Date Published: July 30, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Luna Li, Satinder Dahiya, Sandhya Kortagere, Benjamas Aiamkitsumrit, David Cunningham, Vanessa Pirrone, Michael R. Nonnemacher, Brian Wigdahl.
The human immunodeficiency virus type 1 (HIV-1) promoter or long-terminal repeat (LTR) regulates viral gene expression by interacting with multiple viral and host factors. The viral transactivator protein Tat plays an important role in transcriptional activation of HIV-1 gene expression. Functional domains of Tat and its interaction with transactivation response element RNA and cellular transcription factors have been examined. Genetic variation within tat of different HIV-1 subtypes has been shown to affect the interaction of the viral transactivator with cellular and/or viral proteins, influencing the overall level of transcriptional activation as well as its action as a neurotoxic protein. Consequently, the genetic variability within tat may impact the molecular architecture of functional domains of the Tat protein that may impact HIV pathogenesis and disease. Tat as a therapeutic target for anti-HIV drugs has also been discussed.
The human immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). The HIV-1 genome is about 9.8 kb in length, including two viral long-terminal repeats (LTRs) located at both ends when integrated into the host genome. The genome also includes genes that encode for the structural proteins ([Gag], [Pol], and [Env]), regulatory proteins (Tat and [Rev]), and accessory proteins ([Vpu], [Vpr], [Vif], and [Nef]). The HIV-1 transactivator of transcription (Tat) protein is an early regulatory protein containing from 86 to 106 amino acids in length with a molecular weight of approximately 14 to 16 kDa. Tat is a multifunctional protein that has been proposed to contribute to several pathological consequences of HIV-1 infection. Tat not only plays an important role in viral transcription and replication, it is also capable of inducing the expression of a variety of cellular genes as well as acting as a neurotoxic protein. In this review, the functions of Tat and molecular diversity in Tat are addressed. Moreover, the interaction of Tat with the viral LTR and cellular factors are documented and discussed. Because of its pivotal role in viral replication and disease pathogenesis, Tat and the cellular pathways targeted by Tat could be potential targets for new anti-HIV drugs. Therapeutic strategies that have focused on this topic are also reviewed.
Tat is a 14 to 16 kDa nuclear protein. It is a multifunctional protein, which is essential for the productive and processive transcription driven from the HIV-1 LTR promoter, and is required for overall productive viral replication [1, 2]. It is a 101-amino acid protein encoded by two exons: the first exon encodes amino acids 1 to 72; the second encodes residues from 73 to 101 (Figure 1) . Most clinical HIV-1 isolates of Tat include 101 amino acids, whereas a few isolates contain from 86 to 106 amino acids, with the second exon coding from 14 to 34 residues at the C terminus of the protein . The HIV-1 IIIB Tat used in many in vitro experiments contains 86 amino acids, corresponding to HIV-1 (strain BRU) or a closely related sequence from the HXB2 HIV-1 infectious molecular clone [5, 6]. This 86-amino acid configuration of Tat is the most frequently used form for laboratory investigations; however, it must be noted that it represents a truncated protein when compared to Tat from many clinical isolates. Several studies have established that HIV-1 Tat maintains the 101-amino acid composition as previously reviewed . The more truncated 86-amino acid version of Tat appears to be functional , but functions like modulation of host cell cytoskeleton modifications  and perhaps optimal replication in cells of the monocyte-macrophage lineage have been attributed to the second exon. Also, the fact that most clinical isolates preserve the full 101-amino acid form is indicative of the functional relevance of the second exon in an in vivo setting.
The HIV-1 LTRs are generated during the process of reverse transcription and located on each end of the proviral DNA when the provirus is integrated into the host genome. The LTR is approximately 640 base pairs in length and divided into the unique 5′ (U5) and 3′ (U3) regions as well as the repeat region (Figure 2). LTR sequences include four functional regulatory regions with respect to the control of HIV-1 transcription: TAR element, core promoter, enhancer region, and modulatory region . A multitude of HIV-1 promoter regulatory elements are located within the U3 region of the 5′ LTR and drive the production of HIV-1 mRNA that codes for proteins involved in regulating viral replication as well as the assembly and release of infectious progeny virus. The core region of the LTR is composed of the TATAA box, which is located 29–24 nucleotides upstream of the transcriptional start site, and specificity protein (Sp) binding sites, which are three tandem GC-rich binding sites (−45 to −77) interacting with transcription factors Sp1 through Sp4. The TATAA box binds TATAA-binding protein in association with a number of other proteins that comprise the RNA polymerase II (pol II) transcription complex for transcription initiation and elongation [48–50]. The enhancer element is primarily composed of two copies of 10-base pair binding sites for NF-κBs and related proteins . The modulatory region, which is in the 5′ end of the U3 region, contains binding sites for many factors, including CCAAT/enhancer-binding protein (C/EBP) factors , activating transcription factor/cyclic AMP response element-binding protein (ATF/CREB) , nuclear factor of activated T cells (NFAT) , and a number of other proteins, depending on cell phenotype, differentiation status, and state of activation (Figure 2) [55–58].
HIV-1 transcription involves an early, Tat-independent phase and a late, Tat-dependent phase, and transactivation of the viral genome is a critical step in the viral replication cycle [3, 78]. In the presence of Tat, LTR-mediated transcriptional activity can be enhanced tens or hundreds of fold [79–82], whereas viral replication falls to nearly undetectable levels in the absence of Tat, and short transcripts (30–50 nucleotides) predominate [83–85]. Tat is a unique transcription factor in that it binds to the “UCU” bulge of the TAR, a cis-acting RNA enhancer element within the 5′ end of all viral transcripts. The TAR is located immediately downstream of the transcriptional start site in the HIV-1 LTR, encompassing nucleotides from +1 to +59 [86, 87], and is required for the function of the viral transactivator protein Tat. The Tat-TAR interaction acts to tether Tat and allow its interaction with the basal transcriptional machinery, thus increasing viral transcription and elongation [88, 89]. In a mature transcript, TAR adopts a hairpin structure including a six-nucleotide loop, a trinucleotide pyrimidine bulge, and an extensive duplex structure . U23, in the bulge, is critical for Tat binding [2, 13, 90]; the other two neighboring residues C24 and U25 can be replaced by other nucleotides without affecting Tat binding. Another two regions above the bulge (G26-C39 and A27-U38) and one region below (A22-U40) also contribute to Tat binding [2, 13, 90]. Although the loop structure does not appear to be required for Tat binding, the residues in the loop have been shown to be required for Tat transactivation activity .
The hypophosphorylated form of RNA pol II leads to the production of short RNA molecules (30–50 nucleotides in length including the entire length of the TAR sequence) as a result of premature termination of transcription. However, phosphorylation of the CTD of RNA pol II has been shown to prevent premature termination and promote the efficient elongation and production of full-length HIV-1 RNA transcripts . Phosphorylation of the CTD of RNA pol II has also been shown to be important for the clearance of mediators from RNA pol II  (Figure 3). Transcription factor II H (TFIIH) is a part of the preinitiation complex involved in transcription, and a number of studies have shown that there are combinatorial networks of transcription factors and cofactors, such as P-TEFb, utilized by Tat to activate and repress gene expression [107, 111]. Tat and P-TEFb are recruited to viral preinitiation complexes prior to RNA transcription and are subsequently transferred to nascent RNA after initiation. In addition to regulating HIV-1 gene expression, Tat is known to be involved in dysregulating cellular function and altering cellular gene expression profiles; however, the mechanism by which Tat affects infected cells continues to be explored.
The high level of HIV sequence diversity generated during the course of HIV disease is, for the most part, due to the error-prone nature and low fidelity of reverse transcriptase, poor proofreading by the polymerase, and selective pressures exerted by the host immune response, combination antiretroviral chemotherapy, and perhaps other physiological pressures [3, 102, 138]. The HIV-1 genotypic variants and resultant phenotypes occur as important variables of viral replication during the course of the disease . It has been reported that Tat can tolerate 38% sequence variation without any change in its transactivation potential .
In addition to transactivation of the viral LTR, Tat exhibits a range of biological properties relative to HIV-1 pathogenesis , including the intracellular regulation of host gene expression to facilitate viral production as well as the extracellular detrimental effects on the cells of the immune and nervous systems. HIV-1 induces pathological consequences in a number of end organs including the brain [7, 153]. More than 30% of AIDS patients suffer from some form of HIV-1-induced neurological impairment including HIV-1-associated dementia (HAD) as well as other more subtle minor neurocognitive disorders [154, 155]. Despite the widespread use of highly active antiretroviral therapy and the resultant decrease in the incidence of HAD, the prevalence of HAD and other milder forms of HIV-related neurological disease has become increasingly common problems with respect to the clinical management of HIV/AIDS . In particular, Tat has been implicated in the pathogenesis of HIV-associated neurological disease including HAD via a variety of mechanisms . The neurotoxicity of Tat is further supported by the observation that the mRNA levels for Tat are elevated in brain extracts of patients with HAD . Tat has been shown to act as an intracellular and extracellular mediator of neurotoxicity and to play a critical role in contributing to neurological injury in HAD . Tat protein is secreted by HIV-1-infected cells and acts by diffusing through the cell membrane. It appears to act as a secreted, soluble neurotoxin and induces HIV-1-infected macrophages and microglia to release neurotoxic substances [160–162]. Some Tat variants have been reported to be dysfunctional with respect to LTR transactivation and may contribute to viral latency under certain conditions while still being able to stimulate the transcription of a number of cytokine genes . Tat can also cooperate with cellular factors to enhance the neurotoxic effects on host cells . Tat and cytokines IFN-γ and TNF-α have also been demonstrated to synergistically increase expression of CXCL10 in human astrocytes, which provide an important reservoir for the generation of inflammatory mediators, for instance, CXCL10 as a neurotoxin and a chemoattractant .
The HIV-1 Tat protein has long remained an attractive target for therapeutic intervention owing to its essential role in viral gene expression and activation of the HIV-1 LTR. As discussed before, Tat and the P-TEFb complex bind to TAR to promote efficient transcription of the full-length HIV genome. The expanding knowledge of Tat functional properties and its interactions with other cellular and viral partners has led to the identification of a varied range of compounds that can inhibit different Tat functions. The Tat and HIV-1 transactivation inhibitors fall broadly into the following categories: (1) inhibitors targeting TAR RNA (2), inhibitors targeting Tat protein, and (3) Tat-P-TEFb interaction inhibitors. In this section, we review the current status of the development of therapeutic strategies that target Tat and its functional interactions in the process of HIV-1 transcription (Table 2).
More than two decades of investigations have established the central role of Tat in the activation of HIV-1 LTR. Genetic- and structure/function-based studies have enabled us to understand the functional intricacies of Tat-dependent functions. All of these studies have motivated a number of researchers to use Tat as an important target in combination antiretroviral therapies, but to date none of these have materialized into clinically effective antiviral agents. One crippling factor has been the inability to assess the functions of Tat in relevant systems at a concentration that would be closer to that encountered in vivo. Concentrating efforts in a direction to elucidate the protein-protein interactome established by Tat will go a long way toward targeting specific breakpoints in HIV-1 pathogenesis. Isolation of mutant sequences of Tat from sites like the brain can also be used to identify tissue-specific functions of Tat that may have great bearing on long-term use of Tat inhibitors. Moreover, it would be an important effort to consolidate studies concerning the structural information and functional interactions of Tat across different HIV subtypes and use this information to increase the spectrum of subtypes susceptible to Tat-based therapeutic inhibitors. Eradicating latent reservoirs by the elimination of integrated HIV-1 provirus or irreversibly blocking LTR activation or Tat transactivation activity will provide the next major step forward in controlling the HIV-1 pandemic.