HIV LTR [213].

HDACIs present several advantages as a potential inductive adjuvant therapy in association with efficient cART to purge latent reservoirs. They activate a wide range of HIV-1 subtypes [204] without the toxicity associated with mass T-cell activation, which would generate new target cells for neo-synthesized viruses. HDACIs have been demonstrated to repress the coreceptor CXCR4 in a dose-dependent manner [214]. They act on a broad spectrum of cell types; and therefore, in contrast to agents that specifically induce T cells, they could target the different latent reservoirs (macrophages, dendritic cells and other non-T cells). The most important element regarding the therapeutic goal resides in the fact that HDACIs have been safely administrated to patients for several years in other human diseases: phenylbutyrate in beta-chain hemoglobinopathies such as beta-thalassemia and sickle cell anemia [215, 216] and VPA in epilepsy and bipolar disorders [217, 218]. More recently, SAHA (marketed as Vorinostat) was approved by the Food and Drug Administration (FDA) for treatment of cutaneous T-cell lymphoma [219]. Givinostat and Panobinostat are currently in Phase I/II clinical trials for relapsed hematological malignancies [220, 221]. Givinostat has been shown to be more potent than VPA in terms of reactivation of HIV-1 expression in vitro[222], while Panobinostat has been shown to be at least 10 times more potent than Vorinostat (reviewed in [223]).

The use of HDACIs as HIV-1 inducers has been well characterized in several latency models and in resting CD4+ T cells from cART-treated HIV-1-infected patients [97, 204, 222, 224, 225]. A series of such molecules has been characterized in terms of HIV-1 reactivation [210, 226, 227]. However, some groups have reported that HDACIs have a lower reactivation potential in a primary latency model [228] or in resting CD4+ T cells from cART-treated HIV-1-infected patients [229] compared to infected transformed cell lines. The first study was performed in a model of primary T cells [228], which might not reflect the in vivo situation. Indeed, a recent clinical trial (see below) has shown that Vorinostat reactivates HIV-1 in infected patients [230]. In the second study [229], the concentration of HDACI used was lower than the common concentration used in the numerous studies that have previously tested the HIV-1 reactivation potential of HDACI [226, 231]. Moreover, their finding indicates rather that HDACIs induce virus production but from a restricted fraction of infected cells, in agreement with the coexistence of different latency mechanisms [98, 99, 232].

More recently, many other HDACIs have been evaluated in vitro for their reactivation potential in latentlyHIV-1-infected cells: ITF2357 [222], CG05 and CG06 [233], NCH-51 [234] and MC1293 [235]. In order to identify new compounds, several groups have performed high-throughput screening. In this context, molecules such as AV6 [236] or a range of Merck compounds [95] have been highlighted. In parallel, another study has demonstrated that butyric acid naturally produced from bacteria could promote gene expression of latent HIV-1 [237]. The effects of these different compounds have yet to be confirmed in ex vivo cultures of resting CD4+ T cells from cART-treated HIV-infected patients.

Latency is a multifactorial phenomenon: different levels of transcriptional and epigenetic blocks are involved and probably act in concert to silence HIV-1 transcription. Attacking simultaneously different levels of latency should be more efficient when viral eradication is the objective since the combination of different types of compounds could synergize in the reactivation of latently infected cells. In this context, Van Lint’s group has reported for the first time that HDACIs and NF-kappaB inducers synergistically reactivate latent HIV-1 [204]. Mechanistically, this synergism was associated with a delayed cytoplasmic reappearance of the inhibitory protein IΚBalpha in response to TNFalpha + TSA versus TNFalpha treatment [238], leading to a prolonged intranuclear presence and DNA-binding activity of NF-kappaB [238, 239]. Next, the same group has shown that the non-tumor-promoting NF-kappaB inducer, prostratin, synergistically reactivates HIV-1 production with HDACIs used in human therapies (such as VPA and SAHA) in several postintegration latency model cell lines as well as in ex vivo cultures of in CD4+ CD25- CD69- HLADR- resting T cells isolated from blood of cART-treated patients with undetectable viral load [97]. Mechanistically, HDACIs prolonged and increased prostratin-induced NF-kappaB activation and provoked, in combination with prostratin, a more pronounced nucleosome remodeling in the HIV-1 promoter. This study constitutes a proof-of-concept for the coadministration of two different types of therapeutically promising HIV-1 inducers together with efficient cART as a therapeutic perspective to decrease the pool of latent HIV-1 reservoirs [97]. However, in 40% of the cultures, this group could not detect any viral outgrowth following treatment with prostratin and HDACIs individually or in combination. This could result from a stronger epigenetic control of some integrated proviruses. Latter, Burnett and colleagues reported that the same combination prostratin + SAHA synergistically reactivates several HIV-1 subtypes including A, B, C, D and F in primary CD4+ and Jurkat cell-based in vitro HIV-1 latency models [240]. Another study has shown similar results with the HDACI MC1293 in a latently infected cell line but did not report testing of this HDACI in ex vivo cell cultures from HIV + patients [235]. Interestingly, another non-tumor-promoting PKC inducer, bryostatin, has been shown to activate MAPKs and NF-kappaB pathways and to synergize with HDACIs to reactivate HIV-1 gene expression in latently infected J-Lat cell lines [241].

Histone methylation

While histone deacetylation generally contributes to transcriptional repression, histone methylation can be either linked to transcriptional repression or activation, depending on the site of modification. Methylation of H3 at lysine residue 4 (H3K4) is associated with transcriptional activation, while methylation of H3K9, H3K27, and H4K20 is associated with transcriptional repression (reviewed in [242]). Histone methylation marks (including H3K9 dimethylation (H3K9me2), H3K9 trimethylation (H3K9me3) as well as H3K27 trimethylation (H3K27me3)) have been shown to be associated with HIV-1 transcriptional silencing in different postintegration latency models [66, 68, 207, 243, 244].

The histone methyltransferases (HMTs) Suv39H1 [243], which is primarily involved in H3K9me3, and G9a [244] which is responsible for H3K9me2, have been demonstrated to play a role in HIV-1 transcriptional silencing (Figure 2). Indeed, Benkirane’s group [243] has shown that Suv39H1, HP1gamma (heterochromatin protein 1) and H3K9me3 mediate HIV-1 repression in different cellular models including PBMCs from HIV+ patients. Moreover, Rohr’s laboratory has previously reported a similar mechanism in in vitro HIV-1-infected microglial cells, where Suv39H1 is recruited to the HIV-1 promoter via the transcriptional corepressor CTIP2 leading to a multi-enzymatic complex including HDAC1, HDAC2 and HP1 [207, 211]. In this context, a study has very recently reported increased CTIP2, HP1, MeCP2 and HDAC1 levels in postmortem brain tissue from HIV+ latent cases, most probably due to posttransciptional mechanisms [245]. In addition, G9a-mediated H3K9me2 can also recruit HP1 and therefore participate in the maintenance of HIV-1 silencing [244]. Results from Ding et al. suggest that the G9a-like-protein (GLP) may also play a significant role in the maintenance of HIV-1 latency by catalyzing H3K9me2 in new clonal cell lines where the provirus is integrated in active gene regions and presented an hypomethylation status [246]. Together, their data suggest that histone methyltransferase inhibitors (HMTIs) could represent promising therapeutic drugs in strategies aimed at purging the HIV-1 latent reservoirs.

Two specific HMTIs have been described so far: chaetocin and BIX-01294. Chaetocin, a fungal mycotoxin from Chaetomium minutum[247], acts as a specific inhibitor of Suv39H1 in a S-adenosylmethionine (SAM)-competitive manner [248]. Chaetocin belongs to 3-6-epi-dithio-diketopiperazines, which have been previously reported to have biologic effects including immunosuppressive [249], anti-inflammatory [250] and/or antiviral [251] activities. BIX-01294, a diazepin-quinazolin-amine derivative, functions as a specific inhibitor of G9a [252] in an uncompetitive manner with SAM by binding the G9a SET catalytic domain [252, 253]. Recently, Van Lint’s group demonstrated that the HMTI chaetocin induces HIV-1 recovery in 50% of the CD8+ depleted PBMCs cultures tested and in 86% of the resting CD4+ T-cell cultures from HIV-1+ cART-treated patients with undetectable viral load [98]. They next confirmed the high reactivation potential of HMTIs using BIX-01294, which induced HIV-1 recovery in 80% of the patient resting memory CD4+ T-cell cultures tested [98]. This study and several other recent studies demonstrate that the specific HMTIs chaetocin and BIX-01294 reactivate latent HIV-1 with minimal effects in different latently infected cellular models compared to the effects observed in patient cells [66, 68, 98, 244]. Moreover, the Van Lint’s group has shown that the combinations chaetocin + prostratin and chaetocin + SAHA causes, in most cases, a higher HIV-1 reactivation than these compounds used alone [98], in agreement with the fact that a combination of two different HIV-1 inducers can act on different mechanisms of latency. They also observed this effect using the combination BIX-01294 + SAHA in a smaller group of patient cell cultures. In conclusion, the Van Lint’s group showed for the first time that HMTIs, alone or in combination with HIV-1 inducers, cause HIV-1 recovery in resting memory CD4+ T cells from cART-treated patients [98]. Although chaetocin and BIX-01294 cannot be safely administered to humans, the latter results constitute a proof-of-concept for the use of HMTIs in strategies aimed at reducing the pool of HIV-1 latent reservoirs. Since HMTIs also represent promising compounds in anti-cancer therapies [254–256], other safer HMTIs should be synthesized soon and evaluated for their reactivation potential in cells from HIV-1+ cART-treated individuals.

Suv39H1-mediated trimethylation requires previous demethylation of H3K4 by Lysine-specific demethylase 1 (LSD-1/KDM1) [257]. The Rohr’s and Van lint’s groups have recently postulated that this histone demethylase may be part of the multienzymatic complex recruited by CTIP2 to the HIV-1 promoter [258]. They have reported that LSD1 functionally cooperates with CTIP2 in a synergistic manner to repress both viral replication and transcription in microglial cells, the main HIV-1 targets in the central nervous system [258]. They also demonstrated that CTIP2 and LSD1 interact by coimmunoprecipitation experiments and colocalize in cells with the HIV-1 Tat protein within heterochromatic nuclear structures by confocal microscopy experiments [258]. Altogether, their results support the idea that LSD1 and CTIP2 cooperate to repress the HIV-1 promoter. Recruitment of LSD1 at the HIV-1 proximal promoter is associated with induction of both H3K4 and H3K9 trimethylation in the U1 monocytic/macrophage cell line. Mechanisms underlying LSD-1-mediated increase in H3K4 trimethylation might rely rather on the ability of LSD1 to recruit hSET1 and WDR5, two members of the hCOMPASS complex, at the promoter than on its own enzymatic demethylase activity [258]. These data suggest that the transcriptional repressor LSD1 constitute a potential target (at least in infected cells of the monocytic/macrophages lineage) to induce HIV-1 latency with histone demethylase inhibitors. In contrast, in latently infected T cells, the LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, was shown to act as an activator of HIV-1 transcription through specific demethylation of K51 in Tat [259]. The two studies thus observed opposite effect of LSD1 in microglial cells versus in T cells. This could be due to cell type-specific differences that should be further investigated.

In addition to H3K9 methylation, the Karn’s laboratory has elegantly demonstrated that the HMT enhancer of Zeste 2 (EZH2, a HMT that is part of the Polycomb Repressive Complex 2, called PRC2), the enzyme responsible for H3K27me3, is present at high levels at the LTR of silenced HIV-1 proviruses and was rapidly displaced following proviral reactivation in T cells (Figure 2[66]). This correlates with H3K27 trimethylation of the HIV-1 promoter in latency conditions, inducing a repressive chromatin structure, and with a decreased H3K27me3 level in activated conditions [66]. Recently, the group of Tyagi has reported that CBF-1 (C-promoter binding factor-1) is responsible for the recruitment of EZH2 and other chromatin-modifying enzymes of the Polycomb complex to the HIV-1 promoter [27]. The complex including EZH2 is very important because it can serve as a binding platform for multiple histone- and DNA-modifying enzymes [260, 261]. Treatment of cells with the broad spectrum HMTI DZNep has shown HIV-1 reactivation of silenced proviruses and a significant inhibition of EZH2 activity in a primary T-cell latency model [66]. However, DZNep is not a specific inhibitor of EZH2 but possesses only a greater inhibitory potency for EZH2 than for other HMTs. Indeed, DZNep is an inhibitor of S-adenosyl-L-homocysteine (SAH) hydrolase and thus inhibits indirectly EZH2 through effects on intracellular SAH concentrations and promotes degradation of the PRC2 complex [262]. EZH2 is also implicated in tumorigenesis and correlates with poor prognosis in several tumor types [263–266]. In this context, a recent study identified specific inhibitors of EZH2 methyltransferase activity by a high throughput biochemical screen [267]. In a linked study, a small molecule of this screen has been optimized resulting in a highly selective, potent, SAM-competitive inhibitor of EZH2, named GSK126 [268]. They demonstrated that GSK126 decreases global H3K27me3 levels and reactivates silenced PRC2 target genes in lymphoma. GSK126 has been also evaluated in mice where it was well tolerated and similar results were observed in cell culture [268]. Therefore, it would be interesting to evaluate the GSK126 molecule for its reactivation potential on latent HIV-1 in infected patient cell cultures.

DNA methylation

In mammalian cells, DNA methylation occurs as 5-methyl cytosine predominantly in the context of CpG dinucleotides and is achieved by the specific recruitment of DNA methyltransferases (DNMTs). DNA methylation in transcriptional regulatory regions is generally associated with gene silencing, either by directly blocking binding of transcription factors to their recognition sequences or by indirectly preventing transcription factors from accessing their target sites through attachment of methyl-CpG-binding proteins (MeCPs) that “read” DNA-methylation patterns. These MeCPs recruit HDACs and HMTs, thereby resulting in formation of a closed repressive chromatin structure (for review [269]). During latency, the HIV-1 promoter is hypermethylated at two CpG islands surrounding the HIV-1 transcriptional start site as demonstrated both in J-Lat cell lines and in the Planelles’s primary T cell model of HIV-1 latency ([270], Figure 2). In J-Lat cells, methyl-CpG binding domain protein 2 (MDB2) and HDAC-2 are recruited to the promoter via the second CpG island [270]. Treatment with 5-aza-2’deoxycytidine (5-Aza-CdR, decitabine) decreases cytosine methylation in the two HIV-1 CpG islands, resulting in loss of MBD2 and HDAC-2 from CpG island 2 of the viral promoter region and in partial transcriptional reactivation [270]. The group of Hirsch compared the CpG dinucleotide methylation pattern of the HIV-1 promoter in aviremic versus viremic HIV-1-infected individuals by bisulfite sequencing of DNA from memory resting CD4+ T cells [271]. Analysis of the CpG methylation profile has shown that the HIV-1 5’LTR in 6 patients without detectable plasma viremia contain from 19% to 100% of methylated CpG compared with less than 0.1% for the control group of viremic patients. These results indicate that HIV-1 promoters in the long-term cART-treated aviremic individuals are hypermethylated, in contrast to non-methylated promoters of viremic patients [271]. However, more recently, J. Blazkova now in Fauci and Chun’s laboratory reported novel contrasting results. Indeed, they examined the methylation status of the first CpG island by bisulfite sequencing in resting CD4+ T cells from 11 aviremic patients and did not find significant 5’LTR methylation in any of them [272]. They observed a median frequency of methylated CpG dinucleotides within the HIV-1 5’LTR of 2.4% (range 0-10%) [272]. Both groups used the same technique of DNA methylation analysis and the same system of controls based on Jurkat clones. The major sources of differences between both studies could originate from variability in patients characteristics and cART treatments. The patients from the Hirsh’s study with the most heavily methylated 5’LTRs were long-term infected individuals (infected for 11, 12 and 16 years, with a median of 10 years of ART). Some of them were treated by inefficient therapy at the beginning (see Supplementary Table 3 in [271]). In contrast, the longest treatment period in the Fauci’s study was 6.6 years with a median of 2.9 years. Another difference between both studies is the population of analyzed CD4+ T cells: HIV-1 proviruses analyzed in the Hirsh’s study were extracted from memory CD4+ T cells, while HIV-1 proviruses analyzed in the Fauci’s paper were obtained from resting CD4+ T cells. More stringent mechanisms of maintenance of HIV-1 latency, including CpG methylation, may be needed in the memory CD4+ T cells, which are more prone to proliferate than the resting CD4+ T cells. Since DNA methylation may be a late event that enhances silencing of already-latent viruses rather than contributing to entry into latency, the time of infection in each patient may be an important factor contributing to the differences between both studies. Indeed, substantially longer period of selection of latent proviruses in the memory cell population [271] can result in accumulation of HIV-1 proviruses with methylated promoters in comparison to non-methylated HIV-1 proviruses harbored in the shorter-term-selected population of resting cells [272]. Very recently, Palacios et al. have compared the methylation state of the HIV-1 promoter in PBMCs from two group of infected patients: 1) long-term nonprogressors and/or elite controllers (the LTNP/EC group) who maintain undetectable or low levels of viremia (< 50 or 2000 RNA copies/ml, respectively) without treatment, and 2) aviremic patients in whom cART results in undetectable plasma viremia. Palacios et al. have shown that the 5’LTR of HIV-1 in all aviremic patients exhibited very low or no methylation at all (range of 0 to 1%) compared with that the LTNP/EC group, where the percentage of methylated CpGs in the HIV-1 promoter was higher (median of CpG methylation of 5.4%). However, it is difficult to compare this latter study with the two previous ones [271, 272] because bisulfite analysis was performed on total PBMCs (containing other infected cells such as monocytes in addition to the CD4+ T cells) instead of memory CD4+ T cells. Palacio et al. have observed a weak 5’LTR DNA methylation level in the LTNP/EC group, thereby strengthening the hypothesis that DNA methylation is an epigenetic mark associated with certain forms of latency. Evidently further studies will be necessary to clarify the role of DNA methylation in HIV-1 latency in vivo.

Various anticancer agents including 5-aza-2'deoxycytidine (5-Aza-CdR), an FDA-approved inhibitor of DNA methylation used in humans to treat myelodysplastic syndrome (marketed as Decitabine) [273], were shown to induce HIV-1 transcription in latently infected cell lines [274] and in a doxycycline-dependent HIV-rtTA variant [76]. However the role of DNA methylation in HIV-1 latency is still controversial, and some laboratories have even reported that CpG methylation did not correlate with transcriptional silencing [275]. In addition to the four well-characterized nucleoside analog methylation inhibitors (i.e. 5-azacytidine (5-Aza), 5-Aza-CdR, 5-fluoro-2'-deoxycytidine, and zebularine), there is a growing list of non-nucleoside DNA methylation inhibitors such as procaine, procainamide, hydralazine and RG108 [276]. Only 5-Aza and 5-Aza-CdR are currently FDA-approved and used in cancer therapies. The major hindrance of their usage in humans is their instability in vivo and the toxicity secondary to their excessive incorporation into DNA, which causes cell cycle arrest. These cytosine analogs have also been demonstrated to induce proteasomal degradation of DNMT1 [277]. Today, no clinical trial with HIV-1-infected patients has been performed using DNA methylation inhibitors to reduce the pool of latent reservoirs, but it could an interesting approach.

In addition to their DNA methylation inhibitory activity, 5-Aza and 5-Aza-CdR were reported to exhibit another potential advantage in the context of strategies aimed at curing HIV-1 infection. Indeed, it was reported that the primary antiviral mechanism/activity of 5-Aza (after reduction to 5-Aza-CdR) and of 5-Aza-CdR could be attributed to their ability to increase the HIV-1 mutation rate through viral DNA incorporation during reverse transcription [278]. This results in decreased HIV-1 replication and infectivity through lethal mutagenesis. Clouser et al. have shown that a combination of two clinically approved drugs, decitabine and gemcitabine (2'-deoxy-2',2'-difluorocytidine, a nucleoside analog also presenting ribonucleotide reductase inhibitory activity), reduced HIV-1 infectivity by 73% at concentrations that had minimal antiviral activity when used individually [279]. Recently, the same group has shown that 5-Aza-CdR in combination with gemcitabine, a FDA-approved drug, inhibited disease progression at doses that were not toxic in murine AIDS model, as detected by histopathology, viral loads, and spleen weights [280]. Resveratrol (a ribonucleotide reductase inhibitor) was also shown to inhibit HIV-1 infectivity in combination with 5-Aza-CdR [281].

Combinations of a DNA methylation inhibitor with a HDACI has been previously used in clinical trials as anticancer treatments. A combination 5-Aza-CdR/VPA has been tested in latently infected cell lines but failed to synergistically reactivate HIV-1 transcription [270]. However, in the same experiment, the authors showed that inhibiting provirus methylation led to an almost complete reactivation of latent HIV-1 in J-Lat T cells when combined with the NF-kappaB signaling activator TNFalpha [270]. In another study, 5-Aza-CdR was also shown to synergize with prostratin, which triggers reactivation of latent HIV-1 without broad T-cell activation and inhibits de novo virus infection [144]. Fernandez and Zeichner have shown that 5-Aza-CdR plus TNFalpha activates HIV-1 at least twice as well as TNFalpha alone in almost all J-Lat cell lines tested but the J-Lat 10.6 cell line, in which TNFalpha plus 5-Aza-CdR moderately decreases activation compared to the activation observed after treatment with TNFalpha alone [282]. Surprisingly, 5-Aza-CdR decreased TNFalpha-induced activation of HIV-1 gene expression to an even greater extent in the latently infected cell lines ACH2, J1.1 and U1 than in the J-Lat 10.6 cell lines [282]. In certain cell lines, the authors also observed that 5-Aza-CdR decreases induction of viral expression by the HDACI TSA [282]. These differential responses to epigenetic inducers observed in the various cell lines may be due, in addition to the direct effect of the epigenetic drugs on LTR demethylation, to different indirect effects on cellular genes that directly or indirectly inhibit HIV-1 transcription. Recently, the Van Lint’s group has shown that 5-Aza-CdR in combination with the HDACI SAHA synergistically induces HIV-1 gene expression in several J-Lat cell lines (Bouchat and Van Lint, unpublished data). The reactivation potential of 5-Aza-CdR is currently tested in combination with others HIV-1 inducers in ex vivo cultures of resting CD4+ T cells from cART-treated HIV-infected patients with undetectable viral load (Bouchat and Van Lint, unpublished data). The use of DNA methylation inhibitors coupled with a PKC agonist or with an HDACI could be a further step to purge the latent reservoirs in cART-treated patients.

Tat and P-TEFb

The HIV-1 promoter is a widely used model for mammalian RNAPII elongation control and has provided several insights in the general mechanism of cellular transcription. In the absence of the viral transactivator Tat, basal transcription from the long terminal repeat (LTR) leads to RNAPII pausing after synthesis of a short RNA that includes TAR. The negative elongation factors NELF and DSIF induce RNAPII pausing on the promoter but few molecular details of this process are known (reviewed in [288]). Kiernan and collaborators recently proposed that also RNA contributes to pausing [289]. The recruitment of the Microprocessor complex, Rrp6 exoribonuclease and of the termination factors Setx and Xrn2 induces the cleavage of the short TAR hairpin leading to premature termination by an RNAi independent mechanism. This mechanism is active also on a subset of cellular genes reinforcing the notion that post-initiation events are a hallmark of cellular gene regulation. Indeed, genome-wide mapping studies revealed promoter-proximal pausing of RNAPII on a large proportion of human genes (ENCODE Transcriptome Project, 2009; [290–292]).

Tat, together with P-TEFb, binds the stem loop within TAR, allowing cdk9 to phosphorylate the RNAPII carboxyterminal domain (CTD) and the negative transcription elongation factors NELF and DSIF, licensing RNAPII for productive elongation (Reviewed in [293, 294]). Tat recruits Cyclin T1/cdk9 either from the inactive complex containing the 7SK snRNA, HEXIM1 (or his homologue HEXIM2) (Figure 2), the La-related protein 7 (LARP7), and the 7SK-specific methyl-phosphate-capping enzyme (MePCE) or from the Cyclin T1/cdk9 complex containing the bromodomain-containing protein 4 (BRD4) active on cellular genes. However, other Cyclin T1/CDK9-associated protein complexes that negatively modulate Tat activity have been recently identified [295, 296]. Frankel’s group reported the unexpected findings that Tat assembles into a complex with P-TEFb in its inactive 7SK snRNP form and that this complex is recruited to the HIV-1 promoter before transcription initiation in a TAR-independent manner. Once transcription begins, the nascent TAR hairpin is synthesized and is required to displace the inhibitory 7SK snRNP complex and activate the P-TEFb kinase, which occurs when Tat and CycT1 bind to TAR [297].

Recent advances in live cell imaging allowed direct measurements of RNA biogenesis from the HIV-1 promoter exceeding 50 kb min^-1[298]. The studies of Marcello and collaborators demonstrate that Tat-mediated HIV-1 transcription is highly efficient and able to produce a large amount of pre-mRNA in a short time [299]. Tat is such an effective elongation factor possibly because it is able to recruit the “super elongation complex” (SEC) in addition to P-TEFb. Independent work by the Benkirane and Zhou laboratories using an affinity-purification strategy led to the identification of ELL1 and its homolog ELL2, AFF1 and its homolog AFF4, ENL, AF9 and components of the polymerase-associated factor complex (PAFc) as SEC components of the Tat/P-TEFb complex [300, 301]. ELL1 and ELL2 are well-characterized transcription elongation factors that stimulate the processivity of RNAPII and act in concert with P-TEFb. The SEC was also independently identified associated to the mixed-lineage leukemia (MLL) protein to promote expression of MLL-dependent genes [302]. However, SEC components ENL/AF9/AFF4 interacting with PAFc/RNAPII are able to promote elongation of genes independently of Tat/MLL and were shown to increase HIV-1 basal transcription [303]. Control of SEC by ubiquitination and its reactivation by prostratrin and HMBA (Hexamethylene bisacetamide) represents a novel avenue for developing strategies to control HIV-1 transcription.

HMBA, a hybrid bipolar compound, is a clinically tolerable agent, which was first developed as an anticancer drug [304, 305]. HMBA was previously shown to reactivate viral production in chronically infected cell lines [306, 307]. HMBA activated transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of P-TEFb from its transcriptionally inactive complex with HEXIM1 and 7SK snRNA [308]. Moreover, HMBA triggers Cdk9 recruitment to the HIV-1 5'LTR via an unexpected interaction with the transcription factor Sp1, resulting in stimulation of transcription elongation and viral production [309]. HMBA was shown to induce HIV-1 gene expression in the latently infected T-lymphoid (ACH2, Jurkat cells) and U1 monocytic cell lines and in resting CD4+ T cells isolated from PBMCs which were infected in vitro with HIV-1_LAI[308]. Moreover, Klichko et al. have reported that treatment with HMBA downregulates expression of CD4 in PBMCs, but does not alter expression of the HIV-1 co-receptors CXCR4 and CCR5 [310]. However, unlike mitogen activation, HMBA did not increase cell susceptibility to HIV-1 infection or the expression of cell surface markers of activation [310].

In order to affect the P-TEFb equilibrium, five independent studies have very recently reported that JQ1 (an inhibitor of BET bromodomain BRD4 [311]) is able to reactivate latent HIV-1 [312–316]. The most recent study has also shown that 3 other BRD4 inhibitors (I-Bet, I-Bet151 and MS417) reactivate HIV from latency [316]. The bromodomain containing protein BRD4 and its inhibition of Tat-transactivation is a major impediment to latency reactivation [313]. BRD4 competitively blocks the Tat-SEC interaction on the HIV-1 promoter [313]. The BET bromodomain inhibitor JQ1 dissociates BRD4 from the HIV-1 promoter to allow Tat recruitment of SEC to stimulate HIV-1 elongation. Others studies have shown that JQ1 transiently increases levels of free P-TEFb and BRD4/P-TEFb and SEC/P-TEFb complexes in cells [312]. Indeed, although primarily thought of as an epigenetic modifier, JQ1 also affects the P-TEFb equilibrium directly by removing P-TEFb from the 7SK snRNP or by an indirect effect via stress, as the structure of chromatin changes upon release of BRD4. The BRD4 loss enhances HIV-1 gene expression by increasing Tat/P-TEFb association and RNAPII transcriptional elongation [315]. Importantly, JQ1 was shown to reactivate latent HIV-1 in several cellular models for postintegration latency: in T lymphoid latently infected cell lines [312–315], in the latently infected U1 monocytic cell line [315], in a primary T-cell model of HIV latency [316], and in ex vivo cultures of patient cells [314, 315]. The Montano’s group has reported HIV-1 recovery in one of the three resting CD4+ T cell ex vivo cultures prepared from HIV-1-infected cART-treated patients [314]. The Brass’s group has tested the reactivation potential of JQ1 in ex vivo cultures of CD8+ depleted PBMCs isolated from 19 cART-treated patients with undetectable viral load and has shown that JQ1 alone does not induce HIV-1 recovery but potentiates the actions of several known HIV-1 inducers (PHA, PMA, TNFalpha, prostratin) [315]. This could be explained by the very low level of Cyclin T1 in resting T cells. Li et al. also have reported that JQ1 potentiates HIV-1 reactivation by prostratin and PHA in the J-Lat A2 latent cell line [313]. In addition to BRD4, the study of Ott and Verdin has also identified BRD2 as a new Tat-independent suppressor of HIV-1 transcription in latently infected cells and underscored the therapeutic potential of BET inhibitors in the reversal of HIV latency [316].

Tat itself can be a target for therapeutic intervention at various levels [317, 318]. Recently, the first crystal structure for Tat bound to P-TEFb was reported [319]. This structure resolved at 2.1 angstroms, reveals intimate contacts between Tat and the T loop of cdk9 and marked interactions of Tat and with cyclin T1. This structure could lead to the rational design of a new class of HIV-1 inhibitors.

The “tat code”

The interaction promiscuity of Tat cannot be explained by a single peptide. Similar to the “histone code” [320] that has been proposed to explain how histones interact with several chromatin associated factors, which can activate or repress transcription, Tat can be posttranslationally modified at various residues, thereby creating a variety of isoforms with specific functions, the “Tat code”. Phosphorylation of Tat was the first modification identified, but there has been little follow up on is role in Tat activity. Lysine acetylation has been described at Lys28 allowing high-affinity binding to P-TEFb [321, 322]. Lys28 acetylation is reversed by HDAC6 [323]. Acetylation at Lys50/51 leads to the dissociation of Tat from TAR and promotes the association of the bromodomain-containing p300/CBP associated factor (PCAF) acetyltransferase [321, 322, 324–327]. Lys50 acetylation generates an interaction surface also for the SWI-SNF chromatin-remodeling complex [328]. Deacetylation of Tat is mediated by SIRT1 and possibly controls a late step in Tat transcriptional activity, allowing the recycling of the deacetylated protein for next rounds of transcription [329]. Lys51 is also methylated by the monomethyl-transferase Set7/9 and demethylated by the action of LSD1 [259, 330]. Interestingly, both activities are required for Tat function, possibly controlling different steps of process. To this end, the dwell time calculated by fluorescence recovery after photobleaching (FRAP) of the various components present at the HIV-1 transcription site showed that Tat and Cdk9 behave similarly while RNAPII remains on the gene for a longer time required to reach the end of the gene [331, 332]. These data support the idea that Tat and P-TEFb remain associated in the elongating complex, rather than Tat alone being transferred to the elongating polymerase, but that the complex dissociates from RNAPII before termination of transcription. Finally, polyubiquitination of Lys71 mediated by the proto-oncoprotein Hdm2 does not induce proteasome-dependent degradation but has an activating effect on Tat [333]. Tat interacts also with the 19S PAAF1 component of the proteasome to increase transcription in a non-proteolytic mode [334]. However, the role of Lys71 polyubiquitination in this pathway of Tat activation has not been established yet. Since posttranslational modifications are critical for Tat activity, they represent targets for antiviral therapy. For example, the structure of the Tat/PCAF interaction is being explored for the development of antiviral compounds [335–337].

Recruitment of the SWI /SNF chromatin-remodeling complex to the HIV-1 promoter

Integrated HIV-1 is embedded into host chromatin and shows a characteristic histone signature with the positioning of nuc-1. Remodeling of histones is required for transcription (see above). This could be achieved by specific posttranslational modification of histone tails, such as acetylation and methylation, and by remodeling complexes that require ATP to function. The SWI/SNF family complexes contain the Brahma (BRM) or homologue BRG1 ATPases together with several other components. Two distinct SWI/SNF complexes have been described: BAF and PBAF. The defining subunit of the BAF complex is BAF250a/ARID1a, and those of the PBAF complex are BAF180, BAF200, and BRD7. SWI/SNF complexes participate in the activation of HIV-1 transcription. Kashanchi and collaborators showed that Tat activates HIV-1 transcription in the G1/S phase of the cell cycle [338]. Acetylated Tat binds BRG1 and both are recruited to the 5’ LTR. BRG1 knockdown in latently infected cell lines, such as U1 and ACH2 stimulated with TNFalpha, result in a significant decrease of viral gene expression. Consistently, Verdin and collaborators have shown that knockdown of the core SWI/SNF components integrase interactor 1 (INI-1/SNF5) and BRG-1 suppresses Tat-mediated transactivation and that Tat BRG1 interaction requires Lys50/51 acetylation [328]. They also demonstrated that BRG1 and INI1 cooperate with the p300 coactivator and acetyltransferase to synergistically activate the HIV-1 LTR. The Emiliani’s laboratory demonstrated that BRM is required for proper Tat-mediated activation of the HIV-1 LTR [339]. They showed that Tat fails to transactivate the 2037 integrated LTR if BRM is depleted by siRNA in HeLa cells. Furthermore, acetylated Tat is required for the interaction with BRM. More recently, it has been reported that establishment and maintenance of HIV-1 latency requires the BAF complex, which helps positioning of the repressive nucleosome-1 immediately downstream of the HIV-1 transcriptional start site [340]. Depletion of BAF-specific subunits results in de-repression of HIV-1 latency concomitant with the loss of nuc-1. Upon transcriptional activation, BAF is lost from the HIV-1 promoter, while the PBAF complex is selectively recruited by acetylated Tat to facilitate LTR transcription. Thus, BAF and PBAF, recruited during different stages of the HIV-1 life cycle, display opposing functions on the HIV-1 promoter.

The involvement of SWI/SNF in HIV-1 is more complex since it may involve other mechanisms beyond transcription. The core SWI/SNF component INI1/SNF5 was originally identified as an interactor of HIV-1 integrase [341]. It has been proposed that by interacting with IN, INI/SNF5 interferes with early steps of HIV-1 infection [342]. More recently, it has been shown that HIV-1 integration requires nucleosome remodeling at the integration site and that this activity is mediated by the interaction of INI1/SNF5 with the viral integrase [119]. Finally, an intriguing observation by the Trono’s laboratory, which has not been investigated further, indicated that incoming HIV-1 pre-integration complexes trigger the nuclear export of INI1/SNF5 and of the nuclear body component PML (TRIM19) [343]. The HIV-1 genome appears to associate with these proteins before nuclear export. PML is sequestered in the nucleus in the presence of arsenic while the INI1/pre-integration complex interaction is disrupted and the efficiency of HIV-1 infection is increased [343]. These data suggest a repressive role for INI-1/hSNF5 in HIV-1 transcription.

The “Berlin patient”

A breakthrough in HIV-1 research is the first case of a possible sterilizing cure, “the Berlin patient”. This HIV-1+ patient was treated for acute myeloid leukemia with multiple rounds of chemotherapy and hematopoietic stem cell transplantations from a donor with genetic HIV-1 resistance, a CCR5delta32 homozygous [374]. It is well established that homozygosity for a naturally occuring 32-bp deletion in the chemokine receptor CCR5, the major coreceptor for HIV-1 [375], provides resistance against HIV-1 acquisition [375–382]. The patient stopped cART the day prior to transplant and over 5 years later has not had a rebound in viremia or other indications of viral replication [383]. However, after this intervention, in vitro studies demonstrated that the donor-derived cells were susceptible to infection by X4 virus, but X4 variants have not emerged in vivo[384]. This cleverly designed strategy out of fortunate circumstances led to a patient who remained without viral rebound long after transplantation and discontinuation of antiretroviral therapy. This case has renewed hope for gene therapy in order to specifically eliminate or reduce the expression of HIV-1 coreceptor CCR5. More recently, a recent report has shown that two patients under cART with relapsed Hodgkin’s lymphoma who received a CCR5 −/+ haemopoetic stem-cell transplant, had undetectable proviral DNA and replication-competent HIV 8–17 several months after transplantation (Henrich et al. XIX International AIDS Conference, Washington DC, USA, 22–27 July 2012).

Gene therapy

Gene therapy has been first proposed as a treatment for HIV-1 infection in 1988 by David Baltimore with the term “intracellular immunization” [385]. Several approaches of gene therapy have been proposed in the past. Recently, RNA based strategies (ribozymes, antisense RNA, small-interfering RNA) or protein-based strategies such as zinc-finger nucleases have been implemented (reviewed in [386, 387]). In brief, hematopoetic cells from patients were either transduced ex vivo with a retroviral vector carrying a gene for a ribozyme targeting viral RNA encoding the viral proteins Tat and Vpr [388] or transfected with a vector delivering a ribozyme targeting the CCR5 co-receptor, a short-interfering RNA targeting expression of the HIV-1 proteins Tat and Rev, and a RNA decoy to Tat [384]. The hematopoetic cells or CD4+ T cells can be also treated with a zinc-finger nuclease that is designed to specifically eliminate or reduce expression of the HIV-1 coreceptors CCR5 or CXCR4 (reviewed in [8]). The zinc-finger nuclease can be delivered by adenoviral or retroviral vectors or by nucleofection. These genetically modified cells were then transfused back into the autologous donors. Recently published clinical trials of gene therapy in HIV-1-infected patients are summarized in [8], Table 1.

Elite controllers and VISCONTI patients

Several examples of “natural” functional cure exist. Indeed, a group of rare HIV-1-infected patients spontaneously control HIV-1 replication in absence of cART for prolonged periods of time. These patients called “elite controllers” carry a specific HLA polymorphism and show an effective cytolytic CD8+ T-cell response and an enhanced activity of myeloid dendritic cells [390, 391]. This group represents an imperfect model for a functional cure because their status is linked to a precise genetic background that cannot be reproduced therapeutically and also because they still show disease progression [390]. However, the study of genetics, viral and immune characteristics of elite controllers can teach us a lot about the criteria to be taken into account to achieve a transposable functional cure in naïve or chronic HIV-1 infected patients. In this context, several studies have shown that the reservoir of elite controllers is significantly smaller with low concentration of HIV-1 DNA in different subsets of blood cells and in tissues (reviewed in [390, 391]). The reservoirs of HIV-1 establish early in the infection. Consequently, early treatment may be a potential strategy to reduce or even control the reservoir and to allow preserving immune response. In addition, rare HIV-1-infected patients acquire a controller status after prolonged cART initiated in the acute phase [392, 393] or, even more rarely, in the chronic phase [394, 395]. Treatment initiation during primary HIV-1 infection rather than during chronic HIV-1 infection may: i) further reduce residual viral replication [396], ii) limit viral diversity [397] and viral reservoirs [398], iii) preserve innate immunity and T/B cell functions [399–401], and iv) accelerate immune restoration [402]. Moreover, studies also show that CD4+ T cell counts are higher and that viral rebound occurs later (and at a lower level) after the discontinuation of treatment that began during primary infection compared with treatment that began during chronic HIV-1 infection [403, 404]. In this context, a cohort, called VISCONTI (for Viro-Immunological Sustained Control after Treatment Interruption), is ongoing where patients have been treated with prolonged therapy that was initiated in acute HIV-1 infection and maintain both undetectable plasma viremia and low cell-associated DNA levels despite cART interruption for several years [405]. These individuals are called post-treatment controllers (PTCs). Importantly, a recent and elegant study from the same group has characterized a group of 14 PTCs [406]. These authors found that most PTCs were readily distinguishable from spontaneous HIV controllers in many respects (severe primary infection, unfavorable HLA genotypes, low CD8+ T cell activation status). They have highlighted that the control of viremia following treatment interruption was associated with very low HIV-1 blood reservoirs in the PTCs. Interestingly, five PTCs experienced a progressive decline in their viral reservoir after treatment interruption, which is one of the goals in the search for an HIV cure [406]. Akey additional element might be a low reservoir distribution in cell subsets with long lifespan as naïve and central-memory T cells. They showed that the cell subsets of all the PTCs analyzed ex vivo carried very low levels of HIV-1 DNA [406]. These results argue in favor of early cART initiation and open up new therapeutic perspectives for HIV-1-infected patients. However, arguments against cART initiation during primary HIV-1 infection include the potential for long-term toxicity, the development of resistant viruses and the cost. A low reservoir and a boosted immune response seem to be the two requisite criteria to allow a control of HIV-1 by the host immune system.

Reactivation of latent reservoirs of HIV-1

Early but unsuccessful attempts at purging the latent pool of infected cells were performed using IL-2 and other mitogenic stimuli that mimicked T cell activation (reviewed in [3]). Initial attempts to reduce the frequency of latently infected CD4+ resting T cells in patients on cART have often involved immune activation therapy approaches. These studies were based on the assumption that activation of the latent viral reservoir would result in rapid cell death due to HIV-induced cytopathic effects and that the infections virus released by these activated cells would be contained by the administration of cART. The rational for this is that eradication of latent reservoirs might be feasible through global cellular activation (by using for example IL-2, alone or in combination with an anti-CD3 monoclonal antibody (OKT3) [81, 145, 407]). While these therapies significantly reduced latently infected cell numbers, a rebound of plasma viremia still occurred several weeks after cART treatment interruption. Moreover, induction of generalized immune activation is not really desirable in purging strategies because it leads to severe side effects in patients and produces an abundance of activated target cells for the neo-synthesized viruses. However, this latter problem may be partially resolved by intensification of cART during the treatment period.

There is now interest in the cytokine IL-7 since it is more effective in reversing latency than IL-2 via the JAK/STAT5 signaling pathway [145]. Moreover, IL-7 has been shown to reactivate HIV-1 in primary T cells and seems well tolerated in HIV-infected patients [83, 88]. The administration of IL-7 in cART-treated patients was associated with a small increase or “blip” in plasma HIV-1 RNA [408, 409]. To determine whether IL-7 reduces latent HIV-1 reservoir, a clinical trial, called ERAMUNE01 directed by C. Katlama (ClinicalTrials.gov NCT01019551) is ongoing with HIV-1-infected patients receiving IL-7 in combination with an intensification of cART (Table 1). The outcomes of this trial could help in the development of new and important therapies for HIV-1 eradication, even though some concerns were reported regarding the use of IL-7. Indeed, IL-7 drives homeostatic proliferation of memory T cells and may potentially expand not only uninfected cells, but also latently infected cells by inducing proliferation of all cells, specifically transitional memory T cells [12]. IL-7 has recently been shown unable to reduce the reservoirs size in a model of HIV-1 latency [89] or to reactivate the latent viral pool in patients receiving a single dose to increase their CD4+ T cell count [410]. Therefore, the results of the ERAMUNE01 clinical trial will be of great interest.

Up-regulating cellular transcription to induce HIV-1 gene expression has been proposed as another strategy for reducing latent HIV-1 reservoirs. Recent studies have identified individual compounds that are capable of reversing HIV-1 latency without T-cell activation (described in section 6). Among those, HDACIs are the most studied in anti-HIV-1 clinical trials. In 2005, the Margolis group used VPA, a relatively weak and non-toxic HDACI, in a small pilot study in which a modest decline of the latent reservoir size was observed in three out of four patients [411]. However, latter studies have failed to demonstrate any benefit from VPA in reducing the number of latently infected resting CD4+ T cells [232, 412–414]. Most recently, Routy et al. confirmed that adding VPA to stable cART does not reduce the latent HIV-1 reservoirs in virally suppressed patients [415]. This latter multicentre randomized clinical study based on 56 virologically suppressed patients has shown no significant reduction in the frequency of CD4+ T cells harboring replication-competent HIV-1 after 16 and 32 weeks of VPA therapy 500 mg twice a day [415]. A far more potent HDACI, vorinostat (SAHA), has significant potency in promoting HIV-1 replication in vitro from latently infected cells [224, 225]. Vorinostat is licensed for the treatment of cutaneus T-cell lymphoma and relatively well tolerated in humans. Vorinostat has already entered two clinical trials to evaluate its effect on latent infection in HIV-infected patients: one directed by S. Lewin (ClinicalTrials.gov NCT01365065) at the Alfred Hospital, Melbourne, Australia and the other by D. Margolis (ClinicalTrials.gov NCT01319383) at the University of North Carolina, Chapel Hill, USA (Table 1). The first study currently evaluates the effect of vorinostat at 400 mg per day for 14 days, with a primary end point of change in cell-associated unspliced RNA in CD4+ T cells isolated from both blood and rectal tissue. Results from this study should be available in the next 1–2 years (reported in [226]). In the second study published in July 2012, the Margolis’s group reported that administration to cART-treated patients of a single clinically tolerable dose of vorinostat disrupts HIV-1 latency in blood resting CD4+ T cells. Briefly, these authors first performed validation assays by purifying the resting CD4+ T cells from 16 patients in whom viremia was fully suppressed by cART and by testing the reactivation potential of vorinostat in these ex vivo cultures. Following a 6 h vorinostat exposure, HIV-1 RNA expression and chromatin acetylation level were significantly up-regulated in 11 of the 16 patient cell cultures [230]. Of the 11 eligible/responder patients, 8 patients agreed to receive a single 200 mg dose of vorinostat, followed by a 400 mg dose at 4- to 5-weeks later. In all 8 patients, this exposure resulted in a significant increase in acetylation of total cellular histone H3, temporally associated with an increased (mean increase of 4.8 fold) expression of unspliced HIV-1 gag RNA within resting CD4+ T cells [230]. These results demonstrated that, at least for a period of time in some infected cells, all of the restrictions that limit the expression of latent proviral genomes have been overcome. In contrast, in this trial, the authors observed neither an alteration of residual low-level viremia nor a substantial reduction in the frequency of replication-competent HIV-1 within resting CD4+ T cells [230]. These latter results might reflect that multiple doses of vorinostat, reactivation/anti-latency combination approach and/or additional interventions are required to clear infected cells. Recently, a pilot clinical trial has started with the HDACI panobinostat (called also LBH589) directed by L. Østergaard (ClinicalTrials.gov NCT01680094) at Aarhus University Hospital, Aarhus, Denmark. The primary outcome measure is the change from baseline in HIV transcription in latently infected CD4+ T-cells as quantified by copies of unspliced HIV-RNA in the CD4+ T-cells of HIV-infected patients on suppressive cART. These issues are important goals for future studies as well as the development of HDACI with improved specificity, potency and safety profiles for the selective targeting of latent proviral genomes.

Romidepsin (called also FK288), another HDACI approved by the FDA for the treatment of cutaneous T cell lymphoma, has been shown to be one thousand fold more potent then SAHA at inducing latent HIV-1, one thousand to twenty thousand fold more potent in inhibiting HDAC enzymatic activity and induced HIV RNA expression ex vivo in 12 of 13 HIV + cART-treated patients (Geleziunas et al., 2012, abstract Retrovirology, 9 (Suppl1):O2). The average increase in viral RNA was approximately 8.6-fold (Geleziunas et al., 2012, abstract Retrovirology, 9 (Suppl1):O2). Therefore, the HDACI Romidepsin may be a very promising latent HIV-1 inducer in vivo and its favorable toxicity profiles could allow multiple-dose treatment. A clinical trial is ongoing with Romidepsin and the results are awaited with high interest. Of note, it would be very important to measure the size of the reservoirs during the course of Romidepsin administration in the HIV-1+ patients enrolled in this trial.

PKC inducers also present several advantages in the context of anti-HIV-1 purging strategies (described in section 6.2). In this context, bryostatin, the only clinically available PKC agonist [241] is currently tested with HIV-1-infected patients in a pilot clinical trial directed by S. Moreno, Department of Infectious Diseases, Ramon y Cajal Hospital, Madrid, Spain (Table 1). This ongoing trial evaluates the latent cell reservoir size, residual viremia and cell associated viral RNA after a single dose of bryostatin-1 and results are yet unavailable (Moreno S, 2012, abstract Retrovirology, 9(Suppl 1):I16).

Disulfiram (DSF), a safe and well tolerated FDA-approved drug to treat chronic alcoholism (described in section 6.2), is currently evaluated in a pilot clinical trial (ClinicalTrials.gov NCT01286259) under the direction of S. Deeks at the University of California, San Francisco, USA to assess whether a decrease in the frequency of resting CD4+ T cells harboring replication-competent HIV-1 occurs. This pilot clinical trial involved 14 patients on cART with plasma viral loads below 50 copies HIV-1 RNA/ml of plasma. DSF was administrated for two weeks and the frequency of latently infected cells as well as residual viral viremia was measured during disulfiram administration. Preliminary results from this trial revealed that disufiram administration resulted in a small transient increase in viral load in some trial participants (Spivak et al., 2012, Abstract 19th Conference on Retrovirus and Opportunistic Infections. Seattle, March 5–8). No significant change in the size of the latent reservoir was observed at these intervals.

The hypothesis was that pharmacological reactivation of latent HIV-1 would cause death of the infected cells by viral cytophatic effects or host cytolytic T lymphocytes (CTLs), thereby decreasing the size of the latent pool. Indeed, an overarching question is whether the increases in viral RNA induced either by these compounds will be sufficient to raise the amount of viral protein expression in reactivated cells to levels that lead to viral cytopathicity and/or elimination by cytotoxic T cells. In this regard, the group of Siliciano has recently reported that virus reactivation with vorinostat occurs in latently infected primary CD4+ T cells generated in vitro, but is not associated with death induced directly by viral protein, i.e. viral cytopathic effect [416]. Moreover, unstimulated HIV-specific CTL from cART-treated patients also fail to kill autologous latently infected CD4+ T cells after latent viruses are reactivated [416]. In contrast, antigen-specific stimulation of patient CTLs prior to virus reactivation leads to rapid killing of latently infected vorinostat-reactivated cells [416]. Moreover, monocytes-macrophages and myeloid dendritic cells are more resistant to the cytophatic effects of the virus. Indeed, macrophages have the peculiar capacity to accumulate virions in intracellular vacuolar compartments, as observed originally in vivo in macrophages infiltrating the brain of individuals with HIV-1 encephalopathy and AIDS-associated dementia [417]. However, macrophages could be cleared by host mechanisms if cytolytic T lymphocyte responses are boosted with HIV-1 specific antigens to more efficiently kill infected cells, in which latency has been reversed. Therefore, significant boosting of CTL responses, through therapeutic vaccination or other means, prior to virus reactivation might be an effective strategy for purging the latent pool and thereby eradicating HIV-1 infection. Several approaches of immunotherapy are reviewed in [389].