| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Published online before print August 11, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institut National de la Santé et de la Recherche Médicale Unité 575, Strasbourg, France
1Correspondence: Unité 575 INSERM, 5 rue Blaise Pascal, 67084 Strasbourg, France. E-mail: schaeffer{at}neurochem.u-strasbg.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
Key Words: cellular specificity • LTR • Tat • acetylation • monocytes
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
B and NF of activated T cells (NF-AT) transcription factors, the modulatory region harbors numerous target sequences for a variety of cellular transcription factors such as NF–interleukin (IL)-6, cyclic AMP (cAMP) response element-binding protein (CREB), Ets, and nuclear hormone receptors. These host-cell factors regulate LTR-driven transcription not only by direct binding to their target DNA sequence but also by indirect binding via DNA-bound proteins. Extensive studies have described how cis- and trans-acting elements regulate LTR-driven transcription, but few have focused on the ability of the viral genome to adjust its transcriptional mechanism to each specific cell type. This review highlights the extraordinary capacity of HIV to adapt its transcriptional strategy to different cellular environments. We describe the mechanisms underlying the cell type-specific transcription directed by the HIV-1 LTR in T4 lymphocytes, thymocytes, monocytes, macrophages, and microglial cells. In addition, we present LTR variations in different HIV-1 isolates. New advances suggest that the transcription step of the viral life cycle could be used as a target for an anti-HIV strategy.
|
CELLULAR SPECIFICITY OF HIV-1 GENE TRANSCRIPTION |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
|
REGULATION OF HIV-1 TRANSCRIPTION BY CELLULAR TRANSCRIPTION FACTORS |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
Sp1-mediated regulation of LTR-driven transcription
Transcription factors of the Sp1 multigene family [5
, 6
] modulate HIV-1 gene transcription by direct binding to the three GC boxes adjacent to the TATAA sequence. The Sp1 factor is one of the essential cellular proteins implicated in HIV-1 gene transcription [7
8
9
]. Although Sp1 and Sp4 function as transcriptional activators, the Sp3 protein represses the HIV promoter activity in HeLa cells [10
]. Sp1 and Sp3 proteins are expressed in microglial cells and exert an antagonist action on LTR-driven transcription. In addition, Sp3 is able to repress not only the Sp1 action but also the positive action of the transcription factor chicken ovalbumin upstream promoter transcription factor (COUP-TF), exerted via binding to Sp1 as discussed below [11
].
In U1 monocytic cells, in vivo footprinting analysis confirms the importance of the Sp sites for LTR-driven transcription in living cells [12 ]. It is interesting that monocytic cells that differ in their degree of differentiation present a large difference in the binding of Sp1 and Sp3 to the Sp site III [13 ]. Moreover, recruitment of members of the Sp family is of greater importance in T cells as compared with monocytic cells, as substitution of low-affinity Sp sites in place of the natural site III decreases viral replication in Jurkat T cells and has small effects in U937 monocytes [14 ]. Thus, modulation of HIV-1 gene transcription by proteins of the Sp family depends on the nature of the Sp protein bound to the LTR, on the cell type as well as on the differentiation state of the infected cell.
A number of studies report that the Sp1 protein can serve as an anchor for indirect binding of transcription factors. In microglial and Jurkat T cells, the Sp1 protein interacts directly with COUP-TF to synergistically stimulate transcription [11
, 15
]. Sp1 is also able to functionally interact with NF–IL-6, which leads to a mutual inhibition in microglial cells and to a synergistic transcriptional stimulation in glial cells [16
]. In addition, a cooperative interaction between Sp1 and NF-B, bound to the adjacent sites, is required for optimal HIV-enhancer activation [17
]. In all cell types, an interaction between Sp1 and Tat is required for optimal Tat transactivation [18
, 19
]. Finally, Sp1 is able to recruit the CycT1 subunit of P-TEFb to the LTR, which helps bypass a requirement for TAR/Tat and promotes processive transcription without Tat [20
].
Two adjacent Sp1-binding sites located in the +724/+743 LTR region downstream of the transcription start site also play a positive regulatory role on HIV transcription. Proviruses containing mutations in these sites are totally defective for viral replication in T lymphoid cells [21 ]. Taken together, these observations highlight the capacity of Sp1 to stimulate transcription by acting at distinct sites within the LTR, to recruit cellular factors to the LTR, and to promote Tat-mediated transactivation. Thus, Sp1 appears as a crucial cellular transcription factor for efficient HIV-1 gene transcription.
NF-B-mediated stimulation of LTR-driven transcription
NFs of the B family are involved in the control of a variety of cellular processes, such as immune and inflammatory responses, development, cellular growth, and apoptosis. A large number of stimuli activate the NF-B pathway (for review, see ref. [22
]). Upon induction, NF-B, composed of p50 and p65 (RelA) subunits, is liberated from its cytoplasmic inhibitor IB and translocated in the nucleus where it binds to the two NF-B target sequences within the LTR.
The importance of this factor in unstimulated Jurkat T cells and in microglial cells is revealed by the deletion of the two LTR NF-B sites, which results in a drastic decrease of the level of transcription [11
, 23
]. Similarly, in cells of the monocytic lineage, occupancy of the enhancer by NF-B p50/p65 heterodimers is required for transcription [24
]. However, upon phorbol ester and calcium ionophore activation, NF-B or Sp1 mutant viruses are still able to replicate in CD4+ T cells [25
]. In fact, mutations in the B-binding sites impair virus production in cells with a high basal level of NF-B such as Jurkat T cells [26
]. In CD4+ T lymphocytes, the NF-B pathway is induced by myeloid-related proteins, highly expressed by myeloid cells in the serum of HIV-1-infected patients [27
].
In thymocytes, activation of the NF-B factors is mediated by tumor necrosis factor 
(TNF-) and IL-1 and IL-7, present in the thymic microenvironment, and is required for a high level of transcription. When the B sites are deleted, the HIV provirus fails to replicate, which shows the requirement of NF-B for viral expression in thymocytes [28
].
A new link among cytokine signaling, the dopamine system, and transactivation of the LTR via the involvement of NF-B was reported in Jurkat T cells and peripheral blood mononuclear cells (PBMCs) [23
]. It is interesting that HIV-1 surface proteins can also contribute to support viral replication by stimulating the NF-B pathway. HIV-1 virions incorporate several surface proteins of host origin. Incubation of human T lymphoid cells with viruses bearing host-derived B7-2 proteins potently activates LTR-driven transcription via the NF-B and the NF-AT pathways [29
].
Moreover, NF-B-mediated induction of LTR transcription includes a recruitment of histone acetyl transferase activity [30
, 31
]. As described for Tat and Sp1, NF-B also has the ability to recruit P-TEFb and to stimulate the elongation step of transcription by RNAPII [32
]. This P-TEFb recruitment by Sp1 and NF-B in preinitiation complexes before the synthesis of TAR–RNA and P-TEFb targeting to the LTR can substitute for the lack of transcriptional elongation in the absence of Tat [33
].
NF-AT-mediated regulation of LTR-driven transcription
Similar to NF-B, NF-AT relocalizes to the nucleus following cellular activation. NF-AT proteins are dephosphorylated by the calcium-activated phosphatase calcineurin, undergo nuclear translocation, and then assemble with activated protein-1 (AP-1) [34
]. NF-AT proteins bind the NF-B sites of the LTR region. The two proteins NF-AT1 and NF-AT2/NF-ATc appear to have contrasting effects in different cell types. NF-AT1 acts as an activator of LTR-driven transcription in primary CD4+ T cells, especially in the CD4 memory T cell subset [35
, 36
]. In Jurkat T cells, NF-AT1 appears to regulate NF-B-mediated transactivation negatively, by competing with NF-B for its LTR-binding sites, and to repress Tat-mediated transactivation by interacting with Tat [37
].
NF-AT2/NF-ATc is sufficient to induce a highly permissive state for HIV-1 replication in primary CD4+ T cells [38
]. While in resting, naïve lymphocytes, HIV-1 gene transcription appears repressed, in activated T lymphocytes, the NF-B and NF-AT sites of the LTR mediate transcriptional activation [39
]. NF-AT2 binds to the NF-B sites of the LTR and synergizes with NF-B and Tat in the transcriptional activation and HIV-1 replication [38
]. NF-AT proteins also interact with Ets transcription factors, which results in a cooperative activation of LTR-driven transcription [40
]. Moreover, activation of NF-AT-dependent HIV gene expression appears to be stimulated by the viral Nef protein [41
, 42
].
Lymphoid enhancer-binding factor (LEF-1)/T cell-specific factor-1 (TCF-1)-mediated regulation of LTR-driven transcription
LEF-1/TCF-1 [43
] is described as an activator of LTR-driven transcription in T lymphocytes [44
]. Mutations in the LEF-binding site located in the enhancer region of the LTR (-122/-142) inhibit the transcriptional activity of the HIV-1 enhancer in Jurkat T cells [44
]. In vitro, LEF-1 binds to this LTR-binding site in a nucleosomal context [45
]. Moreover, recombinant LEF-1 protein counteracts chromatin repression, indicating that transcriptional activation by LEF-1 in vitro is a chromatin-dependent process [44
].
CCAAT/enhancer-binding protein (C/EBP)-mediated regulation of LTR-driven transcription
Another example of cell type-specific transcriptional modulation of the HIV-1 genome is documented by studies on the C/EBP family of transcription factors. Two C/EBP-binding sites are centered around positions -170 and -110 of the LTR. The human NF–IL-6 protein (corresponding to the mouse C/EBPß protein) plays a central role in the control of HIV infection in cells of the immune system. The LTR-binding sites and the C/EBP/NF–IL-6 proteins are required for full NF–IL-6 responsiveness and viral replication in promonocytic U937 cells [46
, 47
] in primary macrophages but not in CD4+ T lymphocytes [48
].
Infected macrophages interact with neighboring cell populations, such as endothelial cells, through inflammatory mediators and direct cell contact, which create a microenvironment favorable for HIV replication. In the presence of endothelial cells, the enhancement of viral replication is mediated by C/EBPß, necessary for efficient LTR activity [49 ]. Moreover, after infection with Mycobacterium tuberculosis, monocytes produce a stimulatory 37-kDa C/EBPß transcription factor that enhances HIV-1 replication, whereas macrophages produce an inhibitory 16-kDa C/EBPß that suppresses HIV replication [50 ]. Thus, differentiation of monocytes to macrophages switches the opportunistic infection effect on HIV replication from stimulation to inhibition by affecting expression of the C/EBP proteins. As described in monocytic cells, C/EBP proteins regulate transcription by recruiting coactivators such as histone acetyltransferases to the LTR [51 ].
Two members of the C/EBP family, NF–IL-6 and C/EBP
, are expressed in microglial and glial cells of the central nervous system (CNS). Although NF–IL-6 acts as a potent activator of LTR-driven transcription, C/EBP, which lacks a transcriptional activation domain, acts as a dominant-negative and inhibits NF–IL-6-mediated transactivation. Therefore, the respective level of these two proteins as well as the level of cytokines such as IL-1, IL-6, and TNF-, known to be increased during HIV infection and to increase the level of NF–IL-6, appear critical in the regulation of LTR-driven transcription [40
].
It is interesting that transactivation by NF–IL-6 can bypass a requirement for direct interaction of NF–IL-6 to its DNA-binding sites, through cooperation with Sp1, NF-B, and CREB proteins or via the basal transcriptional machinery [16
, 52
53
54
]. In microglial cells, endogenous NF–IL-6 appears unable to interact with its -170 LTR site, as the upstream stimulatory factor (USF) protein binds to the overlapping E site and competes with NF–IL-6 for binding. In microglial and in Jurkat T cells, NF–IL-6 stimulates transcription via the Sp1 sites of the LTR [16
, 54
]. Functional interactions between Sp1 and NF–IL-6 proteins lead to a mutual repression in microglial cells and to a synergistic stimulation in glial cells [16
]. In T cells, the C/EBP-binding sites and the cooperative action between NF–IL-6 and CREB mediate prostaglandin E2-induced stimulation of LTR-driven transcription [55
]. In cells of monocyte/macrophage lineage, NF–IL-6 also associates and heterodimerizes with CREB [53
].
CREB-mediated regulation of LTR-driven transcription
The importance of the cAMP pathway during the course of HIV-1 infection is well established, as cAMP levels are higher in seropositive patients [56
]. HIV-1 infection of T cell lines [57
] and primary lymphocytes [56
] leads to an increase in intracellular levels of cAMP. This increase in cAMP levels activates the CREB/activating transcription factor (ATF) family through protein kinase A (PKA)-mediated phosphorylation. This phospho-CREB then recruits the adaptor CREB-binding protein (CBP) and basal transcription factors, which lead to increased promoter activation (for review, see ref. [58
]). HIV infection rapidly increases CREB/ATF-binding activity and levels of CREB/ATF proteins in a T cell line infected by HIV-1 [59
]. In latently infected promonocytic U1 cells, the cAMP/PKA and the PKC signaling pathways synergize to increase LTR transactivation and viral replication [60
]. In the same cells, cAMP also potentiates TNF- induction of HIV transcription [61
].
A recognition sequence for members of the CREB/ATF family was identified between the LEF-1 and the NF-B sites [62
]. Two other CREB/ATF-binding sites that also bind AP-1 are located downstream of the transcription initiation site, at positions +92/+102 and +160/+167 of the U5 LTR region [59
]. In cells of monocyte/macrophage lineage, CREB homodimers bind to their DNA site and recruit C/EBP dimers; CREB and C/EBP/NF–IL-6 also heterodimerize and bind to their respective LTR sites. In these cells, sequence variations at the CREB site affect LTR activity [53
]. In contrast, in Jurkat T cells, CREB does not act via its DNA site but mediates cAMP and dopamine-induced transcriptional stimulation through indirect interactions with the -40/+80 region of the LTR. Moreover, functional interactions between CREB and the nuclear receptor COUP-TF lead to synergistic activation of HIV-1 gene transcription in the presence of forskolin, cAMP, and dopamine [15
]. In addition to its effect on the PKA pathway and LTR-driven transcription, cAMP also induces up-regulation of cell-surface CXCR4 coreceptors and renders T lymphocytes more vulnerable to CXCR4-tropic HIV-1 infection [63
].
USF- and Ets-mediated stimulation of LTR-driven transcription
USF stimulates transcription through the TAR–DNA region, the Inr element, and the positions -173 to -157 upstream of the transcription start site of the LTR in vitro [64
, 65
]. USF binds to the E-box overlapping the -170-centered C/EBP region and competes with NF–IL-6 for binding [16
, 65
, 66
]. Although USF activates HIV-1 transcription in a T cell line, it acts as an inhibitor in an epithelial cell line [67
].
The two Ets (-95/-104 and -140/-160)-binding sites of the LTR mediate Ets-mediated activation of HIV-1 gene transcription [68 ]. Interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells [69 ]. Confirming the previous observations, the dominant-negative mutant of Ets-1 markedly suppresses HIV-1 infection in T cells [70 ].
Nuclear hormone receptors mediated regulation of LTR-driven transcription
Various members of the steroid-thyroid-retinoid nuclear receptor superfamily modulate HIV-1 transcription by distinct molecular mechanisms in different cells. The nuclear receptor responsive element (NRRE) is located within the distal -356/-320 region of the LTR [71
72
73
74
]. The retinoic acid receptors (RAR and RXR) stimulate transcription in the presence of their ligand via the NRRE site in choriocarcinoma cells [72
] and in CV1 cells [73
]. In oligodendroglioma cells, RAR and RXR activate transcription in the absence of ligand. Addition of ligands reverses this effect. This retinoid response is mediated by distinct molecular interaction with the LAI and the JR-CSF HIV-1 strains [75
]. In microglial cells, no significant effect of the retinoid pathway could be detected [75
].
Distinct LTR sites are able to mediate the response to RA, independently of the NRRE sequence. In U937 monocytes, RA activation is mediated by the proximal -83/+80 LTR region in the presence of phorbol esters [phorbol 12-myristate 13-acetate (PMA); ref. 76
]. In contrast, RA inhibits HIV gene transcription via the NF-B responsive element in untreated U937 monocytes and HeLa cells [77
]. RA, like arsenic trioxide, induces the remission of acute promyelocytic leukemia, resulting from a fusion of the RAR and the promyelocytic leukemia (PML) genes (for review, see refs. [78
, 79
]). Arsenic activates HIV-1 replication by enhancing reverse transcription and disrupting PML cellular localization [80
, 81
]. The PML protein regulates Tat transactivation by modulating CycT1 availability to the transcriptional machinery [82
]. Thus, as described for arsenic, RA may also impact HIV-1 gene expression independently of LTR-binding sites by targeting PML function.
The LTR is activated by thyroid hormone T3 receptors (T3R) in the absence of ligand in CV1 green monkey kidney cells. This activity is mediated by a direct binding of T3R to the Sp1-binding region, which also functions as a thyroid hormone receptor response element [83 , 84 ]. Recent results suggest that chromatin remodeling, including histone acetylation and chromatin disruption, is important for T3 regulation of the HIV–LTR in vivo [85 ]. In microglial cells, no T3R was detected [11 ], and overexpression of T3R did not alter the LTR activity (unpublished results).
Administration and/or excess secretion of glucocorticoids have been associated with increased HIV-1 replication and AIDS progression. Recombinant glucocorticoid receptors interact with the glucocorticoid receptor responsive element (GRE) located within the -259/-264 region of the LTR [86 ]. In CEM T4 cells, transcription is slightly up-regulated by dexamethazone treatment and in contrast, is repressed in promonocytes [87 , 88 ].
Protease inhibitors used in highly active antiretroviral therapy (HAART) create several metabolic disorders. Agonists of peroxisome proliferator-activated receptor (PPAR) are being studied in HAART-related metabolic disorders. The inhibitory effect of PPAR and PPAR agonists on HIV-1 replication has been shown in monoblastoid U1 cells, PBMCs, and macrophages [89
, 90
]. These results suggest that modulation of PPAR activity by their agonists may improve the metabolic alterations following HAART in conjunction with the desirable decreases in viral replication.
COUP-TF [91 ] is an orphan member of the nuclear receptor superfamily. It is the major species bound to the NRRE region of the LTR in microglial and glial cells of the brain [11 , 92 ], as well as in monocytes THP1 and HL-60 (unpublished results). Distinct cell-type and HIV strain-specific mechanisms govern COUP-TF-induced stimulation. COUP-TF functions as an activator of HIV-1 gene transcription in T cells, microglial cells, and oligodendroglioma but not in astrocytoma [11 , 92 ]. In microglial cells, COUP-TF stimulates LTR-driven transcription independently of its binding to the NRRE region by a direct interaction with the Sp1 protein bound to its LTR site. Similarly, overexpressed COUP-TF in Jurkat T cells also acts via Sp1 [11 , 15 ]. In contrast, in oligodendroglioma cells, the NRRE site is required for COUP-TF action, as mutation of the NRRE sequence, as found in the JR-FL and JR-CSF strains, prevents COUP-TF-mediated transcriptional stimulation [92 ].
AP-1-mediated regulation of LTR-driven transcription
The AP-1 family of oncoproteins Jun and Fos are tetradecanoyl phorbol acetate (TPA)-inducible transcription factors known to interact with the TGACTCA consensus sequence. Three AP-1-binding sites identified in the +92/+167 LTR region downstream of the transcription start site appear important for viral infectivity [21
, 93
]. However, within the modulatory region of the LTR, the two -352/-324 and -306/-285 consensus sites are unable to directly bind AP-1 proteins present in various cell types. AP-1 interacts indirectly with the NRRE region and modulates HIV transcription via members of the nuclear receptor family [75
]. AP-1 components present in glial and HeLa cells but not AP-1 present in Jurkat T cells interact with a TPA-responsive element (TRE)-like sequence located at positions -242/-222 in the LTR of JR-FL and JR-CSF strains and absent in the LAI strain [74
]. This interaction mediates c-Jun-induced transcriptional activation in glial cells. However, no AP-1-induced activation is detected in Jurkat T cells (ref. [94
] and unpublished results). These data show the flexibility of the virus to use, or not, the AP-1 transcription factor, depending of the cell type. Recently, the viral Nef protein has been shown to stimulate the AP-1 pathway [95
].
TAR–DNA-mediated inhibition of LTR-driven transcription
Certain cellular proteins such as lipopolysaccharide (LPS)-binding protein (LBP)-1 and TAR–DNA-binding protein-43 (TDP-43) are able to block HIV transcription. LBP-1 inhibits the binding of the general transcription factor transcription initiation factor IID (TFIID) to the TATA element [96
]. TDP-43 binds to the -18/+80 TAR–DNA sequence of the LTR and inhibits HIV transcription in the absence or the presence of Tat by blocking the assembly of transcription complexes that are capable of responding to Tat [97
, 98
].
Yin Yang 1 (YY1)- and lisofylline (LSF)-mediated inhibition of LTR-driven transcription
The cellular transcription factor YY1 has the ability to activate or repress gene expression. In the context of the HIV-1 LTR, YY1 functions as a repressor of gene transcription and viral replication. The YY1 protein cooperates with LSF (CP-2, LBP-1c, or UBP) in recognition of the -10/+27 LTR region in CEM lymphocytes, U937 monocytes, COS-1, and HeLa–CD4 cells [99
, 100
]. The two factors inhibit HIV-1 gene expression by recruitment of histone deacetylase 1 to the LTR nucleosome 1, which counteracts Tat activation [101
, 102
].
P53-mediated inhibition of LTR-driven transcription
The tumor suppressor p53 regulates cell-cycle progression and transcription of various viral and cellular promoters [103
]. p53 has been suggested to be a host suppressor of HIV-1 activation, as overexpression of wild-type p53 inhibits the basal activity of the HIV–LTR in HeLa cells [103
]. The LTR core region is involved in the p53-mediated repression [104
, 105
]. p53 also associates with Tat and acts as a potent suppressor of Tat transactivation [106
]. In contrast, mutant forms of p53 stimulate expression from the LTR [104
, 105
, 107
]. Inhibition of HIV-1 replication by overexpressed wild-type p53 is detected in Jurkat, SupT1 T cells, and microglial cells (unpublished results).
|
REGULATION OF HIV-1 TRANSCRIPTION BY VIRAL PROTEINS |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
B sites [9
] and a direct interaction with the Sp1 protein [18
, 19
] (for review, see refs. [108
, 109
]). In addition, Tat associates with various coactivators such as CBP/p300 [110
111
112
], pCAF [111
], and hGCN5 [113
], which help Tat activate transcription of integrated viral DNA and derepress the HIV-1 chromatin structure in response to histone acetylation [114
]. Moreover, Tat associates with distinct transcription factors, depending on the cell type and the LTR sequence. In microglial cells, Tat physically interacts and cooperates with COUP-TF to promote NF-B and Sp1-independent transactivation. This interaction leads to an alternative pathway for Tat transactivation, in the case of LTR mutations that abolish Sp1 or NF-B binding [115
]. Tat-dependent transcription is regulated in a cell-cycle-dependent manner. While in the G1 phase, Tat transactivation is TAR-dependent and requires a functional Sp1-binding site; in G2, a second TAR-independent phase of Tat transactivation is observed [116
].
Tat has the ability to modulate the expression of various cellular factors involved in the regulation of the HIV-1 gene transcription. Tat sets up positive up-regulatory loops, which greatly superactivate HIV transcription by activating NF-B in different cell types [117
118
119
] and up-regulating several cytokine genes such as TNF-, TNF-ß, IL-2, and IL-6 (for review, see refs. [120
, 121
]). In macrophages and astrocytes, an exposure to Tat results in prolonged cytokine production maintained by a positive-feedback loop of Tat-induced NF-B activation [122
]. Induction of IL-8 in T cells by Tat is also mediated through NF-B [123
]. Tat activates NF-B through degradation of the inhibitor IB, which requires the function of the cellular interferon (IFN)-inducible protein kinase PKR [124
].
In addition to its action on NF-B, Tat up-regulates the IL-6 gene and interacts with the induced NF–IL-6 protein that activates LTR-driven transcription [125
]. A reciprocal, modulatory interplay takes place between Tat and NF-AT1 in Jurkat T cells: Tat enhances NF-AT1-driven transcription, and NF-AT1 represses Tat-mediated transactivation of the LTR [37
]. Tat induces nitric oxide synthase via the NF-B and C/EBP pathways in glial cells [126
]. Moreover, Tat impairs the Tat suppressor functions of p53 by a dual action: It inhibits the transcription of the p53 gene [106
] and the p300-mediated acetylation of the p53 protein [127
].
Tat affects not only the infected host cells but also when secreted, noninfected, bystander cells. Tat-stimulated microglial cells release proinflammatory molecules and accumulate free radicals [128
]. Tat activates bystander endothelial cell E-selectin expression via a NF-B-dependent mechanism [129
].
Although most of Tat-associated proteins function as positive factors (for review, see ref. [130
]), some of them are able to act as repressors [98
, 102
, 131
]. In microglial cells, Tat function and HIV-1 replication can be repressed by overexpression of the nuclear corepressor COUP-TF-interacting protein 2 (CTIP2), which associates with heterochromatin protein (HP)1 and relocalizes Tat within inactive regions of the chromatin [132
].
Effect of Vpr
The Vpr gene product participates in nuclear transport of the preintegration complex, which empowers HIV to infect and replicate in nondividing cells, particularly in macrophages [133
, 134
]. Vpr is important for efficient viral replication in tissue macrophages but not in nondividing CD4+ T cells [135
].
Vpr functions as a positive regulator of HIV-1 gene transcription, as virion-associated Vpr arrests the cell cycle of infected cells in the G2 phase, which optimizes LTR-directed gene transcription and cooperates with Tat to enhance HIV transcription [136 , 137 ]. The ability of Vpr to activate transcription is mediated by the p300 coactivator [138 ] and by linking the GR to the p300 coactivator in monocytes [139 , 140 ]. The association of Vpr with Tat and CycT1 also stimulates LTR-driven transcription [141 ]. Vpr, expressed de novo or released from virion following entry, also enhances gene expression from integration-incompetent, unintegrated viral DNA generated during infection [142 ]. Moreover, Vpr induces changes in transcriptional patterns through modulation of the state of the target cell (for review, see ref. [143 ]).
Effect of Nef
The viral protein Nef displays multiple functions and acts as a master switch, forcing an environment conducive to dynamic viral production. It has no direct effect on HIV-1 gene transcription but participates in the enhancement of viral expression by up-regulating the expression of factors that positively regulate LTR-driven transcription such as NF-AT [41
], NF-B, AP-1 [95
], signal transducer and activator of transcription-1 (STAT1) [144
], and cdk9 [145
].
|
REGULATION OF HIV-1 TRANSCRIPTION IN DIFFERENT CELL TYPES |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
|
B and NF-AT proteins does the LTR become able to activate expression of the viral genome. Thus, the enhancer region of the LTR that binds cellular proteins such as NF-B and NF-AT is absolutely required for transcriptional activation. In addition, NF–IL-6, CREB, USF, and Ets contribute to full activation.
The thymus is the primary organ in which T cells mature. Depletion of circulating CD4+ T cells partly results from infection of the thymus. Thymic infection leads to thymocyte depletion by apoptosis and also to destruction of T cell progenitors by direct infection [146
, 147
]. In thymocytes, LTR-driven transcription and viral replication require activation of NF-B by various cytokines provided by the thymic microenvironment [28
].
Monocytes/macrophages and dendritic cells (DC)
In the monocyte/macrophage lineage, the NF–IL-6 and/or USF proteins bound to the LTR modulatory region appear essential for HIV-1 gene transcription, in addition to NF-B and Sp1. Moreover, in these cells, the state of differentiation regulates viral expression by affecting the level of CycT1 and thus, the transactivating function of Tat [148
]. In addition, in monocyte/macrophages cells, Vpr is essential for efficient viral replication.
DC residing within epithelial surfaces are likely the first cells infected after mucosal exposure, and DC derived from blood interact with HIV virions crossing the mucosae. DC efficiently process and present viral antigenic determinants to activate specific antiviral T and B cell immunity (for review, see ref. [149
]). The developmental stage of DC influences the extent to which HIV-1 can replicate. Although mature DC and blood monocytes [150
] do not replicate HIV-1, immature DC can be productively infected [151
]. The loss of HIV replication in mature DC is a result of a block occurring mainly at the transcriptional level, which does not affect expression and localization of NF-B, Sp1, and Sp3 [152
].
Microglial cells
In microglial cells, the target for productive infection in the brain, most of the transcription factors that govern LTR-driven transcription have the ability to directly or indirectly interact with the LTR core region. It is interesting that this region is not bound to any nucleosome within the chromatin and thus, may freely interact with cellular transcription factors. Sp1 proteins play an essential stimulating function, as they are able to anchor to the promoter various cellular transcription factors (C/EBP/NF–IL-6, CREB/ATF, COUP-TF) by direct or indirect associations. This unique characteristic leads to HIV-1 strain-independent transcriptional activation. However, as in other cell types, the low level of transcription is enhanced upon stimulation by nuclear relocation of NF-B. Indeed, cultured human embryonic microglial cells internalize HIV-1 but are not permissive to viral replication. HIV replication is only induced when cell cultures are stimulated for 14 days by a combination of cytokines including IFN-, IL-1ß, and TNF-, confirming that activation of the NF-B pathway and cell maturation facilitate HIV-1 replication [153
]. Similarly, adult cultured microglial cells require the presence of a feeder layer of glial cells to promote viral replication [154
].
In resting T cells within the lymphoid compartments, in the DC network, and in microglial cells of the CNS, HIV can adopt a state of proviral latency. Even when viral replication remains undetectable for a long period of time, virus production can be restored from a small proportion of cells. Total virus clearance would require eradication of HIV-1 present within cells that serve as a latent viral reservoir. This reservoir is constituted by cells where the preintegration complex resides in a state of preintegration latency, as well as of cells in which the provirus is integrated in the host genome. Postintegration latency contributes greatly to the chronicity of the infection and to the now-established fact that optimal HAART cannot eradicate HIV (for review, see refs. [155 156 157 ]). Some of the proposed mechanisms for postintegration latency are the lack of activation-dependent host-cellular factors in resting cells [158 , 159 ], an insufficient level of Tat or Tat-associated factors [160 , 161 ], the chromatin structure at the site of the provirus integration [162 ], and inefficient export of RNAs for structural proteins [163 ]. Thus, targeting latency represents a new challenge to viral eradication and requires a basic understanding of the mechanisms underlying proviral expression.
|
VARIABILITY OF THE LTR AND REGULATION OF HIV-1 GENE EXPRESSION |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
|
Variability of the core region
Mutations of the Sp1 sites of the LTR abolish Tat transactivation. In microglial cells, in the absence of Sp1 sites, Tat restores its activity by recruiting the nuclear receptor COUP-TF, which substitutes for the Sp1 protein [115
]. Thus, adaptative interactions of Tat with cellular transcription factors provide a solution to overcome specific LTR mutations or a specific cellular context.
The Y2 strain is a natural variant that harbors a mutation in the Sp1 III-binding site. This mutation has been observed in a long-term, nonprogressor, HIV-positive individual [175 ] and in a rapid progressor [176 ]. The Y2 variant replicates poorly in T cells but replicates well in monocytes, which suggests an adaptation of Y2 to monocytes by specific compensations for the loss of Sp1 binding [14 ].
A cohort of six blood recipients infected from a single HIV-positive and nonprogressive donor remains free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10–14 years after infection. Survival of these patients is directly linked to mutations in the U3 region of the LTR or in the Nef gene [177 ]. Analysis of LTR sequences of 184 clones isolated from nonprogressive patients before and after infection shows that during the nonprogressive phase, the predominant sequences have a large deletion in a Sp1-binding site and adjacent promoter site in the U3 part of the LTR. The transition of a nonprogressive phase to a rapid, progressive infection is impacted by reorganization of the LTR. When the infection became progressive, all viruses had intact Sp1 and promoter sequences and were derived from a minor species present earlier [178 ]. These studies suggest that during the nonprogressive phase, the Sp1 enhancer-promoter deletion likely played a role in attenuating HIV-1 replication.
Variability of the NF-B enhancer region
Analysis of the distinct LTR sequences indicates that each subtype A–G harbors particular DNA-binding motifs. The LTR of subtype C harbors three NF-B motifs instead of the two motifs present in the LTR of other subtypes. Differential regulation of clade-specific B, C, and E LTRs by NF-B has revealed that the NF-B sites of subtypes B and E bind NF-B-related complexes. However, the duplicated B sites of the C subtype do not compete for NF-B binding [179
].
Recently, a variant of a poorly replicative virus has been described in SupT1 cells [169
]. The variant LTR sequence presents a severe loss of NF-B binding directly linked to an adaptative point mutation in the second NF-B-binding sequence. The new nucleotide sequence binds GABP, a member of the Ets family. This conversion of NF-B into a GABP site restores a Tat-responsive activity. This virus adaptation appears so efficient that the GABP variant overgrows the wild-type LAI strain in a mixed infection in SupT1 cells. It is interesting that all 18 subtype E isolates also harbor this GABP-binding site [169
, 180
].
Variability of the LTR modulatory region
The USF-binding site is present only in the HIV-1 subtype B, and the AP-1-binding sites vary from zero to two in different subtypes [180
] and in different virus strains from the same subtype [74
]. A representative subtype D virus circulating in Uganda harbors a duplication of the LEF-1/TCF-1-binding sequence, which results in high infectivity [181
]. This LEF-1-binding site duplication also occurs in 
38% of HIV-infected patients within the Vancouver Lymphadenopathy-AIDS Study [182
]. Sequence variations in the ATF/CREB-binding site also impact transcription factor recruitment and result in more active isolates [183
]. Mutations in the NRRE, which result in the loss of COUP-TF binding in the brain-derived JR-FL strain of the subtype B, do not significantly alter transcription [11
]. CNS-derived JR-FL and CSF strains also harbor a CNS-specific TRE sequence [74
]. Moreover, phylogenetic analysis of nervous tissue-derived HIV LTRs are genetically distinct from that present in other organs such as lung or spleen, indicating that HIV LTR evolution can occur in a compartmentalized manner [184
].
Taken together, the HIV-1 LTR variability indicates an impressive adaptation capacity to the cellular context, which contributes to HIV-1 infectivity.
|
HIV-1 GENE TRANSCRIPTION AS A PUTATIVE TARGET FOR ANTI-HIV STRATEGY |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
B and Sp1 (for review, see ref. [187
]). The major tea catechin epigallocatechin gallate (EGCg) strongly inhibits HIV-1 gene transcription in LPS-activated monocytoid THP1 cells. EGCg also impacts other steps of the HIV-1 replication cycle such as postabsorption entry, viral particle stability, and reverse transcription [188 ]. The bistriazoloacridone temacrazine inhibits HIV-1 gene transcription with a high selectivity regarding any other cellular genes. Temacrazine may impact the binding of factors to positions -1, -2, and +111 of the LTR, as resistance to this compound was generated via mutations at these sites [189 ].
Recent studies demonstrate that engineered zinc-finger transcription factors can inhibit HIV replication in Jurkat T cells. Zinc-finger proteins designed to bind to the -69/-51 Sp1 region of the LTR and fused to the Krüppel-associated box repression domain are efficient inhibitors of LTR-driven transcription [190 ].
Greater specificity might be expected with strategies using compounds designed to inhibit Tat. As Sp1 and NF-B impact the expression of a number of cellular genes, Tat represents an ideal target for inhibiting HIV gene expression. Thus, the precise understanding of Tat-mediated transcriptional stimulatory events is crucial for the development of efficient and specific anti-HIV strategies. Flavopiridol, an inhibitor of cdk and P-TEFb, blocks Tat transactivation and HIV replication [191
]. Fluoroquinoline derivatives that selectively inhibit RNA-binding transactivators are able to repress LTR-driven transcription and replication by targeting Tat [192
, 193
]. More recently, the dominant-negative kinase-inactive mutant cdk9/ hCycT1 chimera was found to inhibit Tat activity and thus, HIV replication in human cells [194
]. Another recent study demonstrates that a chimeric, small nucleolar RNA–TAR decoy, which affects the nucleolar trafficking of Tat, potently inhibits HIV-1 replication in lymphoblastoid CEM cells [195
]. We have recently demonstrated that in microglial cells, overexpression of the nuclear cofactor CTIP2 inhibits HIV-1 gene transcription and replication by relocating Tat to transcriptionally inactive regions of the chromatin via HP1. Active, inhibitory CTIP2-derived peptides could be further tested in an antiviral strategy [132
]. Finally, inhibitors that block the induction pathways of the Tat cofactors cdk9 and CycT1, known to be regulated by extracellular stimuli [196
], could also ultimately interfere with Tat function and affect HIV transcription. Identification of additional molecules that selectively target the Tat transactivation process remains a line of investigation that provides an interesting opportunity to block HIV-1 expression.
The last decade of research on the mechanisms governing gene transcription has highlighted the importance of chromatin structure and remodeling. Multiprotein complexes help activate gene expression by altering chromatin to promote a better access of sequence-specific proteins and of the general transcription machinery to DNA regulatory sequences [197
]. Acetylation/deacetylation events are implicated in chromatin remodeling of the HIV-1 promoter region, as well as in modulating the activity of cellular or viral transcription factors implicated in LTR-driven regulation. LTR-binding transcription factors represent good candidates for the specific targeting of acetyltransferases and deacetylases to the HIV-1 promoter, thereby regulating the acetylation level of histones near the LTR (for review, see refs. [198
, 199
]). In the case of HIV-1, acetylation could also modulate the activity of some components of the basal transcription machinery as well as LTR-binding transcription factors such as Sp1, p53, p65, and p50 subunits of NF-B (for review, see ref. [200
]). It is interesting that Tat itself is subjected to acetylation events (for review, see ref. [199
]). This presently active area of research will help shed new light on the mechanisms of active transcription and postintegration latency of the HIV provirus. As these regulatory mechanisms become better understood, it is likely that new therapeutic targets will become available.
|
ACKNOWLEDGEMENTS |
|---|
Received April 24, 2003; revised July 8, 2003; accepted July 9, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONCELLULAR SPECIFICITY OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...REGULATION OF HIV-1...VARIABILITY OF THE LTR...HIV-1 GENE TRANSCRIPTION AS...REFERENCES |
|---|
