The HIV-1 Transcriptional Program: From Initiation to Elongation Control

Iván D’Orso

doi:10.1016/j.jmb.2024.168690

. Author manuscript; available in PMC: 2025 Apr 13.

Published in final edited form as: J Mol Biol. 2024 Jun 25;437(1):168690. doi: 10.1016/j.jmb.2024.168690

The HIV-1 Transcriptional Program: From Initiation to Elongation Control

Iván D’Orso ^1,^*

PMCID: PMC11994015 NIHMSID: NIHMS2068941 PMID: 38936695

Abstract

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

The Core Components of the HIV-1 Transcriptional Program

Like every single virus on earth, HIV-1 has a life cycle composed of closely orchestrated steps to efficiently replicate and perpetuate the infection. The genetic material integrated into cellular chromatin must be transcribed to produce RNAs and proteins crucial for completion of the viral life cycle. In this section, I start by defining the core components of the HIV-1 transcriptional program including the viral genomic elements (DNA and trans-activation response (TAR) RNA), and the cellular and viral (Tat) transcription factors (TFs) that recognize them. While the collective action of cis elements contained within the viral promoter and cellular TFs facilitate the initial wave of transcription activation, the Tat-TAR axis amplifies transcription for viral replication and latency reactivation. As I will discuss in further detail below, TAR is an RNA structure formed at the 5’ end of viral nascent pre-mRNAs and Tat is an RNA-binder that, unlike cellular TFs, primarily functions by promoting transcription elongation.

A large number of genomic elements and factors participate in the HIV-1 transcriptional program, which consists of three sequential phases (basal, host, and viral) depending on the cell state and composition and amount of expressed TFs [1, 2] (Figure 1). In the basal phase in resting cells, HIV-1 transcription is low-to-undetectable, depending on several properties including the site of HIV-1 integration into the human genome, the reservoir cell type and its state [3]. In the host phase when reservoir cells are exposed to environmental stimulation, cellular TFs facilitate low level of transcription activation that leads to Tat synthesis leading to little viral replication. In the viral phase, when Tat levels accumulate, a constant source of the cellular elongation machinery is recruited to the nascent TAR RNA to assemble transcription elongation competent complexes thereby facilitating sustained transcription activation. Together, this step creates a positive feedback loop, where Tat continuously activates high levels of transcription by alleviating blocks to elongation, thus enabling robust viral replication and pathogenesis. The switch between the host and viral phases is called “transition point” [4] (Figure 1), point at which either Tat takes over to activate transcription exponentially or transcription subdues. Given Tat’s critical role for transcription activation, without Tat the viral phase is never activated. This roadblock prevents the normal activation of the positive feedback loop for sustained HIV-1 transcription activation, causing entry into a state of viral latency.

Despite the importance of Tat to logarithmic RNA amplification (Figure 1), Tat activation is influenced by certain conditions altering reservoir cell state and availability of host cell cofactors [1, 5], indicating that even in the presence of Tat, the viral phase can return to the ground state (basal state) to re-establish latency and promote viral persistence (Figure 1, right panel). This molecular phenomenon also applies to HIV-1 infection of cells in different states of activation or during the effector-memory transition [6], in which cellular relaxation pushes an initial, transcriptionally active provirus into a silent state.

Taken together, there are at least three major features that influence transcription in the three phases of the HIV-1 transcriptional program (reviewed in [2]). First, the site of HIV-1 integration into the human genome [3]. Second, the reservoir cell type (e.g., CD4⁺ T cells, macrophages, microglia in the brain) [7-10] and state of the infected cell (resting or activated, which is certainly not a binary case) [2]. Third, the complexity of the architecture regulating provirus transcription including its genomic elements and the factors that recognize them, which are strictly dependent on both the type and state of the infected cell. In the following sections, I primarily cover the third feature to focus on the core components of the HIV-1 transcriptional program. The first and second features (integration site and cell state) directly contribute to the outcome of the phases of the HIV-1 transcriptional program and thus are briefly discussed in that context. For a more complete review on the roles of integration sites and cell state, the reader should consult recent reviews [2, 11].

HIV-1 Genomic Elements and Cellular Transcription Factors

Our understanding of HIV-1 transcription began with the discovery of viral cis regulatory elements, and the viral and cellular TFs that recognize them. Given the large number of factors that have been reported to regulate the HIV-1 transcriptional program at many different levels, as will be explained in detail below, I provide a Table listing key factors and their potential role(s) to better guide the reader (Table 1).

Table 1. Regulators of the HIV-1 transcriptional program.

List of factors that have been linked to either transcription activation or repression and their known roles in the viral transcriptional cycle. Specific details regarding the evidence linking these factors to their reported function(s) are listed in the manuscript. The reader should be cautious about taking the reported functions for all factors as definitive until indirect effects through the use of more advanced genetic technologies (e.g., acute factor depletion approaches) can be ruled out.

Factor class	Factor (Complex)	Function
Cellular TFs	Sp1	Activator
	TBP	Activator
	NF-κB p65, RelA	Activator
	c-Fos, c-Jun (AP-1)	Activator
	HIF-1α	Activator
	LBP-1	Repressor
	USF1	Activator
	USF2	Activator
	RBF-1	Activator
	RBF-2	Activator
	NF-κB p50	Repressor
	NF-κB RelB	Activator
	C/EBP	Activator
	Ets-1	Activator
	LEF-1	Activator
	COUP-TF	Activator
	CREB family	Activator
	ATF family	Activator
	IRF	Activator
	CBF-1	Repressor
	CTIP2	Repressor
	ESR-1	Repressor
	IFI16	Repressor
	KLF2	Repressor
	KLF3	Repressor
	Oct-1	Activator
	PU.1	Activator
TAFs	TAF1 (TFIID)	Activator
TAFs	TAF110 (TFIID)	Activator
GTFs	TFII-I	Repressor, Activator
	TFIIAα−β, TFIIAγ (TFIIA)	Activator
	TFIIB	Activator
	TFIIF-α, TFIIF-β (TFIIF)	Activator
	TFIIE-α, TFIIE-β (TFIIE)	Activator
	XPB, XPD, p62, p52, p44, p34, p8, MAT1, CCNH, CDK7 (TFIIH)	Activator
Mediator	MED6/7/11/14/21/26/27/28/30 (Mediator)	Activator
Mediator	CDK8 (Mediator kinase module)	Activator
Chromatin regulators, remodeling	BRG1	Repressor
	BAF	Repressor
	PBAF	Activator
	BRD4-S	Repressor
HDACs	HDAC1	Repressor
	HDAC2	Repressor
	HDAC3	Repressor
HATs	CBP, p300	Activator
HATs	PCAF, GCN5	Activator
HKMTs	MLL1	Activator
	MLL2	Activator
	MLL3	Activator
	SETD7	Activator
	SETDB2	Activator
	SETD8	Activator
	Suv420-H2	Activator
	SETMAR	Activator
	SMYD3	Activator
	SMYD5	Activator
	MLL5	Activator
	Suv39H1	Repressor
	SETDB1	Repressor
	GLP	Repressor
	G9a	Repressor
	ASH1L	Repressor
	Suv420-H1	Repressor
	EZH2 (PRC2)	Repressor
	SMYD2	Repressor
HRMTs	CARMA1	Repressor
Elongation factors	HIV-1 Tat	Activator
	CycT1, CDK9 (P-TEFb)	Activator
	SPT4/SPT5 (DSIF)	Activator
	NELF-A/B/C/D/E (NELF)	Repressor
	Tat-SF1	Activator
	KAP1/TRIM28	Activator
	HEXIM1 (7SK snRNP)	Repressor
	MePCE (7SK snRNP)	Repressor
	LARP7 (7SK snRNP)	Repressor
	RHA	Activator
	hnRNP’s (A1, A2/B1, R, Q)	Activators
	PP2B	Activator
	PP1α	Activator
	PPM1A	Activator
	PPM1G	Activator
	DDX21	Activator
	ELL2, AFF1, AFF4, ENL, AF9 (SEC)	Activator
	PAF1, CTR9, CDC73, LEO1, WDR61 (PAF1C)	Activator
	BRD4-L	Activator
Termination factors	CPSF	Activator
Termination factors	CSTF	Activator

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HIV-1 genome and LTRs.

The HIV-1 genome contains 9200-9600 bp of genetic material with two long terminal repeats (LTRs) flanking the 15 genes encoding the structural, accessory, and regulatory (e.g., tat) proteins [12] (Figure 2A). The 5’ LTR is divided into 3 functional domains (U3, R, and U5) that are strategically positioned surrounding the transcription initiation or transcription start site (TSS), with the TSS located in the junction of the U3 and R domains (Figure 2B). The U3 domain can be divided into three regions: modulatory, enhancer, and the core promoter. The R domain contains the sequence that when transcribed produces the TAR RNA stem-loop, located immediately downstream of the TSS. The region downstream of the U5 domain and upstream of the first viral gene (gag) is called the 5’ untranslated region (5’ UTR).

These genomic domains harbor a number of cis motifs that can be recognized by cellular TFs (Figure 2B), which can bind either in a constitutive or inducible manner [13] to dictate the level of transcription activity in each of the three phases of the HIV-1 transcriptional program in a given cell type and state. Throughout this review, I refer to these factors as constitutive TFs (cTFs) and signal-inducible TFs (iTFs) to simply illustrate shared mechanistic similarities of the core transcriptional program despite differences in their specific cell-type expression patterns and mechanisms of action. cTFs include those that bind to the core promoter region to dictate low level of transcription activation in the basal phase including Specificity protein 1 (Sp1) [14] (Table 1). Components of the transcription pre-initiation complex (PIC), such as TATA-box binding protein (TBP) [15, 16] (Table 1), surprisingly assemble at the promoter in an inducible manner and thus belong to the iTF classification.

iTFs typically participate in cell state alterations through the activation of various transcriptional programs including but not limited to nuclear factor-kappa B (NF-κB) [17, 18], nuclear factor of activated T cells (NFAT) [19-21], activator protein 1 (AP-1) [22, 23], and hypoxia-inducible factor-1 (HIF-1α) [24] (Table 1), among many others (Figure 2B). Depending on the cell stimulation, iTFs can function alone or in a cooperative manner with other iTFs and/or cTFs including NF-κB with Sp1 [25], NF-κB with AP-1 [26], and AP-1 with HIF1-α [27], perhaps helping organize the assembly of high-order TF complexes for increased transcription activation, which in other contexts have been referred to as enhanceosome [28].

A common theme among these iTFs is that they are induced through cell signaling events, but activated by unique cues and in a cell-type-universal or cell-type-specific manner depending on their expression patterns. For example, an inflammatory cue such as tumor necrosis factor alpha (TNF-α) selectively activates NF-κB in cells expressing the cognate TNF receptor on its plasma membrane, and an hypoxic environment primarily induces HIF-1α in mostly any given cell type [29]. The main goal of this review is not to provide an extensively detailed survey of the nuances of how cellular TFs operate in response to what signals and in what cell types, but by explaining their common modus operandi and their importance to the virus, as they are central components and required for initiation of the different phases of the HIV-1 transcriptional program (Figure 1). Readers are welcome to consult previous reviews summarizing the complexity of signal- and cell-type-specific TF-mediated HIV-1 transcription activation [30-32]. In brief, cTFs such as Sp1 and several transcriptional repressors such as Leader-binding protein-1 (LBP-1) [33, 34] (Table 1), mainly regulate basal transcription during cell homeostasis and can serve both to maintain the promoter in the virtually off state, and following activator interactions, to reverse the repression and effect a net increase in activity. iTFs cooperate among them and with cTFs to induce low-level transcription during the host phase to produce Tat.

Both cTFs and iTFs recognize their cognate sites on the HIV-1 genome particularly within the 5’ LTR (Figure 2B). The core promoter contains all the necessary elements for efficient transcription initiation in all three phases of the HIV-1 transcriptional program, including the tandem Sp1 binding sites, the TATA box, and initiator [35], and is the minimal region required for Tat trans-activation in the viral phase.

TATA box.

The TATA box, located ~25 bp upstream of the TSS, is recognized by TBP, a component of the general transcription factor IID (TFIID). The TATA box is non-canonical because it bears the sequence CATATAA and thus is also called CATA box, which is likewise used by ~10% of cellular RNA polymerase II (Pol II) promoters [36]. HIV-1 subtype differences in TATA box sequence composition have shown to exhibit reduced transcription relative to others harboring the canonical CATA motif [36], perhaps illustrating important evolutionary tradeoffs for increased viral transcription. For example, most subtypes contain the motif TATAAGC, but in subtype E and some recombinant AG strains, position 3 is mutated (TAAAAGC) and the true TATA box motif starts two nucleotides upstream yielding the sequence CATAAAA while all other subtypes have the sequence CATATAA. Notably, the CATA box of subtypes B and E can be improved for replication fitness by the mutations 1T and 5T, respectively, suggesting that the 5’ LTR is fine-tuned towards a suboptimal level of replication, although this rate may be optimal in the context of an infected individual [36].

Initiator.

Downstream of the TATA box resides a bipartite pyrimidine-rich initiator element (Inr: (G+1GGTCT)) which overlaps with and determines the TSS [35, 37, 38] to stimulate transcription initiation [35]. The activity of the Inr in cell-free transcription assays strictly relies on the correct position of the TATA box, again suggesting cooperation between proteins binding both elements for transcription initiation. Not only the position but also the sequence content of the TATA and Inr elements is critical, as they cannot be functionally substituted by TATA and Inr elements from other gene promoters to stimulate basal transcription and Tat trans-activation in the viral phase [39, 40].

RBEs.

A novel pathway of transcription initiation requiring the Inr and the Inr-binding factor Transcription Factor II-I (TFII-I) was described by Roy and Roeder [41, 42]. TFII-I is related to the helix-loop-helix activator Upstream Stimulatory Factor (USF), a member of the MYC-related family of DNA binding proteins [43]. The Inr contains a highly conserved cis-regulatory element called Ras-responsive binding element (RBE), known to bind the Ras-responsive element binding factor 2 (RBF-2) complex composed of TFII-I and USF1/2 (Figure 2B). Three other highly conserved RBE elements were found upstream of the core promoter, with RBEII and RBEIV bound by the RBF-1 complex, and RBEIII bound by the RBF-2 complex similarly to RBEI. In resting cells, the model poses that TFII-I functions as a repressor and upon cell stimulation RBF-2 phosphorylation favors transcription activation [44, 45]. TFII-I regulates provirus induction in cooperation with USF [46], in agreement with its role as positive regulator of signal-induced transcription [47]. While recombinant USF1/USF2 bind to the RBEIII less efficiently than to its canonical site (E-box), its interaction with RBEIII is stimulated by TFII-I. Thus, USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of the silent viral promoter in response to T-cell activating signals.

Sp1 sites.

Upstream of the TATA element there is a GC-rich region containing three binding sites for the zinc finger-containing TF Sp1 [48]. The multiple Sp1 sites cooperate with the TATA element during basal transcription and Tat trans-activation in the viral phase [14, 49-51]. In other promoter contexts, Sp1 uses a Glutamine-rich domain to engage with a subunit of the TFIID multi-subunit complex, TBP-associated factor 110 (TAF110) [52], but it remains unclear if this and/or other interactions take place between Sp1 and TFIID components in the context of the HIV-1 promoter beyond Sp1-TBP cooperation. Together these previous observations indicate that the topological arrangement of the promoter elements (TATA and Sp1 binding sites) and the factors that recognize them (TBP and Sp1) are critical for the correct organization of the transcription initiation complex, and perhaps for the transition between transcription initiation to elongation for optimal virus transcription.

NF-κB sites.

Immediately upstream of the Sp1-binding sites are two adjacent binding sites for the inducible transcription factor NF-κB [53]. This site is known as enhancer (Figure 2B) and is activated by a plethora of cellular activation signals including inflammatory cytokines [54-60] and mitogenic stimuli such as phorbol esters [61]. Again, enhancer function requires cooperative protein-protein interactions. NF-κB cooperates with Sp1 [25] as well as TBP [62] to perhaps enhance transcription in both the host and viral phases. These discoveries are in line with the fact that sustained NF-κB activity was required for efficient viral transcription due to maintenance of de novo Tat synthesis [18].

The enhancer strongly increases viral promoter activity through NF-κB binding [17, 25]. Most HIV-1 subtypes have two NF-κB binding sites within the enhancer, except subtype C which contains three to four sites to bolster enhancer activity [63]. The p50/p65 NF-κB heterodimer is sequestered and inactivated into the cytoplasm in resting cells. Upon cell stimulation, it translocates into the nucleus where it binds its cognate binding site on the 5’ LTR through a well-established mechanism, which has been thoroughly summarized in previous reports [64, 65] and is not the focus of this review.

In addition to the canonical NF-κB pathway regulated by p50 and p65 (RelA), the non-canonical NF-κB pathway regulated through RelB has also been implicated in HIV-1 transcription activation [66]. Tat itself can also activate canonical NF-κB [18, 67-69] as well as non-canonical NF-κB [66] forming a positive feedback loop to further enhance and sustain virus transcription through highly diverse, direct and indirect mechanisms. Further, the non-canonical NF-κB regulator (RelB) interacts with Tat to promote its recruitment to the viral promoter and transactivate transcription [70].

After cell stimulation, Sp1 acts synergistically with NF-κB via protein–protein interactions to activate transcription [25], also highlighting the importance in the genomic arrangement of the contiguous NF-κB and Sp1 binding sites on the viral promoter (Figure 2B). Sp1 and NF-κB together interact with TBP and select TBP-associated factors (TAFs) [71]. For example, the C-terminal of the NF-κB p65 subunit interacts with TAF1 during HIV-1 transcription activation in vitro [72] and the Glutamine-rich activation domain of Sp1 interacts with TAF110 [52, 73], but its importance for HIV-1 transcription activation in vitro and in cells remains to be determined. Thus, TF cooperativity may help recruit Pol II to the viral promoter and/or induce a chromatin reconfiguration favoring transcriptional activation [74]. Sp1 also cooperates with chicken ovalbumin upstream promoter transcription factor (COUP-TF) to synergically activate viral transcription in microglial cells [75], illustrating how cTFs can accommodate interactions with cell type specific iTFs to achieve an identical outcome: robust transcriptional activation through TF cooperativity.

Additional TF binding sites.

In addition to the cis elements within the core promoter and NF-κB enhancer, the provirus has two additional regions that regulate transcription (Figure 2B). The regions upstream of the enhancer (modulatory region) and downstream of the initiator (leader region), also contain a variety of TF binding sites known to modulate transcription. Several upstream binding sites for CCAAT-enhancer-binding protein (C/EBP), AP-1, E26 Transformation-specific-1 (Ets-1), Lymphoid Enhancer Factor-1 (LEF-1), COUP-TF, and NFAT, among others, have been involved in HIV-1 transcription in a cell type-dependent manner [76-79]. Transcriptional synergy may be mediated by cooperative binding of the factors to the viral promoter because C/EBP has also been shown to physically interact with NF-κB family members [80], again potentially reinforcing the enhanceosome idea [28].

Sequences downstream from the TSS, in the leader region and 5’ UTR (Figure 2B), are also important for transcription [81-85] and provide binding sites for several cellular TFs [85-88] including cTFs (Sp1) and iTFs (e.g., AP-1, CREB/ATF, NF-κB, Interferon regulatory factor -IRF-, NFAT) [30, 85]. The leader region contains three functional AP-1 binding sites that are important for transcription [84-86]. These are recognized by a family of basic-leucine zipper (bZip) TFs composed of members from the JUN, FOS, and ATF protein families that form homo- or hetero-dimers through their leucine zipper domains to recognize their cognate binding sites. Specifically, AP-1 complexes composed of canonical Fos (c-Fos) and c-Jun, and cross-family dimerization of c-Fos/c-Jun and ATF/CREB have been reported [23, 89, 90]. The 5’ UTR may function as a downstream enhancer cooperating with the upstream enhancer and core promoter to ensure maximal transcription activation. This downstream enhancer could bolster viral response to iTFs induced in response to a wide variety of cell activation signals and/or it could also assist in the displacement of nucleosomes during the remodeling of chromatin structure.

Genetic diversity of TF binding sites.

The genetic diversity in the 5’ LTR of different viral clades or subtypes alters the composition and arrangement of cis elements [91]. The resulting sequence variability in these elements could generate new TF binding sites [92] and/or alter the strength of TF binding and epigenetic regulation for transcriptional repression and/or activation, ultimately dictating the efficiency of transcription in the various phases of the HIV-1 transcriptional program. Some LTR variants have stronger or weaker promoter activity, leading to differences in transcription rates, which together can impact viral fitness, replication capacity and disease outcomes [93]. For example, hypermutated LTR variants have shown drastic decreases of transcription activity [94], which may result in decreased viral fitness. Conversely, LTR variants that confer higher transcriptional activity may promote increased viral replication and transmission. Specifically, select variances in Sp1 and NF-κB elements correlate with greater capacity of LTR variants for transcription activation [95], and enhanced transcriptional strength of LTR promoter variants has been shown to minimize noise [96]. Together, LTR diversity can have complex effects on viral transcription, potentially leading to differences in viral replication, pathogenesis, and response to suppressive therapy. Understanding the impact of LTR diversity on transcriptional regulation is crucial for developing effective strategies to combat HIV-1 infection and manage the diversity of viral strains circulating in different populations.

HIV-1 TAR RNA and Tat Protein

Besides the cis motifs and cellular TFs that recognize them, the virus synthesizes a nascent RNA that folds into a 59-nt RNA stem loop structure called TAR (Figure 2C), which is important for coordinating the assembly of transcription elongation complexes through recognition by the Tat protein, and will be described below.

In 1985, the concept of TAR for trans-acting responsive (or target) element was proposed [97, 98], as a region that contains a novel type of regulatory element. Soon after, the identity of the trans-activator gene (tat) (Figure 2A) was reported [99], which illuminated the first glimpses of HIV-1 gene expression control [100, 101].

Tat, one of the 15 proteins of HIV-1 [12], is implicated in viral transcription. Tat contains an activation domain (residues 1-48), a basic region (residues 49-57) and a C-terminal extension (residues 62-86 or 62-101, depending on the isolate/strain) [102, 103] with the smallest, naturally occurring, fully active form of Tat being 72 AAs in length [104] (Figure 2C). While residues 49-57 (RKKRRQRRR) are critical for Tat’s nuclear localization, residues 59-61 (AHQ) act as a nucleolar localization signal [105]. In 1988, Frankel and Pabo described that Tat is a metal-linked dimer [106, 107], with two metal ions (Zn or Cd) bridging cysteine-rich regions in the activation domain of each monomer (7 cysteines per each monomer) (Figure 2C), a previously unnoticed arrangement distinct from the one in zinc finger domain-containing proteins.

In 1987, using a collection of cell-based assays (primer extension, nuclear run-on and RNase protection in COS7 cells in the absence and presence of over-expressed Tat), Peterlin and colleagues proposed an anti-termination role for Tat [108], perhaps motivated by the discoveries with the prokaryotic anti-termination factors [109, 110]. In transient expression assays, Tat largely mediated its effect by increasing the steady-state levels of RNAs containing the TAR sequence at or near their 5’ end. These studies suggested that although the rate of initiation remained unaffected, transcription elongation beyond TAR was only detected in the presence of Tat, providing evidence that Tat functions in a TAR-dependent manner to facilitate transcription. This initial discovery was further expanded with in cell [111, 112] and in vitro [113] data, together substantiating the idea that Tat operates by increasing processivity rather than by preventing a specific termination event [108]. Laspia et al. showed that Tat increased promoter-proximal transcription and suppressed transcriptional polarity, together allowing them to propose that Tat acts through TAR to increase transcription initiation complex formation on the viral promoter and to stabilize transcription complexes during elongation [111].

Using several biochemical and cellular assays, Dingwall et al. first demonstrated that recombinant Tat binds specifically to the TAR element, but not to its antisense RNA version nor TAR DNA sequence [114]; however, attempts to correlate the effect of TAR mutations on in vivo function with their effect on Tat binding in vitro met with mixed success.

It was later in 1989 when Selby and Peterlin revealed that the sequence, structure and position of the TAR stem-loop relative to the TSS and promoter elements were important features for transcription elongation by Tat [115]. Their studies with transient reporter assays, revealed that the helical RNA segments present in TAR primarily serve a structural role in the appropriate presentation of primary sequence information located in and immediately adjacent to the 6-nt terminal loop and the 3-nt bulge of TAR (Figure 2C). Beyond the structure and sequence composition of TAR, an important feature is that it must be in close proximity to the site of initiation for Tat trans-activation to work [115], perhaps consistent with the proposed dual (initiation and elongation) control by Tat [111]. In 1989, Berkhout and Jeang also provided direct evidence that Tat transactivates the viral promoter through a nascent RNA chain and that both its structure and position were critical for transcription activation [116].

In 1990, a series of contemporary papers provided evidence that one of the most important features for Tat trans-activation is the RNA-binding step. First, Southgate, Green and colleagues, replaced the RNA binding domain of Tat with an heterologous one (HIV-1 Rev) and TAR with Rev’s binding site (Rev Response Element: RRE) to demonstrate that a Tat-Rev chimera protein similarly activated an artificial promoter containing a RRE stem-loop in place of TAR [117]. Second, Selby and Peterlin took a conceptually identical approach, but replaced Tat RNA binding domain with the one of the MS2 coat protein, and TAR by the MS2 coat protein RNA target and obtained similar results. These data allowed them to conclude that Tat transactivation can occur independently of TAR RNA and that Tat primarily exerts its transcription effects through a direct protein-RNA interaction [118].

Berkhout and Jeang extended those previous results to demonstrate that efficient Tat activation requires TAR in conjunction with upstream DNA binding sequences including those for the cellular TFs NF-κB and Sp1 [119], because TAR outside its natural promoter context produced a suboptimal Tat response. Similarly, a Tat protein engineered to interact with DNA sites on the 5’ LTR could transactivate through a TAR-independent mechanism, albeit at lower levels, perhaps suggesting that Tat targets DNA bound TFs prior to TAR binding (e.g., to assemble with and/or promote transcription complex assembly to regulate one or more transcriptional steps) consistent with subsequent studies by the Green and Frankel labs [16, 120, 121].

Supporting this model, in 1991, Southgate and Green, demonstrated that Tat can function when bound to upstream DNA promoter elements through its activation domain, but not its RNA-binding domain [122]. Kamine et al. revealed the importance of the Tat activation domain specific recognition of Sp1 for the synergistic activation of synthetic viral promoters [49, 123]. The step of Tat-Sp1 activation is TAR-independent, and is different from the mode of stimulation by acidic transcriptional activators such as VP16 [124], which functions in a Sp1-independent manner. In full agreement, Bohan et al. also provided evidence that in addition to its demonstrated role in RNA elongation, Tat facilitates transcription initiation in vitro by associating with cellular TFs interacting with upstream promoter sequences [125]. The relative contributions of Tat to both transcription initiation and elongation were interrogated in cell-based nuclear run on assays by Laspia et al. who proposed that in addition to stabilizing elongation, Tat also stimulates initiation from the viral promoter [126]. Together these reports strongly indicated that Tat activity requires both TAR RNA and cis elements strategically positioned at the 5’ LTR to assemble and cooperate with promoter-bound cellular TFs (e.g., Sp1, TBP and NF-κB) [25, 49, 123, 124, 127, 128] (Figure 2B).

Notably, the molecular, biochemical, and biophysical features of the Tat-TAR interaction have been studied in detail [106, 107, 121, 129-131] and reviewed extensively [132, 133]. Given this review mainly focuses on the functional aspects of this RNA-protein interaction, I prompt the reader to consult these past literature, if needed.

Marciniak and Sharp provided the first demonstration that recombinantly purified Tat stimulated transcription elongation from the HIV-1 promoter in a TAR-dependent manner (>10-fold) in vitro [113], and also suggested the involvement of a cellular factor in TAR RNA recognition during Tat trans-activation. Using UV crosslinking assays with HeLa nuclear extracts and in vitro transcribed TAR RNA, Marciniak et al. found a ~68 kDa cellular polypeptide that specifically bound to TAR [134]. The binding affinity of TAR loop mutants and deletions altering the overall RNA structure paralleled the reported ability of those mutants to support Tat trans-activation in in cell-based assays, which allowed them to propose that this cellular protein moderates TAR activity in vivo. Their work indicated that the premature transcription termination observed in the absence of Tat occurs at multiple, possible random locations downstream TAR, again consistent with the idea that Tat acts by increasing processivity rather than by preventing a specific termination event [108]. Feinberg et al. extended these studies by treating cell lines containing Tat-defective viruses, which constitutively produced very low levels of virus, with purified Tat protein to increase virus production greater than 30,000-fold, through stimulation of transcription elongation [112], consistent with the in vitro studies by Marciniak and Sharp [113].

Cooperation Between Tat and Cellular Transcription Factors

While early discoveries in the field have provided the critical evidence of a Tat-TAR protein-RNA axis for HIV-1 transcription elongation activation, plenty of evidence also support the requirements of DNA cis elements and cellular TFs in the process of transcription initiation and Tat trans-activation, highlighting the critical importance of a collective DNA and RNA based regulation for robust viral transcription (Figure 2D). In addition, these ideas are consistent with the fact that Tat activity is proportional to the activity on the basal phase dictated by the collection of cellular cTFs assembled on the proviral genome [1, 35, 135]. Taken together, cTFs like Sp1 first regulate transcription in the basal phase and then cooperate with iTFs (e.g., NF-κB and AP-1) to induce low-level transcription during the host phase. As Tat gets synthesized, it engages with cTFs and iTFs to stimulate high-level transcription during the viral phase (Figure 2D). The several reported interactions between Tat and multiple pathway- and sequence-specific TFs points towards a common mechanism to facilitate transcription amplification in the viral phase through interactions with TFs that can be induced or activated in a cell type-dependent or -independent manner through a variety of physiologic ligands.

Below we expand on our collective knowledge of HIV-1 transcription initiation, elongation and potential functional links between initiation and elongation, as well as critical gaps in knowledge to expand our understanding of the HIV-1 transcriptional program.

Transcription Initiation

Transcription initiation is mainly regulated by the formation of the transcription pre-initiation complex and chromatin structure at the 5’ LTR, which are modulated by the binding of sequence-specific TFs alongside the proviral genome. In this section, I discuss the importance of two connected features: the regulation of PIC assembly and chromatin structure to HIV-1 transcription initiation.

Pre-initiation Complex Assembly

Cellular TFs and GTFs.

The recruitment of cellular TFs to their cognate sites is enough to facilitate PIC assembly to initiate transcription. The first step in PIC assembly is the binding of TBP to the TATA box [136], followed by TFIIA and TFIIB prior to Pol II (Figure 3A). The other general transcription factors (GTFs): TFIIF, TFIIE and TFIIH are apparently recruited during and after Pol II [137-140], although the precise mechanistic details as it refers to transcription initiation throughout the three phases of the HIV-1 transcriptional program, are still missing.

Figure 3. — PIC assembly and transcription initiation in the various phases of the HIV-1 transcriptional program. (A) Pathway of PIC formation from the collective knowledge of transcription activation of viral (AdMLP) and human gene promoters [140]. (B-D) Simple scheme depicting PIC assembly and composition status in the basal (B), host (C) and viral (D) phases of the HIV-1 transcriptional program.

In the basal phase, the virtually silent promoter does not appear to assemble a stable PIC [16] because it contains Sp1 alongside several transcriptional repressors that directly or indirectly bind the HIV-1 promoter to inhibit transcription initiation including LBP-1 blocking the TBP-TATA box interaction [33], a p50 NF-κB homodimer bound to the enhancer [141], C-promoter binding factor-1 (CBF-1) [142], COUP-TF interacting protein 2 (CTIP2) [143, 144], estrogen receptor-1 (ESR-1) [145], interferon-γ inducible protein 16 (IFI16) [146], as well as Krueppel-like factor 2 (KLF2) and Krueppel-like factor (KLF3) [147] (Table 1). These transcriptional repressors function by recruiting chromatin-modifying and -remodeling enzymes, which will be discussed in detail in the “Chromatin Structure” section below. Thus, it is the balance between transcriptional activators and/or repressors binding to various regions of the 5’ LTR which ultimately determines the overall transcriptional outcome. In the host phase, cell stimulation elicits the recruitment of iTFs such as NF-κB and AP-1, to perhaps increase the stability and/or content of PIC components in the host phase (Figure 3C), given their ability to engage with TBP and other TFIID subunits [52, 72, 73].

TAFs.

Surprisingly, Raha and Green reported that, Tat stimulated PIC assembly through recruitment of TBP in the absence of TAFs [16] (Figure 3D), unlike other transcriptional activators using TAFs (e.g., VP16, E1A). These data was then validated in two systems harboring integrated proviruses in the absence and presence of cell stimulation (PMA), thus increasing physiologic relevance. While TBP remained undetected at the viral promoter in the absence of Tat, consistent with the lack of PIC formation and Pol II recruitment (Figure 3B), Sp1 and P-TEFb were detected and levels of Sp1 remained constant whereas P-TEFb levels increased with Tat in a TAR-dependent manner, consistent with activation of elongation. These studies illuminated unexpected aspects of HIV-1 transcription control which deserve to be extended using high resolution tools to study the progression of PIC assembly in the multiple phases of the HIV-1 transcriptional program (Figures 3B-D).

Despite the dispensability of TAFs, Raha and Green also showed that the Mediator complex, which represents a fundamental component of the transcription initiation machinery, interacting with and regulating Pol II activity [148], was recruited to the integrated viral promoter in response to but not prior to cell stimulation (PMA) [16] (Figure 3C). The importance of Mediator also was a common discovery among several genome-wide assays profiling cellular factors critical for the virus [149, 150], thus reinforcing the importance of Mediator for HIV-1 gene expression and replication.

Mediator.

Ruiz et al. extended the importance of Mediator to viral transcription [151]. In their studies, silencing of 9 out of the 28 Mediator subunits (MED6, MED7, MED11, MED14, MED21, MED26, MED27, MED28, and MED30) (Table 1) impaired viral replication at a post-integration step without affecting cell viability. Inhibition of transcription initiation was observed by RNAi-mediated silencing of 5 subunits (MED6, MED7, MED11, MED14, and MED28) with MED6 and MED14 having the strongest inhibitory effect in Tat transactivation. Notably, MED14 and Tat associated in cells, potentially indicating the importance of Mediator for both transcription activation prior to and after Tat synthesis (Figures 3C and D), together highlighting the potential importance of HIV-1 promoter–specific Mediator complex interactions. Most recently, the importance of the Mediator kinase module containing CDK8 (Table 1) in promoting HIV-1 transcription was revealed by the use of pharmacologic and genetic approaches [152]; however, it remains unclear whether the Mediator kinase module operates at the transcription initiation and/or elongation steps [153] to either initiate and/or maintain the viral transcriptional program.

Pol II CTD and CTD kinases.

In addition to TBP and Mediator, Tat transactivation of the viral promoter in vitro requires the Pol II C-terminal domain (CTD) and CTD kinases, particularly cyclin-dependent kinase 7 (CDK7), which is part of TFIIH and an integral component of the PIC (Figure 3A). The human Pol II CTD comprises heptad repeats with a general consensus sequence YSPTSPS, which is dynamically phosphorylated in synchrony with the transcription cycle of transcription initiation, elongation, and termination [154, 155]. During transcription initiation, CDK7 phosphorylates the CTD on Ser5 and Ser7 [156, 157], and phosphorylation at these sites is enriched at the 5’ ends of genes in metazoans [155, 158].

Parada and Roeder initially demonstrated that Tat-enhanced HIV-1 transcription in vitro requires both TFIIH and the Pol II CTD [159]. Tat interacts with a functional TFIIH-containing complex through its activation domain and stimulates Pol II CTD phosphorylation by TFIIH in an RNA-dependent manner to promote Pol II processivity. In agreement with Parada and Roeder, Okamoto et al. demonstrated that a pseudo-substrate peptide for CDK7, which selectively blocks CDK7 over CDK9, inhibits Tat activity and HIV-1 replication [160]. Mbonye et al. extended those studies by revealing the importance of CDK7 for Tat transactivation by phosphorylation of the CDK9 T-loop [161], consistent with earlier genetic and biochemical work by Larochelle et al. [162]. While in the basal phase, the levels of TFIIH at the integrated provirus are low to undetectable, consistent with the virtual absence of transcription and lack of stable PIC (Figure 3B), NF-κB recruits TFIIH to promote transcription initiation in the host phase [163] (Figure 3C), and Tat takes over CDK7 to sustain activation in the viral phase.

Supporting a role for Tat in transcription initiation, Kashanchi et al. reported a direct interaction between Tat (residues 36-50) and TFIID composed of TBP and TAFs through a direct interaction with the TBP subunit [127], a discovery that was later confirmed by Veschambre et al. [128]. The Tat-TFIID interaction was later shown to inhibit negative cofactors and stabilize the TFIID-TFIIA complex [164]. In addition to contacting TBP, Tat interacts with other components of the PIC including TFIIE, TFIIF and TFIIH [159] as well as Pol II [165], perhaps to load onto the viral promoter and/or to stimulate one or multiple subsequent transcription steps prior to elongation.

Together, besides interacting with the TAR RNA to promote transcription elongation, Tat binds several factors and protein complexes acting prior to elongation, perhaps illuminating its previously inferred, but not solidly demonstrated importance in transcription initiation, and the precise coordination between initiation and elongation to achieve robust transcription amplification for efficient latency reversal and replication.

Chromatin Structure

As stated in the beginning of this section, the binding of TFs to their cognate binding elements is not only dictated by their presence on the template DNA but also by the chromatin structure established by pioneer factors, as well as chromatin-modifying and chromatin-remodeling enzymes. After integration into the host genome, the HIV-1 provirus adopts a strictly defined chromatin organization contributing to its transcriptional repression through specific nucleosome positioning. The creation of these repressive chromatin structures that impair transcription initiation during reservoir cell homeostasis in the basal phase facilitates both entry into, and maintenance of, viral latency. These repressive nucleosomes must undergo a defined remodeling process involving histone post-translational modifications (PTMs) and nucleosome eviction or displacement prior to transcription activation.

Nucleosome organization and chromatin-remodeling.

While high resolution data on the nucleosome organization at integrated proviruses in multiple clones during latency maintenance and reactivation is still missing, prior studies have revealed a network of nucleosomes and nucleosome-free regions containing DNase I hypersensitive sites (DHSs). Specifically, the 5’ LTR in latent proviruses appears to harbor a specific nucleosomal organization with major DHSs [166]. Three well-positioned nucleosomes (Nuc-0, Nuc-1, and Nuc-2) surrounded by DHSs (1 through 4) corresponding to nucleosome-free regions that are more accessible to TFs, have been mapped in the 5’ LTR and downstream the TSS (Figure 2B). The DHS-4, located in the leader region immediately downstream of the TSS, harbors cis elements for several TFs important for viral transcription including AP-1, IRF, Sp1, ATF, CREB, and NFAT [13, 85]. Another site (DHS-7), which comprehends the pol, vif, vpr, and tat genes (Figure 2A), is part of an intragenic enhancer composed of three functional domains: two regions (+4079/+4342 bp and +4781/+6026 bp relative to the TSS), which exhibit PMA-inducible enhancer action on a heterologous promoter and flank the third domain (+4481/+4982 bp relative to the TSS) [166-168]. These putative intragenic enhancers harbor binding sites for various cellular TFs including Oct-1, AP-1, Est-1, Sp1, and PU.1 [168, 169], and the three AP-1 binding sites facilitate Pol II recruitment to the 5’ LTR in response to cell stimulation (PMA), perhaps working as a bona fide intragenic enhancer during the host phase [170].

In the basal phase, Nuc-1 is positioned downstream of the TSS (+10/+155 bp) [166] to generate a repressive chromatin environment incompetent for transcription activation (Figure 3B). Assembly of positioned nucleosomes requires the cooperative pre-binding of NF-κB and Sp1 [171, 172] and Nuc-1 appears to be maintained by the BRG1-associated factor (BAF) complex, an ATP-dependent chromatin remodeler belonging to the SWI/SNF family [173], whose mechanism of recruitment to the provirus remains unclear. The recent discoveries by Brahma and Henikoff showing that BAF synergizes with promoter-proximal paused Pol II and sequence-specific TFs to evict nucleosomes [174], illuminate a previously unknown regulatory step that may contribute to HIV-1 transcription activation in the host and viral phases.

In the host phase, Nuc-1 is displaced in response to cell stimulation (TNF-α and PMA) [175, 176] in the host phase (Figure 3C) to facilitate chromatin accessibility encompassing the upstream promoter and most of the 5’ UTR associated with transcription activation. Similar chromatin structures were validated in vitro when chromatin assembly occurs in the presence of TFs (Sp1 and NF-κB), again highlighting their cooperative interaction for transcription activation prior to Tat synthesis through modulation of chromatin structure (Figure 3C).

In the host phase, TF binding to the 5’ UTR DHSs also facilitates Nuc-1 disruption because mutation of the cis elements inactivates the viral promoter when it is constrained in a chromatin configuration [177]. Furthermore, the AP-1 binding sites partially overlapping Nuc-1 (Figure 2B), may promote its disruption upon binding by their cognate TFs (AP-1 and/or CREB/ATF) during cell stimulation, consistent with their function as pioneer factors [178, 179] and alteration of chromatin structure [180]. One of the current models is that upon cell stimulation iTFs bind their cognate elements to recruit one or more chromatin-modifying enzymes. For example, the p50/p65 NF-κB heterodimer complex recruits histone acetyltransferases (HATs) such as CBP/p300 to promote histone acetylation and HIV-1 transcription [181]. While CREB site-specific phosphorylation help recruit CBP to alter chromatin structure through site-specific histone acetylation [182, 183], this phenomenon has not been documented with HIV-1, potentially resulting in nucleosome remodeling [184] with the consequent displacement of Nuc-1 to facilitate transcription activation [175, 185].

In the viral phase, BAF is released and the polybromo-associated BAF (PBAF) complex is recruited by Tat to potentially promote, or even sustain, Nuc-1 displacement and chromatin remodeling thereby enabling transcription elongation. In addition to BAF-induced chromatin remodeling, a Tat-independent mechanism by the BRM-containing SWI/SNF complex has been proposed to block transcription elongation [186], perhaps acting in the host phase prior to Tat synthesis. Notably, the short isoform of Bromodomain containing 4 protein (BRD4S), a member of the BET family, acts as a corepressor of HIV-1 transcription by direct binding to the catalytic subunit BRG1 of the BAF complex, thereby preventing Nuc-1 remodeling [187]. Additional functions whereby the various BRD4 isoforms regulate other steps of the HIV-1 transcriptional program are discussed in the sections below.

Like in the host phase, transcription in the viral phase is regulated by histone acetylation through Tat-mediated recruitment of HATs such as p300, CBP and PCAF [188-190]. Additionally, p300 acetylates Tat in the TAR RNA binding domain (lysine 50) (Figure 2C) to reduce formation of a Tat-TAR-P-TEFb ternary complex [191], for which the authors deduced Tat acetylation may help in dissociating the Tat cofactor P-TEFb from TAR RNA and serve to transfer Tat onto the elongating Pol II.

The NF-κB enhancer together with the upstream binding of two other TFs (LEF-1 and Ets-1) also contribute to remodeling of the 5’ LTR chromatin structure [74, 192, 193] and help recruit CBP [181]. LEF-1 is a T-cell-specific TF that promotes assembly and function of enhancer complexes by inducing DNA bending [194-196]. Because activation of latent proviruses is associated with an open chromatin structure in the 5’ UTR [166, 176], iTFs such as AP-1 and NFAT may function to disrupt Nuc-1 independently of, or in concert with, Sp1 binding the core promoter and NF-κB binding at the upstream enhancer, again illustrating the concept of TF co-operativity for synergistic activation of this transcription step.

Unstable nucleosomes such as Nuc-1 can also facilitate transcription by providing the appropriate scaffolding to recruit TFs bound at distant sites into juxtaposition, one of the canonical mechanisms through which enhancers are known to function [197]. Although the HIV-1 genome is constrained in size (<10 kb), as opposed to the human genome (10⁹ kb), Nuc-1 could bring the upstream enhancer and core promoter in close proximity to the downstream enhancer, facilitating protein-protein interactions to occur between the various TFs bound at these remote sites. In this regard, the upstream and downstream NF-κB binding sites can functionally cooperate [198], and NF-κB can physically interact with AP-1 to activate transcription [199], among other critical cooperative TF-TF interactions occurring in the host phase, as explained above.

Chromatin-modifying enzymes.

Previous reports have investigated the roles of chromatin regulators in shaping the chromatin structure at the provirus for transcription activation or repression [200-207]. Several chromatin regulators groups exist including histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs) and histone demethylases (HDMs). Extensive exploitation of histone acetylation and methylation has been reported.

Among histone deacetylation, repressive chromatin-modifying enzymes specifically HDAC1/2 and HDAC3 (Table 1) seem to participate in combinatorial epigenetic silencing of the provirus [208] (Figure 3B). Conversely, activating chromatin-modifying enzymes (HATs) specifically p300/CBP, and PCAF/GCN5 [188-190] have all been linked to HIV-1 transcription activation and latency reversal (Table 1).

Among histone methylation, two families of enzymes have been explored: Histone Lysine Methyl Transferases (HKMTs) [209, 210] and Histone Arginine Methyl Transferases (HRMTs) [211]. These enzymes methylate specific lysine residues on histones (H3 and H4), in addition to non-histone substrates, to modulate the accessibility of TFs to gene promoters and the subsequent changes in transcription. Based on the current data, some HKMTs were proposed to activate latent HIV-1 in basal and/or stimulation conditions (Table 1) including: MLL1 [202, 203], MLL2 [203], MLL3 [203], SETD7 [203], SETDB2 [203], SETD8 [203], Suv420-H2 [203], SETMAR [203], SMYD3 [203], SMYD5 [203], and MLL5 [203], while others were proposed to repress HIV-1 transcription to maintain latency (Table 1) including: Suv39H1 [200, 203], SETDB1 [204], GLP [205], G9a [206], ASH1L [203], Suv420-H1 [203], EZH2 [207], and SMYD2 [203].

The functions of other HKMTs remain inconclusive because of conflictive divergent activities or shRNA-specific phenotypes SETD1A [202], SETD1B [202], MLL4 [202, 203], SETD2 [203], NSD1 [203], NSD2 [203], NSD3 [203], DOT1L [203], EZH1 [203], SETD5 [203]. HRMTs have also been linked to the control of HIV-1 proviral fate. Zhang et al. reported that histone 3 lysine 27 acetylation (H3K27ac) recruited elongation factors and stimulated histone 3 arginine 26 (H3R26) methylation, which subsequently abrogated their recruitment, forming a negative feedback loop. Inhibition of the HRMT responsible for H3R26 deposition (CARMA1) (Table 1) reversed HIV-1 latency [212] providing another example of a potential transcriptional repressor through the control of chromatin structure.

Together, iTFs may function redundantly and/or combinatorially alongside chromatin-modifying and chromatin-remodeling enzymes, to increase chromatin accessibility to favor other steps in the transcriptional cycle (e.g., PIC assembly and or Pol II promoter escape) to promote viral transcription. The switch between cellular and viral (Tat) TFs from the host and viral phases of the transcriptional program may help the provirus maintain its chromatin accessibility for efficient Pol II recruitment to the promoter for transcription initiation and the transition into elongation. Future studies will benefit by examining the detailed, progressive and dynamic chromatin structure changes throughout the HIV-1 transcriptional program at high resolution.

Transcriptional Pausing, Pause Release, and Elongation

Soon after transcription initiation, Pol II undergoes a rate-limiting step of pausing downstream the TSS, before it is released from the pause site to transition into productive elongation. In this section, I describe salient aspects of Pol II pausing at the viral promoter, pause release and elongation, including the discovery of the P-TEFb kinase, and the assembly and regulation (both positive and negative) of transcription elongation complexes (Figure 4).

Figure 4. — Transcriptional pausing, pause release, and elongation in the various phases of the HIV-1 transcriptional program. Depiction of the factors involved in Pol II pausing at the 5’ LTR soon after transcription initiation and the transition to pause release for productive escape into elongation in the basal (A), host (B) and viral (C) phases of the HIV-1 transcriptional program.

Pausing

Pol II must escape the promoter through CDK7-mediated phosphorylation of Pol II in the PIC [213, 214]. Once released from the PIC, Pol II begins transcribing, but pauses at the promoter-proximal region ~20–50 bp downstream of the TSS (reviewed in [215, 216]). The first depiction of Pol II pausing on a genome-wide scale, a feature of active transcription, came in 2007 with the study of Muse and Adelman [217]. Although the high resolution analysis of Pol II pausing at the provirus is still lacking, several studies have provided important mechanistic insights through a combination of in vitro and cell-based assays.

Palangat and Landick carefully examined the phenomenon of pausing at HIV-1 using an in vitro approach [218]. They reported that pausing at the viral promoter partially relies on an alternative RNA structure referred to as “the HIV-1 pause hairpin” that competes with TAR RNA formation. By probing the nascent RNA structure in halted transcription complexes, they found that the transcript folds as the pause hairpin before and at the pause, and rearranges to TAR concurrent with or just after escape from the pause. The pause signal triggers a 2-nt reverse translocation by Pol II that appears to block the active site and be counteracted by formation of TAR. Their studies illustrated how the pause site modulates nascent RNA rearrangement from a structure that favors pausing to one that both recruits Tat and promotes escape from the pause for productive elongation. However, Pol II pausing at high resolution in vivo has not been yet studied so it remains unclear how this in vitro data relate to the in vivo scenario during the phases of the HIV-1 transcriptional program.

NELF and DSIF.

The establishment of paused Pol II is facilitated by nucleosomal barriers near the TSS and negative elongation factors [DRB Sensitivity Inducing Factor (DSIF) and Negative Elongation Factor (NELF)] (Figure 4 and Table 1) [219-223]. Two key aspects of the HIV-1 transcriptional cycle include NELF–mediated Pol II pausing and kinase–induced pause release. Zhang and Henderson provided evidence that the five subunit NELF complex (A/B/C/D/E) [222] associate with the provirus and that its depletion increases viral transcription [224]. Potassium permanganate has been utilized to detect the block in elongation resulting from Pol II pausing by mapping the increased sensitivity of unpaired thymidine’s in the transcription bubble [225-227]. Pol II mapping on the 5’ LTR using potassium permanganate probing revealed that Pol II was paused in a chronically infected cell line in the absence (basal phase) and presence (viral phase) of cell stimulation at ~25-50-nt downstream the TSS and that NELF depletion (NELF-B subunit) released it from the pause site, which correlated with displacement of Nuc-1 and increased H4 acetylation [224]. Jadlowsky and Karn partially extended these studies by providing evidence that RNAi-mediated NELF silencing induced proviral transcription [228], but the use of chronic RNAi in their studies may have altered the expression of NELF-regulated genes that can feedback on the 5’ LTR perhaps eliciting indirect HIV-1 transcription, as originally reported in studies with Drosophila [229].

The NELF-E subunit contains a conserved RNA recognition motif that facilitates Pol II pausing through its association with nascent RNA. Pagano and Lis used in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) required for sequence-specific RNA recognition genome-wide [230]. They found that TAR loop located +30 to +34 bp downstream the TSS (Figure 2C), contains an NBE-like element, required for high affinity RNA-binding, as opposed to the lower stem of TAR, as originally suggested [231, 232]. The NBE-like element assists in the establishment of paused Pol II during transcription activation [230], together supporting a biological role for NELF-E in promoter-proximal pausing at the HIV-1 promoter as well as cellular gene promoters.

Pause Release and Elongation

P-TEFb discovery.

This section introduces the positive transcription elongation factor b (P-TEFb) complex, all the way from its discovery to current mechanistic insights. P-TEFb is composed of the regulatory cyclin T1 (CycT1) subunit and the cyclin dependent kinase 9 (CDK9) subunit (Table 1) (reviewed in [223]) and is critical for promoting transcriptional pause release and elongation from the 5’ LTR.

Early work by Marshall and Price has identified two classes of elongation complexes, abortive or productive depending on their ability to terminate prematurely or fully, respectively [233]. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which they call P-TEF (positive transcription elongation factor), which was selectively inhibited by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) at concentrations that did not alter transcription initiation. While the abortive complexes lacked P-TEF, productive elongation was restored to isolated transcription complexes by the addition of P-TEF after initiation. By fractionating nuclear extracts of Drosophila cells, Marshall and Price identified P-TEFb, composed of two polypeptide subunits with apparent molecular masses of 124 and 43 kDa, which acted after initiation and was essential for the production of DRB-sensitive long transcripts in vitro [234].

Functional homology between the Drosophila and putative human kinases was suggested by transcriptional analysis of a Pol II regulated promoter with HeLa nuclear extracts depleted of the human homolog called PITALRE. The depleted nuclear extracts lost the ability to yield long DRB-sensitive transcripts and this loss was reversed by the addition of Drosophila P-TEFb, strongly indicating that PITALRE is a component of human P-TEFb. Through molecular analysis, they found that PITALRE interacted with the Tat activation domain (Figure 2C), consistent with the work by the Rice lab [235], signifying that P-TEFb was the previously reported Tat-associated kinase (TAK), and suggested that the enhancement of transcriptional processivity by Tat was attributable to augmented function of P-TEFb on the viral promoter. These studies led to the cloning of the human homolog PITALRE of the Drosophila P-TEFb kinase [236], and subsequent work by the Rice lab confirmed that PITALRE indeed was the catalytic subunit of TAK required for Tat trans-activation [237].

Assembly and regulation of Tat:TAR:P-TEFb transcription elongation complexes.

Another foundational study came in 1998, when Wei and Jones described a novel CDK9-associated C-type cyclin (CycT1: 87 kDa) that interacted directly with the Tat activation domain to mediate high-affinity, loop specific binding to TAR RNA [238] (Figure 2C). Tat interaction with CycT1 strongly enhanced the affinity and specificity of the Tat:TAR protein-RNA interaction, which conferred a requirement for sequences in the TAR stem-loop not recognized by Tat alone, collectively demonstrating that Tat redirects P-TEFb (CycT1/CDK9) in close proximity to Pol II through cooperative binding to TAR RNA (Figure 4). Remarkably, ectopic expression of human CycT1 in murine cells, which are refractory to HIV-1 transcription and replication, rescued Tat activity, a discovery that was later confirmed by work in the Zhou and Peterlin labs [239, 240].

The CycT1 cyclin domain was necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro and mediate Tat transactivation in cells [241]. The cyclin domain contains a C-terminal region (residues 250-272) referred to as Tat:TAR recognition motif (TRM) that can simultaneously interact with Tat and CDK9 on TAR RNA in vitro, providing the molecular bases for enhanced Tat:TAR:P-TEFb binding affinity. The interaction between Tat and CycT1 requires zinc as well as essential cysteine residues in both proteins: the seven cysteines in Tat as illustrated by Frankel and Pabo [106] and cysteine 261 by CycT1 to complete the formation of the two zinc finger domains between Tat and CycT1 (Figure 2C). This discovery explained why murine CycT1, which lacks cysteine 261, forms a weak, zinc-independent interaction with Tat that greatly reduces TAR RNA binding in vitro, Tat trans-activation and viral replication in cells. In addition, using RNA-protein photocross-linking and protein foot printing assays Richter et al. later showed that the CycT1 TRM interacts with one side of the TAR RNA loop (particularly U31) and enhance interaction of Tat lysine 50 to the other side of the loop (with G34) [242], providing evidence that TAR RNA functions as a scaffold for two protein partners to assemble the elongation complex. Contemporarily with Garber et al. [241], the studies by Zhou et al. collectively indicated that P-TEFb can mediate Tat trans-activation at multiple levels [243], including by binding to TAR RNA, by phosphorylating the Pol II CTD and by interacting with and phosphorylating Tat cofactors (Tat-SF1) [244, 245] (Table 1).

Despite the requirement of P-TEFb for assembly of a high-affinity Tat:TAR:P-TEFb complex, P-TEFb is incapable of forming a stable complex with Tat and TAR RNA due to a two built-in auto-inhibitory mechanism in P-TEFb [246]. The first auto-inhibition step arises from the unphosphorylated state of CDK9, which establishes a P-TEFb conformation unfavorable for TAR recognition. Kinase autophosphorylation overcomes this inhibition by inducing conformational changes in P-TEFb, thereby exposing the CycT1 TRM for TAR binding. The second auto-inhibitory step constitutes an intramolecular interaction between the N- and C-terminal regions of CycT1 that sterically blocks the P-TEFb:TAR interaction. This inhibition is relieved by the binding of the C-terminal region of CycT1 to the transcription elongation factor Tat-SF1 and perhaps other cellular factors. Upon release from the intramolecular interaction, the C-terminal region also interacts with Pol II and is required for viral transcription, suggesting its role in bridging the Tat:TAR:P-TEFb elongation complex, collectively demonstrating a carefully orchestrated regulatory mechanism for the assembly of the transcription elongation complex at the viral promoter.

Tat:P-TEFb complexes containing CDK9 phosphorylated in its T-loop, and CycT1 phosphorylated in its C-terminal region, enhanced binding to TAR RNA dramatically, together providing evidence that CDK9 phosphorylation is required for high-affinity binding of Tat:P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation regulates Tat transactivation. Specifically, the threonine at position 186 in the T loop of Cdk9 must be phosphorylated for full kinase activation [247, 248]. CDK7 can phosphorylate CDK9 in the PIC without Tat [162] and Tat has been shown to stimulate CDK9 T-loop autophosphorylation in vitro [249], potentially illustrating diverse mechanisms of P-TEFb kinase activation for transcription initiation and elongation. Besides threonine 186, CDK9 phosphorylation at serine 175 within the T-loop enhanced viral transcription in T-lymphocytes [250], perhaps enhancing P-TEFb:BRD4 and P-TEFb:Tat interactions in the host and viral phases, respectively.

Using immobilized DNA templates, Isel and Karn studied the effects of Tat on P-TEFb kinase activity during the transition from initiation to elongation [251]. They found that in the PIC stage, Pol II is rapidly phosphorylated by TFIIH and that Tat does not modulate the rate nor the extent of CTD phosphorylation, despite the fact that P-TEFb subunits are present at equivalent levels in both the pre-initiation and elongation complexes, in agreement with subsequent studies by Raha and Green [16] and D’Orso and Frankel [120] using cell based assays. However, Tat was shown to stimulate additional CTD (Ser2) phosphorylation in elongation complexes in a TAR-binding-dependent manner, consistent with its elongation stimulating capability.

Using a similar, immobilized DNA template assay, Zhou and Brady also analyzed the effect of Tat on P-TEFb kinase activity during initiation and elongation [252]. In agreement with Isel and Karn they found that both TFIIH and P-TEFb associated with the PIC, but at this stage the Pol II CTD was only found to be phosphorylated at Ser5 residues by TFIIH, but not by P-TEFb. Surprisingly, in the presence of Tat the substrate specificity of P-TEFb was altered to phosphorylate both Ser2 and Ser5, again perhaps supporting the idea that PIC composition and/or activity may differ during the basal and activated phases of the HIV-1 transcriptional program (Figures 4A-C) and suggesting that the ability of Tat to increase transcriptional elongation may be due to its capability to modify the substrate specificity of P-TEFb. Despite being one of the canonical Ser2 CTD kinases, P-TEFb can also phosphorylate Ser5 and Ser7 in vitro [253], perhaps explaining the observed change in substrate specificity upon Tat binding [252], which may be beneficial for the virus to sustain the positive feedback loop (Figure 1).

In agreement with prior studies [246, 251], Garber and Jones showed that purified Tat stimulates Pol II CTD phosphorylation by CDK9, but not by CDK7 in vitro [254], but these results contradicted those from Parada and Roeder, who also demonstrated that Tat could stimulate the TFIIH kinase to phosphorylate the CTD [159]; however, an explanation for this discrepancy remains to be elucidated.

Subsequent reports also provided substantiating evidence of P-TEFb mediated Pol II pause release [255]. Ping and Rana used a stepwise Pol II walking approach coupled with a kinase assay to define the dynamics between Tat and the DSIF and NELF elongation factors. DSIF and NELF associates with Pol II complexes early and travel with elongation complexes as the nascent RNA is synthesized in the host and viral phases (Figures 4B and C), but not in the basal phase (Figure 4A), and Tat stimulates DSIF and Pol II site-specific phosphorylation by P-TEFb during elongation, revealing molecular details for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter, consistent with the pioneering in vitro work by Wada et al. [220].

Using a similar transcription elongation assay with arrested elongation complexes containing unphosphorylated Pol II CTD, Kim and Karn also provided direct evidence that Tat activated P-TEFb directly to phosphorylate the Pol II CTD to enhance viral transcription elongation in a well-defined in vitro assay [256]. Extending these studies, Bourgeois and Karn later developed a three-stage Tat-dependent transcription assay that allowed the isolation of active PIC, early-stage elongation complexes, and Tat-activated elongation complexes to study DSIF contributions to the HIV-1 transcriptional cycle [257]. The SPT5 subunit of DSIF was recruited shortly after initiation, in agreement with Ping and Rana [255], and SPT5 and Pol II were simultaneously phosphorylated by P-TEFb soon after Tat contacts TAR RNA. While SPT5 was not required for early elongation, it plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of Pol II pausing at arrest sites, including bent DNA sequences. Fujinaga and Peterlin further demonstrated Tat-induced, P-TEFb phosphorylation of the NELF-E subunit [222], at sites next to its RNA recognition motif, which prevented TAR binding and perhaps promoted its eviction from chromatin to facilitate viral transcription elongation [232] (Figure 3).

Taken together, P-TEFb regulates Pol II pause release by phosphorylating Ser2 residues in the Pol II CTD as well as elongation factors such as the SPT5 subunit of the DSIF complex to enhance processivity and elongation. While in the viral phase Tat directly recruits P-TEFb to the TAR RNA to be strategically positioned nearby the Pol II CTD (Figure 4), in the host phase several P-TEFb molecules could be recruited through different mechanisms including sequence-specific TFs such as NF-κB [258], transcriptional regulators like BRD4 [259, 260] and perhaps be delivered as part of the 7SK small nuclear ribonucleoprotein complex (snRNP) complex through the Kruppel-associated box (KRAB)-associated protein 1 (KAP1) / tripartite motif containing 28 (TRIM28) transcriptional regulator [261] (Table 1) to directly facilitate signal-induced transcription [262]. It remains unclear though, whether these mechanisms function in a concerted or mutually exclusive manner, and thus most if not all proposed mechanisms must be cross-validated through the use of acute factor depletion approaches.

Structure of Tat:P-TEFb and Tat:TAR:P-TEFb complexes.

Our understanding of Tat and P-TEFb-mediated transcription elongation has benefited tremendously by structural data. In an important study, Tahirov and Price solved the crystal structure of Tat bound to P-TEFb, in which Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with CycT1, but also with the CDK9 T-loop [263], extending the initial structural depiction of P-TEFb, its regulation by T-loop phosphorylation and inactivation by flavopiridol [264]. Tat binding induced significant conformational changes in P-TEFb, perhaps related to the mechanism of kinase activation prior to the formation of transcription elongation competent complexes.

The structure of the HIV-1 Tat:TAR:P-TEFb transcription elongation complex has remained elusive. However, Anand and Geyer solved the structure of the CycT1 cyclin box domain in complex with Tat and TAR RNA from the equine infectious anemia virus (EIAV), a distantly-related virus to HIV-1 [265]. The RNA binding domain of Tat was shown to adopt a helical structure whose flanking regions interact with a CycT1-specific loop in the first cyclin box repeat, together coordinating the stem-loop structure of TAR. These data revealed important features in the assembly of the ternary RNA-protein complex, which one day may help solve the human:HIV-1 transcription elongation complex.

Given the importance of Tat site-specific acetylation (lysine 28) for high affinity assembly of the Tat:TAR:P-TEFb complex (by enhancing an interaction with the CycT1 TRM) for viral transcription elongation [266] and replication [190], future structural attempts could benefit from including acetylated Tat, in addition to T-loop phosphorylated kinase on reconstituted RNA-protein complexes and/or maybe adding full-length CycT1 and Pol II [267].

Along this line of thinking, Tat itself has been subject of reversible PTMs through several cellular modifying enzymes including acetylation [191, 268-270], methylation [204, 271-273], and ubiquitination [274, 275]. Interested readers should consult excellent reviews [276], but this research area had lost some traction and recent, up-to-date reviews are missing. Given the large number of lysine’s (28, 29, 41, 50, 51, 71) that can undergo one or multiple PTMs and the several enzymes that have been reported (Table 1), future studies should aim at exploring the dynamic nature of Tat PTMs in the context of the multiple phases of the HIV-1 transcriptional program to better understand their role during latency maintenance and reactivation. A better understanding of their functional importance to discrete events of the HIV-1 transcriptional program can help distill those, like lysine 28 acetylation, important for high affinity assembly of Tat:TAR:P-TEFb ternary complexes to increase the chances of solving their structures at high resolution.

Negative regulation of P-TEFb activity and transcription elongation.

Expanding on the regulation of P-TEFb kinase activity, back-to-back papers reported that the abundant and evolutionary conserved 7SK small non-coding RNA (7SK snRNA) [277] was a negative regulator of P-TEFb activity, contributing to an important feedback loop modulating Pol II transcription [278, 279]. 7SK snRNA prevented general and Tat-specific transcriptional activities of P-TEFb in vitro and in cells by assembling with and inhibiting CDK9 catalytic activity [278]. The interaction between P-TEFb and 7SK snRNA was reversible as the RNA was shown to be dissociated from P-TEFb by stress and transcription activating stimuli including DNA damaging agents (UV) and transcription inhibitors (actinomycin D), which enhanced kinase activity and transcription activation [280-282], providing a mechanistic explanation for their stimulatory effects.

The 7SK snRNA alone was insufficient to inhibit P-TEFb kinase activity [283, 284], which also required the RNA-binding protein HEXIM1 (Table 1) for mediating the HEXIM1:7SK snRNA:P-TEFb interaction to inhibit transcription in vitro and in cells. P-TEFb disassembled from HEXIM1:7SK snRNA in cells undergoing stress response, increased the level of active P-TEFb for stress-induced transcription [283], in agreement with the prior work showing temporary HIV-1 transcriptional activation [280-282]. HEXIM1 was induced upon cell differentiation to cooperate with 7SK snRNA to sequester P-TEFb into a large kinase-inactive HEXIM1:7SK snRNA:P-TEFb complex, to control cell growth and differentiation. Similarly, in resting CD4+ T cells, HEXIM1 levels are extremely low, in agreement with low CycT1 and T-loop phosphorylated CDK9, which increase largely during T cell activation [285], perhaps due to the increased cellular transcriptional demand and most likely contributing to increased viral transcription.

The first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 (KKKHRRRPSKKKRHWKP), were both necessary and sufficient for binding to 7SK RNA in vitro and in cells [286], but insufficient to inhibit the P-TEFb kinase. This motif contained clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in the Tat RNA-binding domain (RKKRRKRRR). These discoveries indicated that a similar RNA-protein recognition mechanism may regulate the formation of the Tat:TAR:P-TEFb and HEXIM1:7SK:P-TEFb protein-RNA complexes, which may help convert the inactive HEXIM1:7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.

The molecular mechanism underlying 7SK RNA-mediated regulation of P-TEFb function was under intense investigation. Barboric et al. used in cell and in vitro assays to propose a model whereby the interplay between 7SK snRNA and oppositely charged (positive and negative) regions in HEXIM1 directed its binding to P-TEFb and subcellular localization, culminating in the inhibition of transcription [287]. A basic, arginine-rich region interacted with adjacent acidic patches in the absence of 7SK snRNA, but was essential for HEXIM1 binding to 7SK snRNA, P-TEFb, and transcription inhibition. The removal of the positive or negative charges from these regions in HEXIM1 led to its sequestration into the 7SK snRNP complex and inhibition of transcription independently of the arginine-rich motif.

Subsequently, the Kiss lab showed that two different 7SK snRNA elements direct P-TEFb and HEXIM1 binding [288]. By characterizing the essential elements of 7SK snRNA directing HEXIM1 and P-TEFb binding, they proposed a model for the assembly of the HEXIM1:7SK:P-TEFb complex. Two structurally and functionally distinct protein binding elements located in the 5’ and 3’ terminal hairpins of 7SK snRNA supported the recruitment of HEXIM1 and P-TEFb in cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5’ hairpin of 7SK snRNA, which is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3’ hairpin of 7SK RNA, which is highly reminiscent of TAR RNA.

In addition to the 7SK snRNA-unbound and -bound P-TEFb complexes, a fraction of 7SK snRNA interacts with a series of RNA-binding proteins including RNA helicase A (RHA) and heterogeneous nuclear ribonucleoprotein (hnRNPs) A1, A2/B1, R and Q [289, 290] (Table 1). Transcription inhibition simultaneously induced the disassembly of P-TEFb from the 7SK snRNP while increasing the levels of 7SK RNA containing the hnRNP proteins. This process is reversible as removal of the transcription inhibitors restores the original levels of the 7SK snRNP and 7SK snRNA:hnRNP complexes. 7SK snRNPs containing mutant 7SK snRNAs lacking the capacity for binding hnRNPs are resistant to stress-induced disassembly, indicating that recruitment of this class of proteins is essential for disruption of 7SK snRNP and P-TEFb release.

Resembling the mode of 7SK snRNA:hnRNPs complex formation during 7SK snRNP disassembly, two studies demonstrated that Tat could manipulate the inhibitory snRNP to obtain P-TEFb for transcription activation in the viral phase (Figure 4C). First, Barboric et al. demonstrated that through its activation domain, Tat prevented the formation of, and also released P-TEFb from, the 7SK snRNP complex in vitro and in vivo [291]. The activity of Tat stemmed from the higher affinity of P-TEFb binding relative to HEXIM1, which allowed for the direct displacement of the kinase inhibitor, together signifying that Tat not only recruits but also increases the active pool of P-TEFb for efficient viral transcription. Second, Sedore et al. extended those studies by showing that HIV-1 infection led to the release of P-TEFb from 7SK snRNP in a Tat-dependent manner [292].

P-TEFb and HEXIM1 are tightly connected to two previously-uncharacterized proteins: MePCE/BCDIN3 (Table 1), which is the 7SK snRNA methyl-phosphate capping enzyme [293], and a La-related protein, LARP7 also referred to as PIP7S (Table 1). MePCE is an evolutionary conserved methyltransferase required for maintaining 7SK snRNA stability [293]. The function of LARP7 was first reported by the Zhou lab [294], and then followed by the Price lab [295]. LARP7 facilitated 7SK snRNP complex integrity to suppress P-TEFb kinase activity and transcription elongation [294]. At difference to HEXIM1 and the hnRNPs, which can be reversibly bound, LARP7 was a stable component critical for 7SK snRNA stability whose loss compromised 7SK snRNA stability in cells, leading to an initial increase in free P-TEFb and elevated Tat transactivation of the viral promoter in reporter assays, but ultimately causes a decrease in total P-TEFb levels [295], highlighting the intertwined connection among all 7SK snRNP components. Barboric et al. showed that not only LARP7, but also MePCE forms a stable, cell-stress resistant particle with 7SK RNA (termed “core” 7SK snRNP) [296], which was later confirmed by Xue et al. [297]. In addition to capping the 7SK snRNA, Xue et al. discovered a capping-independent function of MePCE in stabilizing 7SK snRNA and facilitating the assembly of the inhibitory 7SK snRNP.

Muniz and Kiss used in cell RNA-protein interaction assays to determine the sequence and structural elements of 7SK snRNA directing assembly of the “core” snRNP (MePCE:7SK:LARP7) [298, 299]. MePCE interacts with the short 5’-terminal G1-U4/U106-G111 helix-tail motif and LARP7 binds to the 3’-terminal hairpin and the following U-rich tail of 7SK snRNA, demonstrating that the overall RNA structure and some particular nucleotides provide the information for specific protein recognition. Notably, LARP7 binding was a prerequisite for P-TEFb recruitment, indicating that besides providing RNA stability, LARP7 directly participates in P-TEFb regulation.

Structural studies have more recently revealed that conformational switching on the RNA takes place during 7SK snRNP biogenesis and P-TEFb regulation [300], in agreement with earlier work from the Price lab [301], revealing that the RNA facilitates a role as an RNP chaperone, and that the core snRNP functions as a scaffold for switching between different RNA conformations essential for complex assembly and regulation of P-TEFb sequestration and release. In back-to-back publications, Olson et al. [302] used a single-molecule probing approach to also demonstrate that 7SK snRNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. They proposed that the 7SK snRNA structural equilibrium shifts in response to cell growth and stress conditions and could then be targeted to modulate P-TEFb–mediated HIV-1 transcription.

Positive regulation of P-TEFb kinase activity and transcription elongation.

P-TEFb release factors.

P-TEFb can be released from the 7SK snRNP by several “release factors” including Tat and BRD4 through the P-TEFb–binding region [303], by the action of phosphatases through dephosphorylation of the kinase T-loop including PP2B and PP1α [304], PPM1A [305] and PPM1G [249], and by the action of RNA helicases such as DDX21 [306] (Table 1). However, it remains elusive whether one or multiple redundant mechanisms exist to activate P-TEFb from the 7SK snRNP complex for viral transcription. Because several important reviews have provided detailed information regarding this critical step of P-TEFb activation [223, 299, 307-309], I keep this section focused so we can extend on other critical aspects of the process of viral transcription elongation activation.

One possible mechanism of P-TEFb release from the 7SK snRNP in the host phase is through BRD4, which belongs to the BET family of bromodomain-containing chromatin-binding proteins containing [310]. BRD4 was implicated as a positive regulatory component of P-TEFb and Pol II-dependent transcription [259, 260]. BRD4 interacted with the 7SK snRNA-unbound P-TEFb pool through its bromodomain and facilitated P-TEFb-dependent phosphorylation of Pol II CTD and stimulation of transcription from gene promoters in vivo through an association with acetylated chromatin to maintain functional equilibrium in the cell. In stress-induced cells, the inactive 7SK snRNA-bound P-TEFb complex was quantitatively converted into the BRD4-associated form [260]. This step was necessary to form and recruit the transcriptionally active P-TEFb complex to a promoter, and enable P-TEFb to contact Mediator, a potential target for the BRD4-mediated recruitment mechanism [311].

Release of P-TEFb from the 7SK snRNP is also accompanied by the loss of HEXIM1 through major conformational changes on the 7SK snRNA that block its re-association, thus combinatorial alterations of RNA structure and factor-induced competition mechanisms are at the center of P-TEFb release from the 7SK snRNP prior to activation of transcription elongation. Muniz and Kiss have demonstrated that Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5’ hairpin of 7SK snRNA, which is structurally and functionally indistinguishable from TAR and is imbedded in the HEXIM1-binding elements [312]. HEXIM1 targets a repeated GAUC motif in 7SK snRNA and promotes base pair rearrangements [313]. Interestingly, Tat replaced HEXIM1 on the 7SK snRNA to promote the disassembly of the 7SK snRNP to augment the nuclear level of active P-TEFb to promote viral transcription. Using a structural biology approach, Pham et al. followed up on these molecular studies to demonstrate that Tat interactions with 7SK snRNA and TAR harbor structural mimicry [314].

Besides the discovery that P-TEFb switches from the 7SK snRNP complex to its free, active form, D’Orso and Frankel detected both activating and inhibitory subunits of the snRNP at the integrated viral promoter in HeLa [120] and Jurkat T [261] cell lines, as well as biochemical evidence in a reconstituted system [120], discoveries that were later extended to cellular gene promoters [261, 315]. These pioneering studies suggested that P-TEFb was recruited to the viral promoter in a catalytically inactive state bound to 7SK snRNP, thereby preventing elongation and that the TAR RNA bound Tat and P-TEFb as it emerged on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase, further expanding on the simple concept of Tat physical competition with HEXIM1 for P-TEFb.

These reports have later led to a two-step model where Tat first preassembles at the viral promoter with the 7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation, perhaps consistent with the model of Tat regulation of both initiation and elongation and the presence of P-TEFb in the PIC prior to Tat. To define how the Tat:P-TEFb complex transitioned to TAR to activate the P-TEFb kinase, D’Orso and Frankel combined a comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probed Tat’s activity in different arrangements, genetically defining a transition step in which pre-assembled Tat:P-TEFb complexes switched to TAR RNA through a conserved network of Tat residues [121]. P-TEFb artificially recruited to the nascent transcript was incompetent for transcription, but rather remained inactive due to its assembly with the 7SK snRNP. Tat supplied in trans was then able to displace HEXIM1 from the snRNP and activate P-TEFb, allowing the uncoupling of Tat requirements for kinase activation and TAR binding.

SEC.

Besides the release of P-TEFb from the 7SK snRNP, several Tat:P-TEFb co-activators that further stimulated viral transcription elongation in the viral phase have been identified using an affinity purification - proteomics approach, including the eleven-nineteen Lys-rich leukemia (ELL) protein ELL2 and several frequent mixed lineage leukemia (MLL)-fusion partners involved in leukemia (AFF4, AFF1, ENL or AF9), and the PAF1 complex (PAF1C) [316-318] (Table 1). The increased transcription elongation capacity of P-TEFb when bound to these co-activators prompted the name of Super Elongation Complex (SEC) [319]. Tat stimulated HIV-1 transcription by recruiting a SEC containing P-TEFb, AFF4/1, ELL2, and ENL or AF9 to the viral promoter [318] (Figure 4). Tat interaction with these elongation factors relied on an active P-TEFb kinase, consistent with early work positioning the T-loop phosphorylated kinase on the nascent RNA [246, 254], and that Tat enhanced the interaction between AFF4 and ELL2:P-TEFb to form a bifunctional elongation complex to super-induce viral transcription relative to P-TEFb activation alone.

AFF4 is the central scaffold that recruits the other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and AF9 or ENL act as bridging components that link this complex to P-TEFb and PAF1C. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of the multiple elongation factors and connect the components not only to each other, but also to a larger network of transcriptional regulators [320]. Tat selectively recruits P-TEFb as part of the SEC. Alber and colleagues determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 makes functionally critical, extended contacts with Tat and CycT1, but not with CDK9, and Tat enhances P-TEFb affinity for AFF4 [321]. The crystal structure of Tat bound to P-TEFb and the AFF4 scaffold was later determined to define the mechanism of SEC recruitment to TAR RNA [322]. Interestingly, Tat and AFF4 directly fold onto the surface of CycT1 which partially orders the CycT1 TRM to increase the affinity of Tat:P-TEFb for TAR RNA. The crystal structure (5.9Å resolution) of the TAR RNA containing Tat:P-TEFb:AFF4 complex [323], which revealed that the TAR central loop contacts the CycT1 TRM and the second Tat Zn(2+)-binding loop. Part of AFF4 is stabilized in the TAR complex, despite not binding the RNA, explaining how AFF4 largely enhances TAR recognition by Tat:P-TEFb. Their structure-function analysis supported an integrative binding model, wherein the TAR loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat RNA binding domain.

Besides AFF4, the AFF1 paralog was also shown to be important for P-TEFb binding and kinase activation [324]. AFF1, like AFF4, also increased the affinity of Tat for CycT1 to facilitate P-TEFb release from the 7SK snRNP with the subsequent formation of Tat-SEC complexes for viral transcription activation. Thus, it seems that Tat can exploit AFF family member redundancy by using either AFF1 or AFF4 as central scaffold to assemble the SEC with P-TEFb onto the nascent TAR RNA for enhanced transcription in the viral phase (Figure 4C).

Kuzmina and Taube re-examined the contributions of AFF1 and AFF4 SEC components on viral transcription before and after Tat synthesis [325]. In the absence of Tat (basal phase), AFF4 appears to activate HIV-1 transcription at substantially higher levels than its homolog AFF1, with differential recruitment to the viral promoter and association with PAF1C. In the presence of Tat (viral phase), HIV-1 transcription reached optimal levels despite AFF1 or AFF4 individual depletion; however, their combinatorial depletion completely weakened Tat trans-activation, highlighting their importance for Tat in the viral phase. In their studies, P-TEFb did not seem to be critical for viral transcription before Tat (basal phase), so it remains unclear the observed AFF1/AFF4 dependence if P-TEFb is dispensable.

PAF1C.

Besides the SEC, PAF1C is another important positive regulator of HIV-1 transcription. PAF1C is composed of 5 core subunits (PAF1, LEO1, CDC73, LEO1 and WDR61) and a catalytic subunit (RTF1) that is not stably bound [326]. PAF1C is a positive regulator of transcription that functions by promoting pause release and elongation directly [327-329] and indirectly by recruiting the SEC [318]. Using chronic silencing, the Shilatifard lab proposed that PAF1C functions as both positive and negative regulator of human genes [330, 331]; however, subsequent reports using acute depletion approaches convincingly demonstrated that PAF1C is a positive regulator involved in facilitating Pol II processivity and elongation rate [332, 333].

Gao et al. proposed that competition between PAF1C and the histone methyltransferase Mixed lineage leukemia 1 (MLL1) for the lens epithelium-derived growth factor (LEDGF) protein is what determines HIV-1 proviral fate [202]. While LEDGF has been first functionally associated to HIV-1 integration [334], this study proposed a direct link to transcription activation. In the basal phase, LEDGF appears to suppress transcription by recruiting PAF1C to enforce Pol II pausing and then promotes latency reversal through MLL1 competititve displacement of PAF1C. One caveat of these studies was the use of chronic factor silencing potentially eliciting indirect effects, thus until the results are reproduced using acute depletion systems, these conclusions should be taken with cautious.

Perhaps leveraging the concept of HIV-1 transcriptional repression by PAF1C, Soliman et al. used a docking approach to identify small molecule inhibitors of PAF1C that induce Pol II pause release [335]. The identified compounds did not reactivate latent HIV-1 per se but seemed to potentiate the activity of benchmark latency reversing agents (LRAs). While the implemented approach and generated data are notable, this study contradicts the well-established PAF1C positive regulation of Pol II processivity and elongation [326, 332, 333].

Transcription Termination

Once Pol II finishes elongating, it must be evicted from the template DNA to terminate transcription in both the host and viral phases (Figures 4B and C). Excellent up-to-date reviews about the general aspects of the transcription termination process and polyadenylation have been published and I welcome the reader to consult them if interested in the topic [336, 337]. Briefly, the two LTRs contain identical signals for transcription termination, DNA cleavage and RNA polyadenylation in addition to transcription initiation and elongation. As in eukaryotic pre-mRNAs, the three common signals in the promoter R region are present: i) the polyadenylation hairpin that contains polyadenylation signals (AAUAAA or the AGUAAA variant), ii) the cleavage site, and iii) the G+U-rich downstream element sequence [338], which together constitute the core polyadenylation site. The LTR redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core polyadenylation site at the 5’ end of the transcript, and efficiently utilize an identical signal that resides near the 3’ end. Mechanisms exist to restrict 5’ LTR promoter-proximal polyadenylation site usage and to enhance the activity of the 3’ LTR promoter-distal site [339]. The main determinant of the differential polyadenylation site is the major splice donor site that is immediately downstream of the 5’ LTR, which inhibits cleavage and polyadenylation at the 5’ LTR promoter-proximal site [339, 340]. The pre-mRNA 3’ processing sequences are recognized by the 160 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF), the factor responsible for the recognition of the AAUAAA hexamer and the cleavage stimulation factor (CSTF) [341], together suggesting that polyadenylation site definition involves the recognition of multiple heterogeneous sequence elements in the context of the polyadenylation signal.

Because Pol II is phosphorylated in the CTD to promote pause release and elongation, the CTD must be dephosphorylated for Pol II to become evicted from chromatin for transcription termination. A number of CTD phosphatases have been described including PP1, PP2A, and PP4 [342]. Given the dependence of HIV-1 and signal-inducible genes to P-TEFb, HIV-1 may also be regulated through the PP1-PNUTS complex [343, 344] to slow down Pol II in the 3’-end for transcription termination in the host and viral phases of the HIV-1 transcriptional program (Figures 4B and C). Earlier work supports this model given PP1 phosphatase activity is required for the positive regulation of HIV-1 transcription in the basal [345] and viral [346] phases. Beyond dephosphorylating the Pol II CTD at Ser5 and Ser2 residues, PP1 dephosphorylates CDK9 at the T-loop (threonine 186) to perhaps help coordinate the termination process.

Transcriptional Bursting and Stochasticity

The development of single cell approaches and live cell imaging have propelled our understanding of transcriptional regulation as a whole and have revealed unexpected regulatory features [347]. Transcription is an inherently stochastic process and occurs predominantly in episodes, characterized by the intermittent production of transcripts (called bursts) [348, 349], which are proposed to be a major source of gene expression heterogeneity (noise) [350-353].

The importance of transcriptional bursting and gene expression noise as intrinsic regulatory features of the HIV-1 transcriptional program has been recently summarized [354, 355]. Transcriptional bursts are regulated by a myriad of molecular processes that appear to arise from the stochastic switching of the viral promoter between alternate states (on and off) caused by random binding dynamics of cTFs (Sp1) and iTFs (e.g., TBP/PIC, AP-1 and NF-κB) [356, 357], Mediator, chromatin structure [358, 359], as well as promoter-proximal Pol II initiation, pausing [360], and recycling [361].

iTFs like NF-κB play crucial roles in modulating transcriptional noise by recruiting molecular complexes such as chromatin-modifying and remodeling enzymes that impact various aspects of transcriptional bursting. The chromatin structure can modulate the frequency and magnitude of transcriptional bursting by influencing TF binding, nucleosome positioning, and Pol II function. In latent cells, it has been proposed that the chromatin structure is repressive with lower accessibility [228], and that Pol II promoter-proximal pausing prevents proviral transcription and is essential for latency maintenance [362]. In reactivated cells, the chromatin structure switches to an active state with an increase in accessibility, and Tat activation of pause release and elongation. The site of viral integration into the human genome appears to primarily modulate burst size rather than frequency [363], perhaps supporting the model that virus positions across the genome modulate the quantitative assembly of the transcription initiation machinery, which directly impacts on the transcriptional strength at a given time scale.

Notably, using single-molecule RNA microscopy, Tantale et al. demonstrated that viral transcription is achieved by groups of closely spaced Pol II molecules (named convoys) [364] (Figure 5), as opposed to single isolated enzymes. These Pol II convoys result from a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. Stochastic fluctuations on two independent time scales produce multi-scale bursting: 1) Mediator controlling minute-scale fluctuation (Pol II convoys), and 2) TBP-dependent sub-hour fluctuations (long permissive/non-permissive periods).

Figure 5. — Model integrating established dogmas with recent discoveries and new ideas for future research. Compelling evidence supporting a model in which the provirus acquires a closed genome architecture in which the two LTRs become in close proximity during transcription activation in response to cell stimulation. The core promoter is bound by key TFs such as Sp1 and TBP, and the proximal enhancer is bound by NF-κB, which together may cooperate with iTFs bound at promoter-distal regions in the modulatory and leader regions. Mediator appears critical for activation of the host phase by iTFs such as NF-κB and Tat transactivation through assembly of elongation complexes on TAR. Mediator controls transcriptional bursts by convoys of closely spaced Pol II molecules which might be recycled after transcription termination through potential LTR-LTR long-range interactions.

Using additional single molecule imaging of HIV-1 transcription with the development of quantitative metrics that manage the multiple time scales, Tantale et al. found that Pol II molecules entered a long-lived (>20 min) pause at silent viral promoters to limit transcription (in the basal phase). It was surprising the discovery that Pol II pausing appeared stochastic and not obligatory, with only a small fraction of Pol II molecules undergoing long-lived pausing without Tat, suggesting that the behavior of Pol II pausing at latent proviruses is stochastic (stochastic pausing), thus generating transcriptional bursting, which then contributes to the stochastic activation of HIV-1 [362].

Prior to these past studies with HIV-1, reports in Drosophila and mammalian cells have proposed that while synchronous genes display essentially uniform expression of nascent transcripts in all cells, stochastic genes (like HIV-1) display erratic patterns of de novo activation [365]. Pol II is paused in the promoter regions of synchronous genes, but not stochastic genes [365], in agreement with lack of paused Pol II in the basal phase at the HIV-1 promoter. This discovery argues against the model of Pol II stable pausing at the proviral genome, and may suggest revisiting the contribution of Pol II pausing to the importance of stochastic vs. synchronous HIV-1 transcriptional activation.

While the concept of Pol II pausing has been proposed to regulate entry into HIV-1 latency, pausing alone cannot explain the multi-layer complex regulation of transcription. Since Pol II pausing blocks new initiation [366-368] (Figure 4), there has to be a precise coordination between initiation, pausing, elongation and termination for the acute but robust induction of HIV-1 transcription upon reservoir cell stimulation, and the generation of RNA bursts as a consequence of stochastic transcription may be an important regulatory feature.

Implications of HIV-1 Transcription for Viral Latency and Reactivation

Given the importance of transcriptional regulation to the virus, here I discuss its implications for proviral fate (latency maintenance and reactivation), and briefly deliberate about current and future therapeutic opportunities. As I summarized in prior sections, several factors positively and negatively regulate HIV-1 transcription at the initiation, pausing, pause release, and/or elongation levels (Table 1). It is thus logical to think that the positive regulators of HIV-1 transcription involved in latency reactivation could be targets for permanent silencing strategies [369, 370], while the negative regulators of HIV-1 transcription involved in latency maintenance could be targets for latency reversal for the immune- and/or cell death-mediated elimination of reservoir cells [371-373].

Since the major goal of this review was to immerse the reader in the molecular mechanisms of HIV-1 transcription, and given the large number of reviews primarily focused on targets to manipulate HIV-1 transcription for functional cure approaches [32, 374, 375], here I provide my vision on where the field stands and where it should go to maximize the basic research efforts, rather than describing each isolated attempt and its outcome, but providing a few key examples.

Many transcription complexes are being targeted by a variety of small molecule inhibitors for either latency reactivation or permanent silencing approaches for HIV-1 remission. Potential points for effective therapeutic manipulation include all the transcription steps discussed. Early work has focused in interrupting the Tat-TAR [376, 377] and Tat:P-TEFb [378] interactions, as well as in inhibiting the CDK9/P-TEFb kinase [379], to block the transcription pause release and elongation steps. These approaches to impair the pause release-elongation machinery have later evolved to directly target Tat to more specifically prevent viral transcription elongation. Specifically, an analog of the natural steroidal alkaloid cortistatin A, didehydro-Cortistatin A (dCA) that binds to the Tat RNA-binding domain suppressed Tat activity at sub-nanomolar concentrations [380], potentially offering a new therapeutic tool for HIV-1 remission.

While the rationale for targeting multiple key players of the HIV-1 transcriptional program is reasonable, their translatability awaits to be demonstrated. All these approaches should, in addition to potency, consider selectivity. For example, targeting CDK9 to prevent latent HIV-1 reactivation has been shown to resurrect epigenetically silenced genes and retroelements [381]. If dCA specifically targets Tat, why does the chromatin structure change at the LTR in response to dCA treatment? Is this a direct consequence of Tat inhibition [382], perhaps implying Tat functions prior to TAR binding to regulate chromatin organization at the LTR or does dCA has a second target eliciting this unexpected phenotypic outcome? The recent reports linking cortistatin A to inhibition of the Mediator kinase [383] and its apparent importance to HIV-1 latency reactivation [152], offer previously unanticipated findings to revisit the major targets of dCA for HIV-1 permanent silencing to avoid viral rebound (upon cessation of suppressive therapy).

The targeting of two transcription elongation regulators have revealed unexpected outcomes. PAF1C is a positive regulator of HIV-1 transcription [329, 332], but small molecules inhibitors of PAF1C enhance the latency reactivation potential of benchmark LRAs [335]. More recently, it was proposed that release of P-TEFb from the SEC promotes HIV-1 latency reversal [384]. Given the importance of the SEC to HIV-1 transcription activation, it remains unclear how release of P-TEFb from the SEC can exert a positive, direct role on HIV-1 transcription to promote latency reversal.

Contrary to the many examples to block HIV-1 transcription elongation, the transcription initiation step has not been a primary subject to therapeutic manipulation. While TFs are not easily druggable, the transcription initiation machinery contains various enzymatic components that could be leveraged for rationale approaches. Although the exact PIC composition promoting HIV-1 transcription initiation and latency reactivation remains to be determined, there are several known components that could be therapeutically exploited. Examples include the TFIID complex, composed of a protein kinases, ubiquitin-activating and conjugating enzymes, and HAT; the TFIIH complex, composed of an ATPase, helicase, transcription-coupled nucleotide excision repair, E3 ubiquitin ligase and CDK7 [385] and the Mediator kinase module. Of course, targeting any individual or combination of these PIC components will not provide specificity as some or all of them are used for basal and/or signal-induced transcription activity. However, the exact similarities and differences by which constitutive and inducible genes (including HIV-1) in reservoir cells are transcriptionally activated remain incompletely understood. Filling this void in knowledge is essential to determine any potential sources of specificities to improve and leverage the gained information for alternative therapeutic approaches.

Targeting the epigenetic machinery such as chromatin readers and chromatin-modifying enzymes involved in transcription repression has also been exploited (recently reviewed in [201, 386]). While scientifically sound, this modality has been convoluted by the pleiotropic functions of the targeted chromatin regulators including transcriptional activation and repression, cell cycle progression, and DNA damage response, among others. This is the example of BRD4, a positive regulator of HIV-1 transcription [259, 260], also involved in cell cycle progression [310, 387, 388] and chromatin insulation from DNA damage signaling [389]. While early work has revealed that BRD4 inhibition with JQ1 and other inhibitors of the Bromodomain and Extra-Terminal (BET) family of proteins (iBET) reactivates latent HIV-1 [390] by antagonizing inhibition of Tat activation [391], later work has shown that inhibition is due to a short isoform of BRD4 that engages with the repressive SWI/SNF chromatin-remodeling complex [187]. The studies with BRD4 as prime example of chromatin regulator, illustrate their complexity in the regulation of multiple biological processes and the importance of basic science research to first understand biology.

The magic transcriptional target to eliminate the HIV-1 reservoir may not exist, but the ideas in the field to inactivate Tat to selectively target the virus are well rounded. Given the many examples in this review regarding Tat acting through the transcription initiation and elongation machineries to coordinate the logarithmic RNA amplification required for efficient latency reactivation or viral replication, Tat seems one if not the best therapeutic target for the permanent, durable silencing strategies towards HIV-1 remission.

The list of factors “potentially” involved in HIV-1 transcription and latency grow day-by-day. It is critically important for the field to truly distill this list to make sure academia and industry investments yield fruitful efforts in our battle towards a functional cure. Changes in proviral fate upon genetic or chemical perturbations of the factor under scrutiny does not directly link that factor to regulation of the HIV-1 provirus, as alterations to reservoir cell state (e.g., due to stress, autophagy, innate immunity, etc.) can indirectly influence the highly responsive LTR promoter. Thus, our collective efforts in the field should be placed in precisely defining how the various transcription players operate to identify the best targets and in improving the definition of direct vs indirect mechanisms. These are two critical points to establish solid evidence between target engagement and the reported phenotypic outcome(s): latency reactivation or permanent silencing. The benefit of this basic science exercise is to rule out indirect effects through which the small compounds under investigation can alter HIV-1 proviral fate thereby making wrong mechanistic assumptions that can complicate their future translatability.

One common theme about all approaches to block the activity of negative regulators as part latency reversal regimes is the unexpected, heightened latency reversal response. Thus, how specifically targeting a repressor involved in latency maintenance with an LRA immediately switches to high levels of reactivation achieved by physiologic signals that engage on known agonists of T cell activation (e.g., PKC) or reservoir cell inflammation (e.g., NF-κB)? Are the reported LRAs selective enough or do they alter reservoir cell state thus largely leading to latency reversal through mechanisms unrelated to inhibition of the main target reported? Given these lingering issues, the field should not be using small compounds to interrogate the basic biology of HIV-1 transcription until they are thoroughly characterized to make sure the reported phenotypic outcomes solely or primarily derive from the reported target engagement and not from indirect effects (e.g., alterations of reservoir cell state biology influencing HIV-1 proviral fate). This should be the first step before any attempts of druggability and translatability can be initiated. Although critically important a thorough discussion of these points is beyond the scope of this review focused on the molecular aspects. Together, I hope this discussion helps to establish community-accepted practices to guide future basic and translational efforts for HIV-1 cure.

Future Perspectives

I have provided a critical overview of the research spanned over the past four decades providing the fundamental knowledge to understand the molecular bases of the HIV-1 transcriptional program. In this final section, I summarize on where the field is, the current challenges and bottlenecks to future major advances, and areas that deserve more attention from the scientific community to bolster our basic knowledge of the core HIV-1 transcriptional program to both provide new biology and open up new therapeutic opportunities.

Integrated initiation and elongation control.

Early discoveries in the field have provided the critical evidence of a Tat-TAR protein-RNA axis important for transcription elongation. Extensive evidence also support the requirements of DNA cis elements and cellular TFs in the process of transcription initiation and transition to elongation, highlighting the critical importance of an integrated DNA- and RNA-based regulatory mechanism to both induce and maintain the HIV-1 transcriptional program. This complex program is regulated by the sequential and combinatorial action of cellular TFs and Tat, in concerted action with the initiation and elongation machineries to maintain high levels of transcription initiation so that Tat can do its job at sustaining elongation. Studies of PIC assembly and composition have revealed mixed outcomes. While the Roeder lab revealed that HIV-1 transcription by Tat functions with TAFs, the Green lab proposed that HIV-1 transcription operates in a TAF-independent manner; however, both assays have significant experimental differences. Future studies should help define the progression in PIC assembly in the basal, host and viral phases at integrated proviruses to reveal how HIV-1 transcription initiation truly operates and identify similarities and differences among HIV-1 and signal-inducible human genes that could open new therapeutic opportunities. Thus, learning more about two major areas: 1) transcription initiation and 2) the transition from initiation to elongation should be important priorities for basic research and to leverage those discoveries for therapeutic opportunities.

Despite the large number of environmental signals that can activate reservoir cells to promote HIV-1 transcription, a minimalistic model can be proposed that summarizes the complexity of all possible cell signaling-transcriptional programs. In this model, iTFs recognize their cognate binding elements to function individually and cooperatively, highlighting the importance in the genomic arrangement of the contiguous TF binding sites on the viral promoter. These factors cooperate with Tat, and with other machineries of the transcriptional apparatus including Mediator, the PIC, and the Pol II complex, perhaps helping organize the assembly of high-order TF complexes for increased transcription activation in the three-dimensional space (Figure 5).

Numerous evidence support an integrated initiation-elongation model. First, the TAR RNA must be in close proximity to the site of transcription initiation for Tat to normally promote elongation [111, 115]. Second, the topological arrangement of promoter (Sp1 and TATA boxes) and enhancer (NF-κB) cis elements are critical for the correct organization of the transcription complex for initiation and for Tat to facilitate elongation. Mutations in the core promoter dramatically reduce transcription initiation rates in vitro and dampen Tat elongation activity proportionally [35], consistent with cellular data indicating that the site of integration alter basal transcription and response to Tat transactivation [135]. Third, the TAR-independent cooperativity between Tat and promoter-bound cellular TFs such as Sp1 [392, 393], TBP [127, 128, 164], other PIC components [159], and Mediator [151], as well as other cellular TFs interacting with upstream 5’ LTR promoter sequences [125], may allow Tat to sustain elongation in the viral phase, again consistent with the idea that Tat requires both TAR RNA and cis elements strategically positioned at the 5’ LTR to assemble with a select transcriptional apparatus prior to TAR recognition (Figure 5), and may point towards the importance of synchronizing early and late transcription steps for efficient virus transcription activation. Fourth, beyond its canonical role in regulating PIC assembly, the TATA box was shown to mediate recruitment of P-TEFb [394, 395], highlighting the importance of the promoter, and not only TAR, in modulating transcription elongation. These evidence suggest that something unique about TBP and P-TEFb at the PIC state signals for a composite surface for efficient recruitment of elongation transcription complexes, albeit kept in a primed stage until TAR binding, perhaps bound to the 7SK snRNP [120].

Obtaining rigorous mechanistic details urges the use of advanced genetic tools to reveal a factor’s primary function that is not convoluted by indirect effects. As opposed to approaches that allow for long-term gene depletion or RNA silencing, approaches leveraging the use of degron of E3 ubiquitin ligases for the acute depletion of viral or host factors have several advantages including recording of primary phenotypes to delineate causality in gene control, before confounding secondary effects manifest [396, 397]. The recent work by Randolph et al. exemplifies the utility of the dTAG system, which leverages the potency of cell-permeable heterobifunctional degraders (dTAG-13) [396], to study HIV-1 transcription [398] and has helped solidify the precise function of host factors solving controversial results in the field. Further, with the advent of genome engineering tools, the proviral genome can be precisely edited with point mutations to study the function(s) of viral genomic elements and/or factors in the different steps and phases of the HIV-1 transcriptional program. The recent work by Hyder et al. illustrates the use of CRISPR-Cas9 to mutate the translation initiation codon of tat to dissect out Tat’s contribution to the host and viral phases [4].

Three-dimensional topology of the provirus genome and transcriptional enhancers.

Proudfoot and colleagues have provided evidence of transcription-dependent HIV-1 provirus looping in which the 5’ and 3’ LTRs come in spatial proximity (Figure 5) [399]. An outstanding question is how this process is regulated and whether looping formation has any functional implications in the process of HIV-1 transcription. Although the nature of cellular and viral factors that help regulate the organization of the proviral genome in the three-dimensional space remains unknown, the large number of transcription initiation factors interacting at multiple elements within the core promoter and 5’ LTR may aid novel communication mechanisms between upstream regulatory factors and the general transcriptional machinery.

What are the roles of the promoter-distal (upstream and downstream) modulatory, leader region and intragenic enhancers [166-168] in this process and beyond? Several binding sites for cell-type specific iTFs involved in HIV-1 transcription exist both upstream and downstream of the core promoter [76-79], which perhaps function as upstream and downstream enhancers to facilitate the establishment and/or maintenance of long-range chromatin-chromatin interactions for robust Tat transactivation.

The 5’ UTR located downstream of the TSS may function as an enhancer (downstream enhancer) working together with the upstream enhancer and core promoter to ensure maximal activation of the viral promoter to maybe regulate the three-dimensional topology of the provirus genome. This downstream enhancer could bolster viral response to iTFs induced in response to a wide variety of cell activation signals and/or it could also assist in the displacement of nucleosomes during the remodeling of chromatin structure [30]. NF-κB family members bind as hetero-dimers to the enhancer and certain combinations of NF-κB subunits (p65-p50) are preferred for Tat trans-activation [400] to increase the rate of RNA synthesis during cell stimulation [55, 56]. Furthermore, Tat relies on NF-κB promoter binding activity to withstand HIV-1 transcription during sustained cell stimulation (TNF-α) [18]. Collectively, the cooperativity between promoter- and/or enhancer- bound TFs may help recruit Pol II to the viral promoter and/or induce a chromatin reconfiguration favoring transcription initiation and the transition to elongation in the host phase [74] and/or the maintenance of the initiation-elongation program by Tat.

What role does Mediator play in the process of HIV-1 transcription activation? Given that only a few Mediator subunits appear to promote viral transcription, why does a minimalistic Mediator, as opposed to a more complex Mediator [401], operate at the viral promoter? Although Mediator does not appear to play an architectural, but functional, role regulating promoter-enhancer communications at human loci [402], what is the role of Mediator in HIV-1 transcription initiation and elongation in the various phases of the viral transcriptional program? Master TFs and Mediator operate together to establish super enhancers at key cell identity genes [403], thus it is possible that HIV-1 has co-opted a similar regulatory principle in which iTFs and Mediator establish multiple contacts between them and with the basal transcriptional apparatus to first initiate (in the host phase) and/or then maintain (in the viral phase) an active transcriptional foci (Figure 5).

Although the past four decades have a revealed a critical volume of knowledge, several salient questions remain. How do all above transcription components (iTFs, Mediator, PIC, and Pol II) assemble in the context of the proviral chromatin structure? How is the nucleosome architecture established and regulated at the provirus in the various phases of the HIV-1 transcriptional program? Does the chromatin structure in the basal phase restrict the binding of cellular TFs prior to cell stimulation? Does remodeling of the chromatin structure in the basal phase promote the binding of cellular TFs after cell stimulation? Or does TF binding to chromatin accessible and/or nucleosomal sites facilitate chromatin remodeling? What is the role of the various putative upstream and downstream enhancers and what role, if any, the three-dimensional chromatin structure play in the process of activation in the various phases of the transcription cycle?

Given the above gaps in knowledge, there is a need for rigorous basic science to move the field forward and provide a sustained impact in the field. I hope future research will help hone our understanding of the HIV-1 transcriptional program by further elucidating previously unexplored or incompletely understood facets.

Acknowledgements

I am grateful to Angela Diehl for critical discussions and assistance in preparing the Figures, Christian Forst for comments, Jasmin Bakker for editorial assistance, and the three anonymous reviewers for their constructive criticisms to improve the presentation of this review. The research reported in this publication was supported by NIAID grant under awards number R01AI114362 and R21AI175042 to Iván D’Orso and the UTSW Simmons Comprehensive Cancer Center grant under award number P30CA142543 to Carlos Arteaga.

Footnotes

Declaration of competing interest

None.

Data availability

No data was used for the research described in this article.

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PERMALINK

The HIV-1 Transcriptional Program: From Initiation to Elongation Control

Iván D’Orso

Abstract

The Core Components of the HIV-1 Transcriptional Program

Figure 1.

HIV-1 Genomic Elements and Cellular Transcription Factors

Table 1. Regulators of the HIV-1 transcriptional program.

HIV-1 genome and LTRs.

Figure 2.

TATA box.

Initiator.

RBEs.

Sp1 sites.

NF-κB sites.

Additional TF binding sites.

Genetic diversity of TF binding sites.

HIV-1 TAR RNA and Tat Protein

Cooperation Between Tat and Cellular Transcription Factors

Transcription Initiation

Pre-initiation Complex Assembly

Cellular TFs and GTFs.

Figure 3.

TAFs.

Mediator.

Pol II CTD and CTD kinases.

Chromatin Structure

Nucleosome organization and chromatin-remodeling.

Chromatin-modifying enzymes.

Transcriptional Pausing, Pause Release, and Elongation

Figure 4.

Pausing

NELF and DSIF.

Pause Release and Elongation

P-TEFb discovery.

Assembly and regulation of Tat:TAR:P-TEFb transcription elongation complexes.

Structure of Tat:P-TEFb and Tat:TAR:P-TEFb complexes.

Negative regulation of P-TEFb activity and transcription elongation.

Positive regulation of P-TEFb kinase activity and transcription elongation.

P-TEFb release factors.

SEC.

PAF1C.

Transcription Termination

Transcriptional Bursting and Stochasticity

Figure 5.

Implications of HIV-1 Transcription for Viral Latency and Reactivation

Future Perspectives

Integrated initiation and elongation control.

Three-dimensional topology of the provirus genome and transcriptional enhancers.

Acknowledgements

Footnotes

Data availability

References

Associated Data

Data Availability Statement

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