Just as the host cell genome is comprised of individual genes, the 3D chromatin environment of the nucleus is comprised of “2D” elements. Of particular interest are nucleosomes and their covalent modifications, which regulate accessibility to local gene regions by promoting the formation of open or closed chromatin. The HIV-1 genome is one such local region, spanning approximately 9,200 to 9,600 nucleotides in length (reviewed in [70]). Because HIV-1 integrates into the host chromosome as a provirus, it is not surprising that the same factors that regulate cellular gene expression also maintain the axis of latency and reactivation.

The coding regions of the HIV-1 genome are flanked by identical 5’ and 3’ long terminal repeats (LTRs), which regulate gene expression and enable synthesis of mature mRNAs from viral genes (reviewed in [70]). The LTR contains 3 regions of interest (5’ to 3’): U3, R, and U5 (reviewed in [16, 71]). Despite having identical sequences, however, each LTR has a different function. The 5’ LTR acts as the viral promoter and contains binding sites for critical TFs, the TSS and trans-activation response element (TAR) sequence [72, 73]. By contrast, the 3’ LTR terminates transcription and encodes the polyadenylation signal [74]. In addition, the provirus encodes Tat, which accumulates in the nucleus and facilitates proviral gene expression through several mechanisms (reviewed in [75]), including binding the TAR and recruiting factors that promote transcriptional processivity, chromatin-modifying factors, and other 2D epigenetic regulators described below.

The role of the 2D chromatin environment in HIV-1 latency and reactivation has been investigated in-depth over the last several decades. In this section, we summarize much of the research characterizing the enzymes, small molecules, and other factors that have been found to play a role in regulating the latent state of the HIV-1 provirus.

The positions of nucleosomes on the proviral 5’ LTR have been well-characterized in several cell lines, as well as primary models of HIV-1 latency (Fig. 3). In early studies, DNase I hypersensitive sites (HS or DHS) were identified at the 5’ and 3’ LTR. Of note are HS2, HS3 (collectively known as DHS1), and HS4 (also known as DHS2) (Fig. 3B, see T cell [immortalized]). Additional sites have also been identified on the 5’ LTR, called HS1, and within gene coding regions in the monocytic cell line U1 (Fig. 3B, see Macrophage [immortalized]). Interestingly, the hypersensitive sites of the 3’ LTR do not correspond to where they are found on the 5’ LTR [76]. At the 5’ LTR, the regions separating the hypersensitive sites are occupied by conserved nucleosomes, termed nuc-0 and nuc-1 (Fig. 3B); nuc-1 is of particular interest, as it overlies the R-U5 region just downstream of the TSS. Nuc-2, -3, and − 4 have also been identified [77], but they are less well-characterized and may not be as highly conserved (Fig. 3B, compare T cell [immortalized] and T cell [primary]) [78, 79].

The 2D chromatin environment of the 5’ LTR is altered by cell type and activation state. (A) Nucleosomes on unintegrated HIV-1 DNA are positioned slightly upstream of where they are found on the integrated provirus. Additionally, nucDHS occupies the hypersensitive site between nuc-0 and nuc-1, preventing transcription factors from binding and stimulating gene expression prior to integration. (B) After integration and during latency in most immortalized cells, nucDHS is evicted and nuc-0, -1, and − 2 occupy their canonical positions as HS2, HS3, and HS4 are formed between them. HS1 is also found in immortalized macrophages slightly upstream of nuc-0. In J-Lat 11.1 cells, HS2 and HS3 are slightly protected from digestion by MNase, indicating a poorly-positioned nucleosome in this region. In some primary T cells, nuc-0 remains slightly upstream of where it is found in other models, possibly leading to HS2 being larger than what is seen in immortalized cells. Additionally, nuc-2 is evicted. Across cell types, nuc-1 consistently occupies the R region, blocking the transcriptional start site (TSS) and TAR to block processive transcription. (C) When infected cells are activated with the appropriate stimulus, nuc-1 is quickly destabilized and repositioned to increase accessibility to the TSS (indicated by an arrow). In accordance with nuc-1 remodeling, HS3 also becomes larger and more sensitive to digestion with DNase I. In immortalized macrophages, HS1 undergoes similar alterations

After the viral particle uncoats in the nucleus, histones are deposited onto the linear, unintegrated HIV-1 DNA; that is, integration into the host genome follows nucleosome assembly [80]. This timeline may be conserved among retroviruses, as inhibition of reverse transcriptase and nuclear import, but not integrase activity, prevented histones H2B and H3 loading in both HIV-1 and murine leukemia virus [80, 81]. In a Jurkat model of HIV-1 infection, nuc-0 and nuc-2 were only found at their known positions after integration; on unintegrated HIV-1 DNA, they were instead upstream of these sites (compare Fig. 3A and B). In primary cells, however, nuc-0 sliding was not observed, and nuc-2 was evicted after integration (Fig. 3B, see T cell [primary]) [80]. These differences in nucleosome positioning may suggest that host factors at the integration site influence the epigenetic landscape of the provirus. Recently, an additional 5’ LTR nucleosome was identified on unintegrated HIV-1 DNA at DHS1, termed nucDHS (Fig. 3A). While its role has not been fully characterized, nucDHS may inhibit pre-integration transcription by establishing a repressive chromatin environment and reducing RNAPII recruitment. In support of this hypothesis, treating Jurkat T cells with histone deacetylase inhibitors early in infection stimulated HIV-1 transcription despite low levels of integrated HIV-1 at this stage. Upon integration, however, nucDHS was quickly evicted, exposing HS2 and HS3 in the provirus [80].

To date, the chromatin environment of the downstream coding regions of the provirus and 3’ LTR is poorly characterized. Recently, a nucleosome positioned in the U3 region of the 3’ LTR was identified, and may serve to regulate the expression of the HIV-1 antisense transcript Ast [82]. A “poorly-positioned” nucleosome at DHS1 has also been observed in J-Lat 11.1 cells (Fig. 3B, see J-Lat 11.1), indicated by incomplete protection from MNase digestion, but this has not been found in other systems to date [78, 79]. Whether this nucleosome is a result of a failure to evict nucDHS remains to be investigated. Additionally, ATAC-seq data has shown low levels of accessibility between the 5’ and 3’ LTRs in resting cells [83], indicating that other structures may be present.

Changes in the 5’ LTR hypersensitive regions and nucleosomes following stimulation have been well-established. Indeed, the R-U5 region in U1 and ACH2 cells became more sensitive to DNase I and MNase digestion following treatment with PMA. In particular, HS3 became larger after stimulation with phorbol esters or histone deacetylase inhibitors; in U1 cells, HS1 also becamse more sensitive to digestion (Fig. 3C) [76, 77, 84]. Likewise, ATAC-seq data indicates increased accessibility at the R-U5 region in activated cells [83]. Taken together, these studies suggest that nuc-1 is destabilized during cell activation, establishing a more favorable chromatin environment for transcriptional machinery to assemble (Fig. 3C). Notably, inhibition of RNAPII does not prevent this process from occurring, indicating that nuc-1 remodeling is independent of, but critical to, efficient transcription from the provirus [77].

In eukaryotes, nucleosome positioning is highly conserved and mediated by the activity of chromatin modifying complexes (CMCs). In humans, there are two broad categories of CMCs: complexes that affect nucleosome structure through covalent modifications, and those that use the energy of ATP hydrolysis to directly reposition the nucleosome (reviewed in [85]).

The SWItch/Sucrose Non-fermentable (SWI/SNF) family belongs to the latter category of CMCs and is of particular interest due to its role in HIV-1 latency and reactivation alike. In humans, there are two members of this family: BRG-/BRM-associated Factor (BAF) and Polybromo-associated BAF (PBAF) (reviewed in [85]). BAF may use Brahma-related Gene 1 (BRG1, also known as SMARCA4) or Brahma (BRM, also known as SMARCA2) as a core catalytic subunit, while PBAF only uses BRG1 [86]. Additionally, there are other subunits that are exclusive to one member or the other. PBAF contains BAF180, BAF200, and Brd7, but lacks BAF250 [85, 86], while BAF does contain BAF250 and lacks the other PBAF-associated subunits. Otherwise, they share core components, such as BAF155/170, INI-1/BAF47, β-actin, and others (reviewed in [85]).

Notably, the sites occupied by nuc-0, -1, and − 2 are not what would be expected by DNA sequence alone [78, 79, 87]. Using the NuPoP algorithm to predict nucleosome positioning based on the LTR sequence [88], Rafati et al. found that the 5’ LTR nucleosome positions were actually negatively correlated with histone affinity [78, 79], indicating a role for external factors in establishing the canonical epigenetic landscape of the provirus. Indeed, the BAF complex has been shown to be essential to this process early in infection, in the absence of Tat. Depletion of BRG1 and BAF250, but not BRM or BAF180, was associated with de-repression of the proviral promoter, and BAF250-deficient infected Jurkat T cells were unable to maintain latency. In these cells, the canonical epigenetic landscape of the 5’ LTR was disrupted and the nucleosomes instead assumed the positions that would be predicted by the sequence’s histone affinity; that is, accessibility at nuc-1 increased while DHS1 and DHS2 became more protected [78]. Both BAF200 and BAF250 have also been shown to be present at nuc-1 in resting cells; however, after the cells were activated, BAF250 was removed [89]. Taken together, in the absence of Tat, the BRG1-containing BAF (BRG1-BAF) complex facilitates HIV-1 latency through the positioning of nucleosomes at the 5’ LTR (Table 1) [78].

Bromodomain-containing Protein 4 (BRD4) is a member of the Bromodomain and Extraterminal Domain (BET) family of proteins, and has long (L) and short (S) isoforms. BRD4L is known to inhibit Tat-mediated transactivation by binding with Positive Transcription Elongation Factor b (P-TEFb) [90, 91], but BRD4S seems to exert its own repressive action through recruiting the BRG1-BAF complex to the proviral promoter. Indeed, BRG1 and BRD4S immunoprecipitated with one another from the 5’ LTR, but treating the cells with the BET inhibitor JQ1 disrupted this interaction. Additionally, knockdown of BRD4S reduced BRG1 occupancy at nuc-1 and induced HIV-1 RNA production. Taken together, these results indicate that BRG1-BAF is recruited to the proviral promoter by BRD4S [91] and has a repressive effect on HIV-1 transcription (Table 1).

Interestingly, while BRG1-containing BAF complexes are associated with repression of HIV transcription early in infection, BRM-containing BAF complexes seem to facilitate proviral transcription later in infection. In the presence of Tat, BRM and BAF155 were enriched at nuc-1, and overexpression of BRM in a HeLa model of infection could increase transactivation by Tat. In agreement with this, knockdown of the gene had the opposite effect. In the absence of Tat, however, BRM-deficient cells had no difference in basal promoter activity when compared to BRM-sufficient cells [92], indicating the synergistic relationship between Tat and BRM-containing BAF complexes (BRM-BAF). However, in a model of latency utilizing SW13 and C33A cells, some clones had greater dependence on BRM-mediated chromatin remodeling than others, while BRM-deficient ACH2 cells had impaired virion production when compared with controls [93]. Importantly, though, BRG1 and BRM have been demonstrated to have differential expression between cell and tissue types [94]. Taken together, it is possible that the host cell type and integration site of the provirus determine the degree to which proviral reactivation depends on BRM (Table 1). Further studies will be necessary to elucidate how BRM-BAF may play a role in maintaining latency in different anatomical reservoirs.

The other member of the SWI/SNF family, PBAF, has also been implicated in the activation of proviral gene expression. In cells treated with PMA, depletion of BAF250 did not affect recruitment of PBAF subunits to nuc-1 [78, 95], but BAF200 was present at the promoter in both latent and active cells [89, 95, 96], indicating a role for PBAF in HIV-1 reactivation. Indeed, in several latently-infected T cell lines as well as stably-infected HEK293T cells, BRG1 knockdown inhibited viral gene expression and virion production [89, 97]. Mahmoudi et al. also demonstrated through immunoprecipitation that Tat is associated with Integrase Interactor 1 (INI-1, also known as SMARCB1), β-actin, and BRG-1 [98]. Interestingly, in contrast to the findings implicating BRM-BAF in proviral reactivation [92], they did not find Tat to be associated with BRM [98].

While this discrepancy may be due to the use of different cell lines and integration sites, it may also be attributed to the acetylation state of Tat in these systems. BRG1 contains a C-terminal bromodomain with a binding motif for acetylated proteins [98, 99], and Tat may be acetylated at Lys-50 or -51 (Tat-ac) by the cellular acetyltransferase p300 [98]. Tat-ac actually failed to interact with BRM, and was associated with BAF200 (PBAF-specific) [92, 96]. By contrast, immunoprecipitates of BAF250 (BAF-specific) were enriched with unmodified Tat [96]. Taken together, it would appear that PBAF’s capacity to act on the proviral nucleosomes relies on acetylated Tat, while BRM-BAF utilizes unmodified Tat (Table 1). Indeed, Tat-mediated transactivation was abrogated in cells deficient in BAF200 [96]. Likewise, Lys-50 and − 51 mutants of Tat were also unable to undergo Tat-mediated transactivation. In support of this, the region occupied by nuc-1 has been shown to have increased sensitivity to digestion in the presence of acetylated Tat [97]. Tat’s association with BRG1 was also increased in the presence of p300, but this relationship was abolished when Tat or the catalytic site of p300 were mutated [98]. Interestingly, in the absence of INI-1 and p300, Tat alone still had weak transactivation activity [98], possibly due to its interaction with BRM-BAF in the unmodified state [92, 93].

Other components of the SWI/SNF complex have also been implicated in HIV-1 latency and reactivation. During integration, the core subunit INI-1 directly interacts with integrase and stimulates its activity in vitro [100]. Interestingly, INI-1-deficient, stably-infected HeLa cells also had impaired, but not completely abrogated, transactivation capability. After the reintroduction of INI-1 in the presence of Tat, however, transactivation was rescued. The Rpt1 and Rpt2 domains of INI-1 were further demonstrated to be required to achieve this effect [101]. While these domains are required for the formation of a functional SWI/SNF complex [101], Rpt1 has also been shown to mimic the TAR structure in silico and in vitro [102]. The degree to which this affects the ability of SWI/SNF complexes to interact with Tat remains to be studied, however, and may provide a unique target in altering the latent state of the provirus.

The effects of the core subunit BAF53 are also at least partly dependent on the activity of Tat. BAF53-deficient HEK293T and J1.1 cells had increased virion production, and these virions additionally had improved reverse transcriptase activity. To this end, BAF53 was evicted from the 5’ LTR in active cells. Taken together, BAF53 seems to be inhibitive to proviral gene expression in a resting cell, possibly early in infection. In the presence of Tat, however, the Cyclin T: CDK9 complex could more effectively phosphorylate BAF53, which prevented its association with actin [89]. This may have the overall effect of preventing the formation and binding of BRG1-BAF, which in turn permits the binding and activity of other SWI/SNF complexes and TFs to facilitate proviral gene expression.

Another chromatin remodeler, Facilitates Chromatin Transcription (FACT), has also been implicated in HIV-1 latency. While FACT normally removes and re-deposits the H2A-H2B dimer of histones to facilitate transcription [103], it may also have a repressive role in the context of HIV-1 latency. Knocking down FACT components SUPT16H or SSRP1 in NL4-3-infected HEK293 cells enhanced viral replication and Tat-mediated transactivation. Additionally, while both FACT components were associated with the 5’ LTR, only SUPT16H co-precipitated with Tat. In agreement with these findings, FACT-deficient J-Lat A2 cells and infected primary CD4 + T cells underwent spontaneous latency reversal, and were also sensitized to the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Taken together, these results indicate that FACT is repressive to proviral gene expression (Table 1) [104]. However, certain FACT-targeting compounds called curaxins have been shown to have variable effects on HIV transcription [105, 106]. Further studies will be necessary to elucidate the precise relationship between FACT and the 5’ LTR nucleosomes, and the mechanism by which it may repress or facilitate proviral gene expression.

Overall, these results highlight the importance of the 2D chromatin environment in maintaining the axis of latency and reactivation in the HIV-1 provirus. Interestingly, SWI/SNF-mediated reactivation is improved when coupled with covalent modifications to the 5’ LTR histones’ tails, particularly acetylation [84, 89, 95, 97]. Thus, while direct nucleosome repositioning is highly important in regulating proviral latency and reactivation, additional mechanisms are also occurring at other levels.

Besides direct repositioning of nucleosomes on the chromosome, one of the key mechanisms eukaryotic cells use to regulate gene expression is through post-translational modification (PTM) of the histone tails. As we have described previously, these PTMs alter the charge of the histone and therefore the strength of its interactions with DNA. Generally, positive charges are associated with tighter interactions and transcriptional repression; by contrast, neutral and negative charges are associated with transcriptional activation, as the negatively-charged DNA is repelled from the histone core (reviewed in [25]). Unsurprisingly, proviral latency and reactivation are influenced in large part through PTMs of the 5’ LTR nucleosomes. Currently, acetylation and methylation are the best-characterized in both latent and reactivated HIV-1 infection (reviewed in [73, 107]).

Histone acetylation is mediated by two classes of enzyme: histone acetyltransferases (HATs), and histone deacetylases (HDACs). The former functions as the “writers” of histone acetylation, depositing the negatively-charged acetyl groups on the ε-amino groups of the histone tails’ lysine or arginine residues; the latter are “erasers” in that they remove these acetyl groups and restore the repressive chromatin environment (reviewed in [