Published online before print December 10, 2007
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B-dependent control of HIV-1 transcription by the second coding exon of Tat in T cells
,1
,2
* University of Heidelberg, Department of Hematology/Oncology, Heidelberg, Germany;
Department of Virology, IFR 133, EA 3186 Franche-Comté University, Besancon, France; and
Laboratoire de Virologie Moleculaire, Service de Chimie Biologique, Institut de Biologie et de Medecine Moleculaires, Universite Libre de Bruxelles, Gosselies, Belgium
2Correspondence: Department of Virology, Franche-Comté School of Medicine, Hôpital Saint-Jacques, 2 Place Saint-Jacques, F-25030 Besançon Cedex, France. E-mail: gherbein{at}chu-besancon.fr
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B activation, long terminal repeat (LTR) transactivation, and viral replication in T cells. C-terminal deletion and substitution mutants made with the infectious clone HIV-89.6 were assayed for their ability to transactivate NF-B-secreted alkaline phosphatase and HIV-1 LTR-luciferase reporter constructs for low concentrations of Tat. A mutant infectious clone of HIV-89.6 engineered by introducing a stop codon at aa 72 in the Tat open-reading frame (HIV
tatexon2) replicated at a significantly lower rate than the wild-type HIV-89.6 in phytohemagglutinin-A/IL-2-stimulated primary peripheral blood lymphocytes. Altogether, our results suggest a critical role for the glutamic acids at positions 92, 94, and 96 or lysines at positions 88, 89, and 90, present in the second encoding Tat exon in activating NF-B, transactivating the HIV-1 LTR and enhancing HIV-1 replication in T cells.
Key Words: LTR stimulation • viral reservoir • AIDS pathogenesis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B proteins [9
, 10
]. Tat also binds with high affinity and specificity to cyclin T1, an event that is believed to induce the elongation process of RNA polymerase II [11
]. The 87-kDa cyclin T1 binds to the cyclin-dependent kinase 9 (CDK9; PITALRE) to form a complex called positive transcription elongation factor b (p-TEFb) [12
13
14
]. According to one model, although Tat/p-TEFb complexes bind to TAR, CDK9 phosphorylates the carboxy-terminal domain of RNA polymerase II, and that enhances the level of viral gene transcription [15
]. In addition, specific protein 1 transcription factor (Sp1) has been reported to display an activity redundant to NF-B, particularly in many simian LTRs, which have single or no NF-B binding sites. Thus, cyclin T1 could interact with the Sp1 serine/threonine- and glutamine-rich "A" activation domain to reconstitute Tat/TAR-independent transcriptional activity [16
]. In an engineered HIV containing a noncognate Tat-TAR pair that neither interacts nor efficiently replicates (HIV-1 TAR and bovine immunodeficiency virus Tat), viral revertants were isolated in which TAR had become mutated to generate a functional HIV Tat-binding site, supporting the view that incremental changes to TAR structure can provide routes for evolving new Tat-TAR complexes and maintain active viral replication [17
]. The overall architecture of the LTR promoter is remarkably conserved among different viral isolates, but some degrees of variation in the number and type of binding sites are evident in the different subtypes [18
]. These variations, although small, modulate virus replication in a cell type-specific manner [19
]. The transcriptional strength of the LTR could be increased easily by duplication of binding sites, yet such viruses are generally not observed. However, defective viruses can regain replication competence by duplication of existing binding sites [20
]. This body of evidence implies that transcription of the HIV LTR is optimized for the needs of viral replication and that increased transcription rates are not beneficial, unless in the context of a replication-impaired mutant. HIV requires specific and controlled levels of transcription, and the strength of the wild-type LTR promoter represents an evolutionary optimum in terms of viral fitness [21
].
HIV/SIV Tat proteins are specified by two coding exons. Full-length HIV-1 Tat has been shown to have several roles, including the modulation of host gene expression [22
23
24
25
26
], activation of resting T cells [27
], induction of chemokine production [28
], and direct suppression of antigen-specific CD8+ T cell immune response in vivo [29
]. Although previous data suggest that the second coding exon of Tat is largely devoid of function [30
], the second coding exon of Tat has been reported to be involved in the cellular uptake of the exogenous Tat protein [31
]. The human translation elongation factor 1- binds to the second exon of HIV-1 Tat, resulting in a dramatic decrease of the efficiency of the translation of cellular, but not viral, mRNAs [32
]. Also, the suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences has been reported to result in a strong overall inhibition of reverse transcription in vitro [33
]. Tat induction of apoptosis is separated from the transactivation function of Tat, requires expression of the second exon of Tat, and is associated with the increased activity of caspase-8 [34
, 35
]. In lymphoid CD4+ T cells, Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK [36
, 37
]. The second coding exon of Tat has also been shown to have a propitious effect on HIV-1 replication in tissue culture [38
, 39
]. Moreover, a recent report indicates that the second coding exon of Tat contributes to chronic SIV replication in vivo [40
]. Thus, in macaques, a CTL response to the second coding exon of Tat appears to durably control SIV replication. When SIV mutated in an attempt to escape this second Tat-exon-CTL, the resulting virus was less replicatively fit and failed to populate the host in vivo. Thus, the second coding exon of Tat could play an important role in HIV-1 replication. As the function encoded by the second exon of Tat is currently reassessed, we investigated the role of the second coding exon of HIV-1 Tat in NF-B activation and LTR-mediated transcription. We observed a NF-B-dependent control of HIV-1 transcription by the second coding exon of the Tat protein in T cells, resulting in enhanced viral replication in primary PBLs.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and AP-1 consensus sequences were obtained from Santa Cruz Biotechnology. TNF was purchased from R&D Systems (Minneapolis, MN, USA).
Cell lines and primary PBLs
The studies were performed with the Jurkat lymphoid T cells obtained from the American Type Culture Collection (Manassas, VA, USA). The Jurkat cells were cultivated in RPMI 1640 supplemented with 10% FBS. Where specified, Jurkat cells were stimulated with 16 nM PMA and 1 µM ionomycin. Purified PBLs were prepared from peripheral blood of healthy donors as described previously [41
]. For purified PBL preparation, Ficoll-Hypaque (Pharmacia, Uppsala, Sweden)-isolated PBMCs were incubated for 2 h on 2% gelatin-coated plates. Nonadherent cells, >98% that were PBLs, as assessed by CD45/CD14 detection by flow cytometry analysis (Simultest Leucogate, Becton Dickinson, San Jose, CA, USA), were harvested after Ficoll-Hypaque isolation and adherence. PBLs were cultivated in RPMI with 10% (v/v) FBS supplemented with human recombinant IL-2 (20 IU/ml) following treatment with PHA (5 µg/ml) for 48 h.
Generation of a Tat-deleted, infectious molecular clone derived from HIV-1 89.6
Primary PBLs were infected with HIV-1 89.6 or a mutant infectious clone derived from HIV-1 89.6 by introducing a stop codon at aa 72 in the Tat open reading frame (ORF; HIVtatexon2). A point mutation (C>T) was introduced into a molecular clone of HIV-1 89.6 [42
] to change codon 72 of the Tat ORF from CAG to TAG. The mutation was introduced with the use of the transformer kit (Clontech, Palo Alto, CA, USA) with the following two oligonucleotides: 5'-CTCTATCAAAGTAGTAAGTAGTAC-3' (Tat mutation) and 5'-GTGCCACCTGATATCTAAGAAACC-3' (selection primer). The presence of the mutation was verified by sequencing, and a fully resequenced SalI-StuI fragment containing the mutation was subcloned back into p89.6. Supernatants from CEMX174 cells transfected with this DNA were harvested, and their RT activity was measured. To confirm that virus stock had not reverted to wild-type, virus stocks were centrifuged, and purified RNA was used in RT-PCR to amplify a fragment containing the mutated tat gene. This PCR fragment was cloned with the TA cloning kit (Invitrogen, San Diego, CA, USA), 10 individual clones were resequenced, and all contained the original mutation in the tat gene [24
].
Generation of Tat-expressing plasmids
A PCR-amplified cDNA fragment corresponding to the first exon (Tat72) or both exons (Tat101) of the HIV-1 tat gene was amplified with the use of the HIV-1 cDNA clone pCV1 as a template and primers containing an additional BamHI site for cloning. The amplified fragment was cloned into the unique BamHI site of the episomal vector pRep9 (Invitrogen) under the control of the Rous sarcoma virus promoter. As pCV1 cDNA encodes a truncated, 86-aa form of Tat, a mutation TAG[>]TCG, changing codon 86 from a stop to a serine, was introduced in the 3' primer used in the PCR reaction to reopen the Tat reading frame to its full length. The pcDNA3.1-based expression vectors for HIV-1-89.6 have been described elsewhere [24
]. Deletion and point mutation constructs were generated by standard PCR and cloning procedures. Tat constructs were verified by DNA sequencing.
EMSAs
To measure NF-B activation, EMSAs were carried out as described previously by Varin et al. [43
]. Briefly, the Jurkat cells were transfected with increasing amounts of pTat101 and pTat72 constructs (0, 10, 100, 500, 1000, 5000 ng). Twenty-four hours later, nuclear extracts prepared from these cells were then incubated with 32P-end-labeled, 45-mer, double-stranded NF-B oligonucleotide 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (bold indicates NF-B-binding sites; Santa Cruz Biotechnology), and the DNA–protein complexes formed were analyzed on a 6% native polyacrylamide gel. The specificity binding was examined by competition with unlabeled oligonucleotide, with a mutated NF-B oligonucleotide 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3' and with a heterologous, unlabeled AP-1 oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (Santa Cruz Biotechnology). The dried gels were visualized, and radioactive bands were quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) using ImageQuant software.
Microwell colorimetric NF-B assay
Microwell colorimetric NF-B assay was performed using a Trans-AM NF-B family transcription factor assay kit (Active Motif, Carlsbad, CA, USA). Briefly, nuclear extracts were incubated in a 96-well plate coated with an oligonucleotide containing the NF-B consensus-binding site (5'-GGGACTTTCC-3'). Activated transcription factors from extracts specifically bound to the respective immobilized oligonucleotide were detected using the antibodies to NF-B p50, p65, RelB, and c-Rel subunits, followed by a secondary antibody conjugated to HRP in an ELISA-like assay. Absorbance was read within 5 min on a multilabel reader (Perkin Elmer, Tarku, Finland) at 450 nm.
Reporter gene expression assays
To examine NF-B-driven gene expression by the Tat protein, cells were transfected with the secreted alkaline phosphatase (SEAP) expression plasmid (Clontech) for 24 h before transfection with pTat72, pTat101, and C terminus-truncated (pTat75, pTat80, pTat86, pTat89, pTat95) or point-mutated Tat constructs. After 24 h, cell culture-conditioned medium was harvested and analyzed for alkaline phosphatase activity, as described previously [43
]. SEAP activity was assayed on 96-well flat-bottom microtiter plates suitable for plate luminometers (Victor Wallac, PerkinElmer, Wellesley, MA, USA). This reporter system was specific, as TNF-induced NF-B activity was inhibited by overexpression of the IB
mutant, IB-DN, which lacks Ser-32 and Ser-36 [43
, 44
].
To examine LTR-driven gene expression by the Tat protein, Jurkat cells were cotransfected with increasing DNA concentrations (0, 10, 100, 1000, 5000 ng) from pTat72, pTat101, or point-mutated Tat plasmids and with 20 µg p-LTR-Luc or 20 µg p-LTR-NFBmut-Luc using the electroporation system, according to the manufacturer’s instructions (BioRad, Hercules, CA, USA). Similar experiments were performed using an isogenic, TAR-deleted pLTRTAR-Luc [5
]. Forty-eight hours later, luciferase activity was measured in cell lysates, as reported above. Values were normalized to protein concentrations.
Western blot analysis
Cytoplasmic extracts of transfected Jurkat cells were used to examine IB phosphorylation by the Western blot procedure, as described previously [43
]. The gels were vizualized and bands quantified by a Molecular Images System GS 505 (BioRad) using Multianalyst software.
Infections and p24 assay
PHA/IL-2-activated PBLs were cultivated in six-well plates at a density of 3.6 x 106 cells/well and were left uninfected or were infected with increasing concentrations of HIV-1 89.6 or HIVtatexon2 (1, 5, 10, 15 ng p24 antigen per 3.6x106 cells), as reported previously [41
]. After one night of exposure to virus at 37°C, cells were washed three times with PBS to remove the unadsorbed inoculum and reincubated in fresh culture medium at 37°C. Culture supernatants were collected for up to 28 days and assayed for p24 antigen using a microELISA assay (Organon Teknika, Biomerieux, Boxtel, Netherlands).
Statistical analysis
Figures show the means of independent experiments and SD.
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B activationB activation by EMSA following transfection of Jurkat cells with pTat101 or pTat72 plasmids, derived from HIV-1 89.6, a viral infectious clone isolated from a HIV-infected individual (Fig. 1
). Following transfection, the expression levels of Tat101 and Tat72 proteins were similar, as measured using Western blotting (data not shown). Transient transfection of Jurkat cells with pTat101 revealed a dose-dependent activation of NF-B by EMSA, with maximum activation at 
100 ng (Fig. 2A
and 2B
). Similarly, transient transfection of Jurkat cells with pTat72 resulted in a dose-dependent activation of NF-B by EMSA, with maximum activation at 500 ng (Fig. 2A
and 2B)
. Interestingly, full-length Tat101 was more efficient in NF-B activation than truncated Tat72, especially when low amounts of plasmids were transfected, e.g., 10–100 ng (Fig. 2A
and 2B)
. The gel-shift band was specific, as formation of the complex was blocked with an unlabeled oligonucleotide (Fig. 2C)
. An interference by anti-p50 or anti-p65 antibody alone and also by a mixture of anti-p50 and anti-p65 antibodies was observed (Fig. 2D)
, indicating that the NF-B complex is composed of p50 and p65 subunits. To rule out the possibility that a TNF inducer, such as LPS, produced the activity, we confirmed the absence of endotoxin in all our reagents (data not shown). The phosphorylated form of IB, which is required for IB degradation, appeared when Jurkat cells were transfected with pTat72 or pTat101 plasmids (Fig. 2E)
. The full-length Tat101 induced IB phosphorylation in Jurkat cells more efficiently than the truncated Tat72, especially when low amounts of plasmids were transfected (Fig. 2E
and 2F)
.
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Figure 1. Schematic representation of wild-type (Tat101), truncated, and point-mutated HIV-1 Tat constructs. A series of C-terminal deletion and substitution Tat mutants was made, starting from HIV-189.6 Tat [42
].
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Figure 2. NF-B activation in Jurkat cells transfected with pTat101 and pTat72 plasmids. (A) Dose response of NF-B activation following transfection with pTat101 and pTat72 plasmids. Jurkat cells (2.5x106) were transfected with indicated concentrations of HIV-1 pTat101 and pTat72 plasmids for 24 h at 37°C, and then nuclear extracts were assayed for NF-B activation by EMSA as described in Materials and Methods. Data shown are representative of three independent experiments. (B) Dose response of NF-B activation following transfections with pTat101 and pTat72 plasmids. Jurkat cells (2.5x106) were transiently transfected with indicated concentrations of HIV-1 pTat101 and pTat72 plasmids for 24 h at 37°C, and then nuclear extracts were assayed for NF-B activation by EMSA, measured as fold induction versus untreated cells using Molecular Images System GS 505 (BioRad) Multianalyst software (upper panel) or by Microwell colorimetric NF-B assay (lower panel), as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown. (C) Specificity of NF-B activation by transfected HIV-1 pTat101 and pTat72 plasmids. Nuclear extracts from Jurkat cells transfected with pTat101 and pTat72 (500 ng) were incubated for 15 min with increasing concentrations of unlabeled NF-B oligonucleotide (0, 2, 9, 18 pmol), NF-B-mutated oligonucleotide (0, 2, 9, 18 pmol), and heterologous (AP-1) oligonucleotide (0, 2, 9, 18 pmol) and then assayed for NF-B activation. (D) Interference of NF-B activation by pTat101 and pTat72 in transfected Jurkat cells. Nuclear extracts from Jurkat cells transfected with pTat101 or pTat72 (100 ng) were incubated for 20 min with anti-p50, anti-p65, anti-p65 + anti-p50, anti-cRel, anti-RelB, or anti-p52 antibodies and then assayed for NF-B DNA-binding activity. Data shown are representative of three independent experiments. (E) Dose response of IB degradation and IB phosphorylation detected by Western blotting. Jurkat cells (2.5x106) were transfected with different concentrations of pTat101 and pTat72 plasmids. β-Actin was used as a loading control. (F) Dose response of IB phosphorylation quantified by a Molecular Images Systems GS 505 (BioRad) using Multianalyst software as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown.
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B activation at a much higher rate than Tat72, especially when low amounts (10–100 ng) of plasmid were transfected. Therefore, we assessed whether NF-B activation triggers gene expression in Jurkat cells transfected with pTat72 or pTat101 plasmids. We examined the effect of Tat72 and Tat101 on NF-B-driven SEAP expression in Jurkat cells. Tat72 and Tat101 enhanced SEAP expression in a dose-dependent manner (Fig. 3A
). Tat101 and Tat95 increased NF-B activation at a much higher rate than Tat72 (P<0.05), especially when low amounts (10–100 ng) of plasmid were transfected (Fig. 3A
and 3B)
. A series of C-terminal deletion Tat mutants was made starting from HIV-1 89.6 (Fig. 1)
. Transfections with Tat proteins, sequentially truncated at the C terminus through aa 72, indicated a critical role for the most distal region of the second coding exon of Tat (aa 86–101; Fig. 3B
). To further delineate the region(s) of the second coding exon of Tat involved in NF-B activation, when low amounts of plasmids were transfected, a series of C-terminal point-mutation Tat constructs was made with HIV-89.6 (Fig. 1)
and assayed for their ability to transactivate NF-B reporter constructs. Among all point-mutated Tat constructs, substitutions of glutamic acids at positions 92, 94, and 96 (E92-94-96) or lysines at positions 88, 89, and 90 (K88,89,90) with alanines dramatically impacted the activity of the 101-aa Tat protein, bringing it close to that of the Tat72 protein (Fig. 3C)
. NF-B activation, induced following Tat transfection, was not observed when a plasmid containing a mutated NF-B site, p-mut-NF-B-SEAP, was used, instead of NF-B-SEAP (Fig. 3B
and 3C)
.
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Figure 3. Regions of the second exon of Tat involved in NF-B-driven reporter gene expression. (A) Dose response of NF-B-driven reporter gene expression following transfections with pTat101 and pTat72 plasmids. Jurkat cells (2.5x106) were transiently transfected with a NF-B-driven [wild-type (wt)] or a mut-NF-B-driven SEAP expression plasmid for 24 h before transfection with indicated concentrations of pTat72 and pTat101 plasmids. Twenty-four hours later, cell culture-conditioned medium was harvested and analyzed for alkaline phosphatase activity as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown. (B) Jurkat cells (2.5x106) were transiently transfected with a NF-B-driven or a mut-NF-B-driven SEAP expression plasmid for 24 h before transfection with 100 ng pTat72, pTat101, and C terminus-truncated pTat constructs. Twenty-four hours later, cell culture-conditioned medium was harvested and analyzed for alkaline phosphatase activity as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown. *, Tat72 versus Tat95 and Tat72 versus Tat101: P < 0.05; **, Tat72 versus Tat86: P = not significant. (C) Jurkat cells (2.5x106) were transiently transfected with a NF-B-driven or a mut-NF-B-driven SEAP expression plasmid for 24 h before transfection with pTat72, pTat101, and C terminus point-mutated pTat constructs (100 ng). Twenty-four hours later, cell culture-conditioned medium was harvested and analyzed for alkaline phosphatase activity as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown.
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B-binding sites are present in the HIV-1 LTR [45
]. We assessed whether NF-B activation triggers LTR-mediated gene expression in Jurkat cells transfected with pTat72 and pTat101. To examine the effect of the second exon of Tat on NF-B-dependent LTR stimulation, Jurkat cells were transiently transfected with a target plasmid that contains the luciferase reporter gene under the control of the HIV-1 LTR promoter, pLTR-Luc. As activation of NF-B, specifically depending on the second coding exon of Tat, was observed preferentially for low amounts of transfected plasmid, Jurkat cells were transiently transfected 24 h later, with increasing concentrations of pTat72 or pTat101 plasmids (10 ng–5 µg), and luciferase activity was measured in cell lysates. Transfection with pTat72 and pTat101 plasmids resulted in increased LTR activation over control Jurkat cells transfected with the empty vector (Fig. 4A
). The transfection of Jurkat cells with the pTat101 plasmid resulted in a significant increase of LTR activation over pTat72-transfected cells, especially when low amounts of plasmid were used, e.g., 10–100 ng (Fig. 4A)
. The LTR activation induced by transfected Tat protein was not observed when a plasmid containing a mutated NF-B site, LTR-mut-NF-B-Luc, was used instead of pLTR-Luc (Fig. 4A)
. These data indicate that following transfection with low amounts of tat plasmid (10–100 ng), the second exon of Tat protein is critical for optimal NF-B-mediated HIV LTR activation. Then, the C-terminal substitution Tat mutants derived from HIV-89.6 (Fig. 1)
were assayed for their ability to transactivate HIV-1 LTR-luciferase reporter constructs. Among all point-mutated Tat constructs tested, E92-94-96 or K88,89,90 with alanines, did impact dramatically the activity of the 101-aa Tat protein, bringing it close to that of the Tat72 protein (Fig. 4B)
. We then assessed the role of TAR in Tat-mediated LTR stimulation following transfection with low amounts of tat plasmid. We observed that following transfection with low amounts of tat plasmid (100 ng), the LTR stimulation mediated by the second coding exon of Tat was NF-B-dependent and to a much lesser extent, TAR-dependent (Fig. 4C)
. By contrast, following transfection with higher amounts of tat plasmid (e.g., 500 ng), the LTR stimulation was primarily TAR-dependent and was mostly mediated by the first coding exon of Tat (Fig. 4D)
. To further delineate the respective role of NF-B dependence and TAR dependence in Tat-mediated LTR stimulation, we calculated the percentage of NF-B dependence and TAR dependence following transfection with increasing concentrations of pTat72 and pTat101 plasmids. Our results indicate that at low concentrations of pTat101 (10–100 ng/ml), the Tat-mediated LTR stimulation was mostly driven via NF-B, although a modest TAR dependence could also be observed (Fig. 4E)
.
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Figure 4. Regions of the second exon of Tat involved in LTR stimulation. (A) Jurkat cells (2.5x106) were transiently transfected with 20 µg p-LTR-Luc or with 20 µg p-LTR-mut-NFB-Luc. Twenty-four hours later, transfected cells were mock-transfected or transfected with different concentrations of pTat72 or pTat101. Twenty-four hours later, luciferase activity was measured in cell lysates. Mean values (±SD) of three independent experiments are shown. RLU, Relative light units. (B) Jurkat cells (2.5x106) were transiently transfected with 20 µg p-LTR-Luc or with 20 µg p-LTR-mut-NFB-Luc. Twenty-four hours later, transfected cells were mock-transfected or transfected with 100 ng pTat72, pTat101, or C terminus point-mutated pTat constructs. Twenty-four hours later, luciferase activity was measured in cell lysates. Mean values (±SD) of three independent experiments are shown. (C) Jurkat cells (2.5x106) were transiently transfected with 800 ng p-LTR-Luc, 800 ng p-LTR-mut-NFB-Luc, or 800 ng p-LTR-TAR-Luc. Twenty-four hours later, transfected cells were mock-transfected or transfected with 100 ng pTat72, pTat101, or C terminus point-mutated Tat constructs. Twenty-four hours later, luciferase activity was measured in cell lysates. Mean values (±SD) of three independent experiments are shown. (D) Dose response with TAR-deleted constructs. Jurkat cells (2.5x106) were transiently transfected with 800 µg p-LTR-Luc, 800 µg p-LTR-mut-NFB-Luc, or 800 ng p-LTR-TAR-Luc. Twenty-four hours later, transfected cells were mock-transfected or transfected with indicated concentrations of pTat72 or pTat101. Twenty-four hours later, luciferase activity was measured in cell lysates. Mean values (±SD) of three independent experiments are shown. (E) Respective roles of NF-B and TAR in Tat-mediated LTR stimulation. Following transient transfection of Jurkat cells with the indicated concentrations of pTat72 and pTat101 plasmids, the respective NF-B and TAR dependences (depd) of Tat-mediated LTR stimulation were calculated as follows: TAR dependence = (Tat-mediated LTR stimulation–TAR-mediated LTR stimulation)/(Tat-mediated LTR stimulation); NF-B dependence = (Tat-mediated LTR stimulation–NF-B-mediated LTR stimulation)/(Tat-mediated LTR stimulation). Mean values (±SD) of four independent experiments are shown.
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tatexon2). This mutation does not affect the sequence of Rev, which is encoded in another ORF in the same region [24
]. Replication of wild-type HIV-1 89.6 was superior to the replication of HIVtatexon2 in PBL cultures (Fig. 5A
). The delay in virus growth between HIVtatexon2 and HIV-1 89.6 was 10 days and 3–5 days when PBLs were infected at low m.o.i. (1 ng p24 per 3.6x106 cells) and high m.o.i. (15 ng p24 per 3.6x106 cells), respectively (Fig. 5A)
. Tat in the virus emerging at later time-points (e.g., at Day 28 postinfection following infection with 1 ng p24 HIVtatexon2) reverted to wild-type virus, as determined by the sequencing of the tat gene (data not shown). Moreover, the preferential replication of HIV-1 89.6 versus HIVtatexon2 was mostly observed at an early time after in vitro infection (Day 3 vs. Day 15; Fig. 5B
).
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Figure 5. Enhancement of HIV-1 replication in acutely infected PBLs by the second coding exon of Tat. (A) Primary PBLs were left uninfected or were infected with HIV 89.6 or HIVtatexon2 at a low multiplicity of infection (m.o.i.; 1 ng p24 per 3.6x106 cells) or at a high m.o.i. (15 ng p24 per 3.6x106 cells), and p24 was measured in culture supernatants at different times postinfection, as described in Materials and Methods. Mean values (±SD) of three independent experiments are shown. (B) Primary PBLs were left uninfected or were infected with increasing amounts of HIV 89.6 or HIVtatexon2 (1, 5, 10, 15 ng p24/3.6x106 cells), and p24 was measured at Days 3 and 15 postinfection in culture supernatants, as described in Materials and Methods. p24 fold induction following infection with HIV 89.6 versus HIVtatexon2 is shown. Mean values (±SD) of three independent experiments are shown.
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B activation, LTR transactivation, and HIV-1 replication in primary T cells. This functional advantage as a result of the second coding exon of Tat is lost when higher amounts of Tat are expressed within the cell. Our data indicate an important role for the second coding exon of Tat in the NF-B-dependent regulation of the HIV-1 transcription in T cells.
Here, we report that full-length Tat and Tat 1 exon proteins activate NF-B and induce IB phosphorylation and degradation in T lymphoid Jurkat cells. Although activation of NF-B was observed with Tat101 and Tat72, the second coding exon of Tat was shown to be critical for an optimal NF-B activation following transfection with low amounts of tat plasmid. In agreement with our results, increased NF-B activation has been described previously in lymphoid cells transfected with Tat-expressing plasmids or treated with exogenous recombinant Tat [46
]. Using high levels of Tat expression, the N-terminal region of Tat, which contains the short core domain (aa 21–40), has been shown to be sufficient for NF-B activation, LTR transactivation, and enhanced HIV replication in vitro [47
]. Therefore, the second coding exon of Tat could be dispensable for optimal NF-B activation when high amounts of the Tat protein are produced within the infected cell. This phenomenon might be observed in activated T cells where sustained NF-B activation occurs. In support of this hypothesis, in PMA/ionomycin-activated Jurkat cells, we did not observe a higher NF-B activation following transfection with full-length Tat101 versus transfection with Tat72, even at low plasmid concentrations (data not shown). Altogether, these data indicate that the second coding exon of Tat could be critical to obtain optimal NF-B activation when low amounts of Tat protein are present within the infected cell. This could occur at the early stage of the viral life cycle when low amounts of Tat protein are present within the infected cell [48
]. Following T cell activation and sustained viral production, the second coding exon of Tat might be no more useful and might even be harmful by leading to an hyperactivation of NF-B. The molecular mechanisms underlying the silencing of the second coding exon of Tat still have to be unveiled. Nevertheless, recent studies have shown that Tat activation can be inhibited by the overexpression of the host factors Yin Yang 1 and late SV40 transcription factor, which recruit histone deacetylase 1 to the LTR [49
]. Also, recruitment of Tat to heterochromatin protein 1 via interaction with transcription factor-interacting protein 2 (CTIP2) has recently been shown to inhibit HIV-1 replication via inhibition of Tat-mediated transactivation [50
]. The N-terminal region of Tat was shown to be sufficient for interaction with CTIP2 in vitro [50
]. We cannot exclude that the second coding exon of Tat could, in a similar way, restrict NF-B activation and further LTR transactivation by binding transcriptional repressors yet to be defined [51
]. The two-exon but not the one-exon form of Tat has been reported to suppress viral RT activity, especially during the late stage of the disease [52
, 53
]. In agreement with this hypothesis, we observed an inhibitory effect of the second coding exon of Tat versus Tat72 on NF-B activation and LTR stimulation, especially following transfection with a high amount of plasmids (superior to 1000 ng DNA per 2.5x106 cells). Transcription is part of a tightly coordinated HIV gene expression program that includes mRNA splicing, nuclear export, translation in the cytoplasm, virion assembly, and budding. Too high transcription rates may disturb this well-balanced process. Furthermore, cellular cofactors are needed at multiple stages of the virus life cycle. It is conceivable that overactivation of HIV transcription may lead to exhaustion of one or more factors present in limiting amounts, resulting in a decrease in infectivity [54
]. Transient induction of cyclin T1 during human macrophage differentiation has been shown recently to regulate HIV-1 Tat transactivation function, and this may play a critical role in the establishment of latency and subsequent reactivation of HIV-1 in infected macrophages [54
].
We observed that motifs of E92-94-96 and K88,89,90 at the C terminus of Tat are critical for an optimal NF-B activation, when low amounts of plasmid are transfected. In agreement with our results, using unrestricted HIV genome constructs replicating in primary cells, such as PBMC, 80 clones were sequenced from the Tat 2-exon virus, and 18 nucleic acid changes were found. A small fraction—four changes—of the total was located in the second coding exon of Tat. Furthermore, two of the four changes were conservative for amino acid, especially mE92E [55
]. Therefore, some regions of the second exon of Tat, e.g., E92, could be privileged in never exhibiting changes and could presumably be critical for replication. Moreover, a CTL epitope in the second exon of HIV-1 Tat (B58 CTL epitope) overlaps the regions of the second coding exon of Tat, especially the lysine-rich domain K88,89,90, which we demonstrated to be critical for NF-B activation and LTR stimulation [40
]. In macaque infected with SIVmac239, a CTL response to the second coding exon of Tat was observed, which appears to durably control SIV replication [56
]. Moreover, when SIV mutated in an attempt to escape this second Tat-exon-CTL, the resulting virus was less replicatively fit and failed to populate the host in vivo. Thus, successful, long-term suppression of viremia by CTL presumably might require immune targeting to epitope(s), in which any mutation is associated with a significant loss of viral function or fitness [56
57
58
59
], as it may be the case for several epitopes present in the second coding exon of Tat.
Our results indicate that the second coding exon of Tat stimulates the HIV-1 LTR in a NF-B-dependent manner, when low amounts of plasmid are transfected. In agreement with our data, Tat has been reported to transactivate the LTR by activating NF-B [60
]. Two distinct mechanisms could be involved in this phenomenon. First, Tat interacts with protein kinase R and the activated protein kinase R phosphorylates IB and allows the nuclear translocation of NF-B [61
]. Second, Tat has been reported to interact directly with the NF-B sequence, resulting in LTR stimulation [62
].
In agreement with previous results [38 ], our data indicate that the second coding exon of Tat enhances HIV-1 replication in primary PBLs, especially at low m.o.i. Moreover, among three human individuals infected with 1-exon Tat HIV, the opening of the HIV IIIB Tat stop codon in one of the patients has been correlated with the emergence of low CD4+ T cell count and rapid progression to AIDS [40 ]. In macaques infected with SIVtat1ex and whose virus mutated quickly to SIVmac239, all have uniformly higher viral loads, lower CD4+ cell counts, and more rapid progression to simian AIDS than their SIVtat1ex counterparts [40 ]. Collectively, these findings implicate a contributory function in the second coding exon of Tat to in vivo replication and pathogenesis during the chronic phase of infection.
In conclusion, our results show that the second coding exon of Tat plays a critical role in the activation of NF-B, the transactivation of the HIV-1 LTR, and in enhanced HIV-1 replication in T cells, especially when low amounts of Tat protein are expressed within the cell. This observation suggests a critical role for the second coding exon of Tat in AIDS pathogenesis via enhancement of HIV-1 replication, especially at early stages of the disease when optimal conditions are required to favor the spread of the disease. A better understanding of the mechanisms underlying the replication of HIV-1 in T cells and especially the role of regulatory proteins such as Tat is likely to lead to new therapeutic approaches and to the design of HIV-1 vaccine candidates.
Received June 15, 2007; revised October 31, 2007; accepted November 1, 2007.
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B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat J. Virol. 68,3971-3981 delineates a mechanism for HIV-1-mediated shut-off of host mRNA translation Biochem. Biophys. Res. Commun. 244,384-389[CrossRef][Medline]B and stimulates HIV-1 transcription in promonocytic cells and primary macrophages J. Biol. Chem. 280,42557-42567B by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-B-inducing kinase. Identification of a novel TRAF6 interaction motif J. Biol. Chem. 274,7724-7731 B, activator protein-1, c-Jun N-terminal kinase, and apoptosis J. Immunol. 164,5156-5166B by the Tat protein of human immunodeficiency virus type 1 J. Virol. 70,4427-4437[Abstract]B enhancer sequence: role in viral and cellular gene expression Nucleic Acids Res. 32,1270-1278
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