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The Journal of Immunology, 2006, 176: 4182-4190.
Copyright © 2006 by The American Association of Immunologists

Transactivator of Transcription from HIV Type 1 Subtype E Selectively Inhibits TNF Gene Expression via Interference with Chromatin Remodeling of the TNF Locus1

Shahin Ranjbar, Ricardo Rajsbaum and Anne E. Goldfeld2

CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transactivator of transcription (Tat) protein is essential for efficient HIV type 1 (HIV-1) replication and is involved in the transcriptional regulation of the host immune response gene, TNF. In this study, we demonstrate that Tat proteins from representative HIV-1 subtype E isolates, but not from subtypes B or C, selectively inhibit TNF gene transcription and protein production in CD4+ Jurkat T cells. Strikingly, we show that this repression is due to a tryptophan at residue 32 of Tat E and is secondary to interference with recruitment of the histone acetyltransferase P/CAF to the TNF promoter and with chromatin remodeling of the TNF locus. This study presents a novel mechanism by which HIV-1 manipulates a host immune response gene that is important in its own replication. Moreover, these results demonstrate a new mechanism by which the TNF gene is regulated via chromatin remodeling secondary to viral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The HIV type 1 (HIV-1)3 transactivator of transcription (Tat) protein is essential for viral replication (1). Once it is bound to the TAR, an RNA stem-loop structure located from position +1 to +46 of the HIV-1 long terminal repeat (LTR), Tat promotes efficient transcriptional elongation of viral transcripts (2).

Tat can be released from HIV-1-infected cells, and can be taken up by uninfected cells (3, 4, 5), and is therefore capable of also influencing the transcription of host genes in HIV-1-uninfected cells. Most studies analyzing the effect of Tat on either HIV-1 or host gene expression have been performed using Tat from B subtypes, although the C and E viral subtypes are the most prevalent subtypes globally (6) and differ from B subtype viruses in the sequences of several important viral genes, including Tat (7).

TNF is a host gene whose expression is influenced by Tat that is of particular relevance to HIV-1 biology. The binding of TNF protein to its cognate receptors triggers signal transduction pathways, resulting in activation of MAPK pathways and in the nuclear translocation of NF-{kappa}B (8) and subsequent LTR-mediated viral transcription in infected cells (9, 10, 11, 12). Studies analyzing effects of Tat upon TNF activation have largely been performed in cells from the monocytic lineage, where HIV-1 Tat B has been shown to augment TNF gene expression in monocytic, astrocytic, and dendritic cells (13, 14, 15, 16). However, TNF is a major immediate early gene product of Ag-activated T cells and causes T cell septic shock, and TNF levels in activated T cells are equivalent to those levels induced in monocytic cells and dendritic cells activated via pathogen recognition receptors such as TLRs (Refs.17 and 18 and data not shown).

The key checkpoint in TNF protein production in T cells, as well as monocytic cells, is the transcriptional activation of the gene (18, 19, 20). Furthermore, TNF gene expression is regulated differently in the monocyte/macrophage and T cell lineages via the recruitment of inducer and cell type-specific secondary order DNA-protein complexes, or enhanceosomes (17, 21, 22). However, in both cell types histone acetyltransferases (HATs) (23) and chromatin remodeling (24) play a critical role in enhanceosome formation and are required for TNF gene activation.

In this study, we show that Tat from HIV-1 subtype E isolates interfere with chromatin remodeling of the TNF locus and with the recruitment of p300/CBP-associating factor (P/CAF) to the TNF promoter, resulting in lower levels of TNF gene expression and protein production in T cells. We localize these effects to residue 32 in exon 1 of Tat E and to the proximal TNF promoter. These results demonstrate a novel mechanism by which HIV-1 manipulates the host immune response and a specific gene critical in its own biology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Viruses

HIV-1Bal, HIV-192TH03, HIV-193TH64, HIV-193TH51, HIV-193TH53, and HIV-198IN17 were obtained from the National Institutes of Health AIDS Research and Reference Reagents Program, Division of AIDS, National Institutes of Allergy and Infectious Diseases (Bethesda, MD). HIV-1LAI and HIV-198IN22 were obtained from the Centralized Facility for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC; South Mimms, U.K.).

Plasmids

The –200 TNF-Luciferase reporter gene (TNF-Luc) has been described previously (19). To construct HIV-1 Tat expression vectors, a pool of PHA (Sigma-Aldrich)-stimulated normal donor PBMC were infected with the above viruses for a short period of time, after which total RNA was extracted using RNeasy mini kit (Qiagen), and cDNA was generated. Sets of primers were designed to amplify the Tat gene from subtypes B, C, or E. The PCR products were subcloned into the pcDNA3.1 expression vector (Invitrogen Life Technologies) using BamH1 and Xho1 restriction sites.

Sequence analysis

Sequence analysis was performed by cycle-sequencing and dye terminator methods with an automated DNA sequencer (Brigham and Women’s Hospital Sequencing Center, Boston, MA). Between 10–20 Tat molecular clones were sequenced from each isolate, and the Tat amino acid sequences were aligned and analyzed by using CLUSTAL W (<http://www.ebi.ac.uk/clustalw/>).

Cell culture and transfection

The cells Jurkat E6-1 (CD4+ T-lymphoid) and U937 (monocytic) cell lines were obtained from the Centralized Facility for AIDS Reagents, the NIBSC (South Mimms, U.K.), and were grown at 37°C, 5% CO2, in RPMI 1640 supplemented with 10% FCS, L-glutamine (2 mM), penicillin (1 U/ml), and streptomycin (100 µg/ml). Transfections were performed using an Effectene transfection kit (Qiagen) according to the manufacturer’s protocol. After 16 h, cells were stimulated with 20 ng/ml PMA (Calbiochem) plus 4 µg/ml PHA (Sigma-Aldrich), or plus 1 µM Ionomycin (Calbiochem) for 8 h as indicated in the figures, and luciferase assays were performed according to the manufacturer’s instructions (Dual Luciferase Reporter assay system; Promega) using a Dynex luminometer, with Renilla luciferase (pRL-TK) as an internal control.

Stable Tat transfectants

Jurkat cells were transfected with pcDNA3.1 vectors expressing Tat from B subtype HIV-1LAI, or E subtype HIV-193TH64 (wild-type (wt) (TH64)) or W>G mutant (TH64mut) under the control of the CMV promoter, and cells were grown in RPMI 1640 supplemented with 10% FCS, under the continuous selective pressure of 800 mg/ml G418 (neomycin) (Sigma-Aldrich). As a control, Jurkat cells were also stably transfected with the pcDNA3.1 vector alone and grown and selected under same condition.

TNF measurements

A total of 1 x 106 cells/ml was cultured in 12-well plates in RPMI 1640 supplemented with 10% FCS, and cells were untreated or stimulated with 4 µg/ml PHA plus 20 ng/ml PMA. Supernatants were collected at various time points, and TNF levels were measured by ELISA using OptEIA Kit (BD Pharmingen).

DNaseI hypersensitivity (DH) analysis

DH analysis of the human TNF locus was performed, as previously described (24), in Jurkat cells stably expressing subtypes B Tat (LAI), E (TH64), or empty vector (pCDNA3.1). Briefly, 50 x 106 cells were either untreated or stimulated with 4 µg/ml PHA and 20 ng/ml PMA for 4 h, after which nuclei were isolated in Nonidet P-40 lysis buffer and digested with 0, 2, or 4 µg/ml DNaseI. DNA was phenol/chloroform-extracted, ethanol precipitated, and digested with BamHI. Samples were resolved on 1% agarose gels, transferred to nitrocellulose membranes, and hybridized with probes corresponding to the 5'-flanking region of the TNF gene.

Formaldehyde cross-linking and chromatin immunoprecipitation (ChIP)

A total of 40 x 107 cells was untreated or stimulated with PHA (4 µg/ml) and PMA (20 ng/ml) for 4 h, after which the cells were treated with formaldehyde (1% final concentration) for 30 min at room temperature. Fixed chromatin was sonicated, extracted, and purified as described previously (19, 25), followed by immunoprecipitation of 12 µg of chromatin with anti-P/CAF, and anti-GCN5, anti-AcH3 (Lys9/14), and anti-AcH4 (Lys8) Abs (Santa Cruz Biotechnology). Normal rabbit serum was used as a negative control. Immunoprecipitated chromatin was reversed cross-linked and amplified with primers specific to the TNF promoter as described previously (19).

Quantitative PCR

RT-PCR was used to determine TNF and Tat levels of the cells used for ChIP analysis. The reaction conditions were 95°C for 10 min to activate the DNA polymers followed by 40 cycles of 95°C for 20 s and 60.5°C for 1 min. The results were normalized using beta-actin as an internal control.

Statistical analysis

Results are expressed as a mean ± SEM. Comparison between two groups was performed using the unpaired Student t test with the aid of Microsoft Excel software. p < 0.05 was considered significant.

Three-dimensional modeling of Tats

Model building. The three-dimensional models of Tat LAI, TH64wt, and TH64mut were constructed by homology modeling using the Swiss-Model protein modeling server (<http://www.expasy.org/spdbv>) (26, 27, 28, 29). HIV-1BRU Tat (Protein Data Bank (PDB) entry code 1JFW) showed the highest homology to HIV-1LAI. Tat BRU therefore was selected as a template to model Tat LAI, TH64wt, and TH64mut. Templates and target sequences were aligned using Clustal W (30) and optimized in the Swiss-Pdb Viewer version 3.7b1 (26, 27, 28, 29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Sequence analysis of Tat from subtypes B, C, and E isolates

We isolated Tat from representatives of HIV-1 viral isolates from subtypes B (HIV1LAI and HIV1Bal), C (HIV198IN22 and HIV198IN17), and E (HIV192TH03, HIV193TH51, HIV193TH53, and HIV193TH64), and sequenced 10–20 Tat clones from each isolate and identified the most dominant clones (Fig. 1).


Figure 1
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  		FIGURE 1. Comparison of Tat amino acid sequences from representative viral isolates. Sequences from subtype B (HIV-1LAI, clone LAI; and HIV-1BAL, clone BAL), subtype C (HIV-198IN17, clone IN17; and HIV-198IN22, clone IN22), and subtype E (HIV-193TH64, clones TH64 wt and TH64 W>G; HIV-192TH03, clones TH03 wt and TH03-{Delta}2; HIV-193TH51, clones TH51 wt and TH51 W>R; and HIV-193TH53, clone TH53 wt) are shown. Amino acids identical with those of consensus HXB2 (B subtype) sequences are shown as a dash (-). Amino acids at position 32 are shown in block.

 
Comparison of Tat amino acid sequences among isolates from the three subtypes showed several variable regions, and these variable regions were generally consistent within specific subtypes and different between subtypes. One variable domain was in a cysteine (C)-rich region between aas 20–40, which in subtype B has been shown to interact with the HATs P/CAF (31) and GCN5 (32). The seven C residues in this motif are conserved in the B isolates and the E isolates examined. By contrast, consistent with other studies (7, 33), a serine replaced the C at residue 31 in the Tat C clones (Fig. 1). Notably, this serine at residue 31 in Tat C has been correlated with a significant reduction of monocyte migration to the brain (33).

We detected the occurrence of another putative subtype-specific amino acid located at residue 32 in the HAT-binding region of exon 1. In the subtype B Tats, we detected a phenylalanine (F) at this position. We note that other analyses of B subtype Tats revealed a leucine (L) at this same position (7). By contrast, in the subtype C we detected a tyrosine (Y), and in the subtype E isolates we detected a tryptophan (W) at this position (Fig. 1). W at position 32 has been reported in the Los Alamos Database in all E and G subtypes and in certain AG recombinant viruses, but has not to date been reported in Tats from other subtypes (7). Interestingly, we also detected Tat clones from subtype E isolates with naturally occurring variations at position 32, where the W was replaced with either a glycine (G) or an arginine (R). A Tat E clone, which included only the first exon of Tat, was also detected (Fig. 1). The frequency of these three variant Tat forms was <4% of the total pool of Tat isolates analyzed (data not shown).

Another region of Tat, which has been shown to interact with P/CAF (34) and p300/CBP (35), occurs between aas 50–60. Notably, this HAT interaction domain was also distinct in E isolates as compared with the B and C isolates examined. In the E Tats, there was a lysine (K) at position 53 and a histidine (H) at position 54, whereas there are two arginines (R) at these positions in both the subtype B and C isolates analyzed.

Another major difference between Tat from the B, C, and E isolates examined was in the acetylation sites of the Tats from the different subtypes. Potential acetylation sites at Lys28, Lys50, and Lys51 have previously been described in the three subtypes (32, 36, 37). However, we also detected additional lysine residues at positions 24, 40, 53, 63, and 96 that could serve as potential acetylation sites in Tat E, and these are not present in Tat B or C (Fig. 1). Taken together, our data demonstrate that the Tats from subtype B, C, and E isolates that we analyzed differ in sequences that have previously been shown to interact with a variety of HATs, and in sequences which may influence acetylation of Tat itself.

HIV-1 Tat regulates TNF gene expression in a subtype- and cell type-specific manner

To determine whether these subtype-specific Tat sequence differences had an impact upon Tat regulation of TNF gene expression, we cotransfected expression vectors encoding Tats from subtype B, C, or E with a TNF-Luciferase (Luc) reporter gene containing –200 to +93 nt relative to the TNF cap site, into a CD4+ T (Jurkat) cell line and stimulated the cells with PMA/PHA. Although cotransfection of B or C Tat isolates enhanced TNF reporter activity, the three Tat E expression vectors significantly inhibited TNF-Luc reporter activity in stimulated Jurkat cells (p < 0.01) (Fig. 2A). Because previous studies of Tat/TNF interactions had been performed in monocytic cells, we next tested the effect of Tat B, C, and E upon TNF reporter activity in U937 monocytic cells. Notably, the Tat B expression vectors significantly increased TNF activity in monocytic cells (p < 0.05), consistent with previous results (14, 16), as did the C Tat expression vectors (p < 0.01) (Fig. 2B). By contrast, the Tat E expression vectors had no appreciable effect upon TNF reporter gene activity in U937 monocytic cells stimulated by PMA and ionomycin (Fig. 2B). Taken together, these results demonstrate that the effect of Tat E upon TNF gene expression is distinct from the effect of Tat from B and C isolates tested in both a T cell and a monocytic cell line.


Figure 2
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  		FIGURE 2. Regulation of TNF transcription by Tat is viral subtype-specific. Expression vectors encoding subtype B, C, or E Tat (sequences shown in Fig. 1) or empty vector, pCDNA3.1 (0.4 µg/ml) were individually cotransfected with 0.25 µg/ml TNF luciferase reporter gene (TNF-Luc) into Jurkat (A) or U937 cells (B). The cells were transfected with the specific Tat expression vector, the TNF luciferase reporter gene, and a control Renilla-luciferase plasmid. Sixteen hours after transfection, cells were left untreated or stimulated with PHA (4 µg/ml) plus PMA (20 ng/ml), in the case of Jurkat cells (A and C), or PMA (20 µg/ml) and ionomycin (1 µM), in the case of U937 (B). The cells were harvested 8 h later, and luciferase assays were performed. As a control for transfection efficiency, relative luciferase values of the reporter genes were normalized to Renilla luciferase activity. C, LTR-luciferase reporter genes were cotransfected with Tat expression vectors isolated from the homologous viral isolate into Jurkat cells that were mock or PHA/PMA-stimulated for 8 h and processed as described above. The LTR molecular clones were constructed by inserting nucleotides –455 to +86 of HIV-1 LTR fragment from the isolates indicated from subtype B (HIV-1LAI), C (HIV-198IN22), and E (HIV-193TH64). Results are shown as relative value and are representative of three separate experiments. Results are shown as mean + SEM. *, p < 0.05; **, p < 0.01.

 
As a positive control for the ability of Tat E to activate transcription in Jurkat cells, we next tested the ability of representative Tat B, C, and E expression vectors to activate transcription of their autologous LTR in stimulated Jurkats. Cotransfection of Tat E and LTR resulted in a significant augmentation of LTR activity. LTR activation by Tat E was, however, lower than the increase observed using the B and C Tats on their own autologous LTRs in PMA/PHA-stimulated Jurkat cells, consistent with the lower LTR Luc activity of the E LTR in activated cells in the absence of Tat (Fig. 2B). Thus, the repressive effect of Tat E on TNF gene expression in Jurkat T cells is specific because it functions as a strong activator of its own LTR.

Naturally occurring variations define important residues in E subtype Tat that influence TNF gene expression

One of the naturally occurring variant Tat E proteins we identified was a mut of Tat TH64 that had an amino acid change in the P/CAF/GCN5 interaction domain, encoding a G at position 32 instead of the W (TH64 mut) found at this position in all wt E subtype isolates reported to date (Fig. 1 and see Ref.7). Remarkably, cotransfection of the W>G variant (TH64 mut) resulted in significantly higher levels of TNF reporter activity (p < 0.05) as compared with wt TH64 Tat in Jurkat cells. These levels did not, however, reach those levels observed when the subtype B (LAI) Tat expression vector was cotransfected with the TNF reporter gene (Fig. 3A).


Figure 3
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  		FIGURE 3. Amino acid Trp (W) at position 32 in subtype E Tat plays and important role in TNF suppression. A, B subtype LAI Tat or E subtype Tat TH64 wt or the TH64 W>G mut was cotransfected with TNF promoter-Luc reporter. Mutation of W>G significantly (*, p < 0.05) relieved TH64wt Tat repression of TNF reporter activity. B, E subtype Tat represses TNF transcription in a dose-dependent manner. Following stimulation with PHA (4 µg/ml) and PMA (20 ng/ml), TNF reporter activity was augmented with increased concentrations of B subtype Tat and was further decreased with increased concentrations of wt E subtype Tat. C, W>G mutation at position 32 of subtype E Tat partially relieves the repression of TNF reporter activity compared with subtype B Tat. Results are shown as relative TNF-Luc value and are representative of three independent experiments. As a control for transfection efficiency, relative luciferase values of the reporter genes were normalized to Renilla luciferase activity. The values are shown as mean ± SEM. We note that the activity of the different Tats relative to each other on TNF reporter activity is consistent between the experiments shown in A at the same concentration of Tat added, which was 0.5 µg in A, as compared with when we added 0.5 µg of the particular Tat in the experiments displayed in B and C.

 
To determine whether overexpression of Tat TH64 could overcome its repressive effect upon TNF reporter activity, we next cotransfected increasing amounts of the Tat TH64wt or Tat TH64mut expression vectors with the TNF reporter gene into Jurkat cells and compared their ability to enhance or repress TNF gene expression as compared with Tat LAI in activated cells. As shown in Fig. 3, B and C, TNF reporter gene activity was approximately doubled when the reporter gene was cotransfected with the Tat LAI expression vector at the highest Tat concentration (0.8 µg/ml), and Tat TH64 wt had the opposite effect upon TNF reporter activity, which was ~4-fold less at the same Tat concentration (Fig. 3B). Notably, however, increasing concentrations of the Tat TH64 mut expression vector resulted in TNF reporter levels that were ~60% of what was observed when LAI Tat was cotransfected with the TNF reporter (Fig. 3C). We also obtained similar results using wt E Tat TH51 and the variant Tat TH51 (W>R) mut (data not shown). Another of the naturally occurring variant Tat proteins we identified was a mut of Tat TH03, which had a stop codon at the end of the first exon of Tat after residue 66 (Tat TH03{Delta}2), but contained the wt W at residue 32. This variant allowed us to test whether the first exon of Tat with the W at position 32 was sufficient to cause repression of TNF. Indeed, cotransfection of Tat TH03{Delta}2 also significantly inhibited TNF reporter activity (data not shown).

Taken together, these results demonstrate the following: 1) W at residue 32 is specifically involved in Tat E repression of TNF gene expression; 2) the TNF proximal promoter spanning from –200 to +93 nt relative to the TNF cap site is sufficient for this repression; and 3) the first exon of E subtype Tat is sufficient for TNF repression in PMA/PHA-stimulated Jurkat T cells.

Tat B and Tat E cause differential chromatin remodeling of the TNF locus

Modification of repressive chromatin structures resulting in increased DNA accessibility, as demonstrated by the occurrence of DH sites, is required for transcriptional activators to interact with regulatory elements and for transcription to proceed (Reviewed in Refs.38 and 39). Activation of TNF gene expression in stimulated Jurkat cells is associated with chromatin remodeling and the appearance of an inducible DH site in an area of the TNF promoter rich with activator recruitment (21). This TNF promoter DH site in fact coincides with an in vivo footprint in this region of the TNF promoter in activated Jurkat cells (24). Furthermore, TNF gene activation is dependent upon coactivator proteins (20, 22, 23). Given that the change of a W to a G at position 32 is in Tat’s interaction domain with P/CAF and GCN5, we thus speculated that interference with chromatin remodeling and recruitment of coactivator proteins might play a role in Tat E’s inhibition of TNF gene expression.

We thus first investigated whether Tat E repression of TNF gene expression involved chromatin remodeling. To perform this experiment, we stably transfected Tat E TH64wt or Tat B LAI into Jurkat cells and then performed a DH analysis upon the endogenous TNF gene. Remarkably, the Tat LAI and Tat TH64 Jurkat cell transfectants displayed different TNF DH patterns upon PHA/PMA stimulation. Strikingly, the inducible DH site mapping to the TNF promoter, previously demonstrated in PMA/PHA-stimulated Jurkat cells in the absence of Tat (24), was present in the LAI transfectants but was undetectable in the TH64 Tat transfectants (Fig. 4, arrow 1, compare lanes 6 and 12). By contrast, there was increased cleavage of a constitutive DH site mapping to the 3'UTR region of the gene in the Tat E TH64 transfectants mapping above this band as compared with the LAI transfectants (Fig. 4, arrow 3, compare lanes 6 and 12), further strengthening the specificity of the inhibition of the inducible DH site in the TH64 Tat E transfectants.


Figure 4
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  		FIGURE 4. Tats from subtypes B and E differentially remodel the chromosomal region surrounding the human TNF locus. DNA from unstimulated (UN) or PHA plus PMA-stimulated Jurkat cells stably expressing Tats from subtype B HIV-1 (LAI) or HIV-1 E (TH64wt). Arrow 1 shows an inducible DH site mapping to the TNF promoter region in the Tat-expressing cells. The experiment shown is representative of six independent experiments. The location of Tat-specific DH sites is indicated by arrowheads and is summarized in the map at the bottom. The genetic map of the human TNF locus including the LT-{alpha} and LT-beta genes was compiled from GenBank accession nos. AF129756, D00102, X01394, L11015, and AF000424 and drawn to scale.

 
As a control, we also analyzed endogenous TNF protein levels and kinetics in stimulated Jurkat-Tat transfectants by ELISA. As shown in Fig. 5A, following stimulation, high levels of endogenous TNF were detected by 3-h poststimulation in the pCDNA3.1 control transfectants, and these levels were further enhanced by ~25% in the Tat LAI transfectants. There were no detectable TNF levels in unstimulated cells (data not shown). Consistent with our transient transfection results, TNF levels were significantly inhibited in the TH64 transfectants as compared with the pCDNA3.1 control transfectants (p < 0.001). Furthermore, TNF levels in Jurkat cells stably transfected with the Tat E TH64 mut were significantly increased (~4-fold; p < 0.05) relative to the repression of endogenous TNF protein in the TH64wt transfectants at all time points tested. We note, however, that TNF levels in the TH64mut transfectants were only ~30% of the TNF levels achieved in the control transfectants (Fig. 5A). As shown in Fig. 5B, there was no detectable Tat mRNA in the pCDNA3.1 control transfectants, and we detected equivalent levels of Tat RNA in the LAI, TH64 wt, and TH64mut transfectants. Thus, Tat from the HIV-1LAI and HIV-193TH64 isolates cause distinct patterns of chromatin remodeling of the TNF locus and functionally produce different levels of TNF protein. Furthermore, the DH findings are consistent with our transient transfection analyses and indicate that the Tat TH64 inhibitory effect upon TNF gene expression localizes to the proximal TNF promoter.


Figure 5
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  		FIGURE 5. Tat from subtypes B and E cause different TNF protein levels in Jurkat cells following cellular stimulation. A, Jurkat Tat stable transfectants (1 x 106 cells) were stimulated with PHA (4 µg/ml) and PMA (20 ng/ml), and supernatants were collected 2, 5, 8, and 11 h later, and TNF levels were assessed by ELISA. B, Tat mRNA expression levels in Jurkat-Tat stable transfectants were equivalent. Cells stably transfected with the pCDNA3.1 empty vector are shown as control. beta-actin was used as an internal control.

 
Tat E interferes with the recruitment of P/CAF and GCN5 to the TNF promoter and with histone acetylation

To determine whether Tat E might influence recruitment of P/CAF or GCN5 and/or acetylation of the TNF promoter in Jurkat cells in vivo, we next performed ChIP assays using chromatin from mock or PMA/PHA-stimulated Jurkat cells stably transfected with pCDNA3.1, Tat LAI, Tat TH64wt, or Tat TH64 mut, and Abs to P/CAF, GCN5, and acetylated residues in histones H3 (AcH3) and H4 (AcH4), and primers to amplify the TNF promoter (Fig. 6A).


Figure 6
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  		FIGURE 6. Differential HAT recruitment to the TNF promoter in Jurkat cells stably transfected with Tat from subtypes B and E. A, ChIP of mock or PMA/PHA-stimulated Jurkat cells stably transfected with the pCDNA3.1 control vector. Following 4-h stimulation, cells were treated with formaldehyde to cross-link endogenous protein and DNA. Sonicated and purified chromatin was immunoprecipitated with the indicated Abs, and a fixed amount of DNA isolated from the immunoprecipitated material was amplified with primers specific for the TNF promoter. An increase in the relative amount of the amplified TNF promoter-specific PCR product indicates binding of the protein to the endogenous TNF promoter. Control amplifications with the chromatin used as input for the immunoprecipitations are shown at the bottom. B, ChIP of PMA/PHA-stimulated Jurkat cells stably transfected with Tats from subtypes B (LAI) and E (TH64wt and TH64 mut). Cells were stimulated and ChIP performed as in A. The experiments displayed are representative of four independent experiments.

 
In control pCDNA3.1 transfectants, we detected inducible recruitment of P/CAF and GCN5 to the TNF promoter and H3 acetylation upon stimulation with PMA/PHA, and we found that AcH4 levels were constitutive and not increased by cellular stimulation (Fig. 6A). Thus, in the absence of Tat, in stimulated Jurkat cells, P/CAF and GCN5 are recruited to the TNF promoter, and H3 is acetylated in vivo.

We next compared the recruitment of P/CAF and GCN5 to the TNF promoter and AcH3 and AcH4 levels in stimulated Jurkat cells stably transfected with Tat LAI, TH64wt, or TH64 mut. As shown in Fig. 6B, the Tat E TH64 wt transfectants displayed a dramatically different pattern of HAT recruitment to the TNF promoter as compared with the Tat B LAI transfectants. P/CAF was not inducibly recruited to the TNF promoter in the Tat TH64 wt transfectants, and GCN5 and AcH3 levels were ~50% less than their levels in the Tat LAI transfectants (Fig. 6B). In the case of the mut Tat E TH64 mut transfectants, P/CAF levels were restored to ~50% of the levels observed in the Tat B LAI transfectants. Furthermore, GCN5 levels were restored to those observed in the LAI transfectants, and AcH3 levels were not increased above the levels observed for the TH64wt transfectants (Fig. 6B). Thus, Tat E TH64wt differentially inhibits the recruitment of P/CAF and GCN5 to the TNF promoter and H3 acetylation in vivo. Although GCN5 levels in the TH64mut transfectants were equivalent to the levels observed in the LAI transfectants, P/CAF levels and AcH3 levels remained compromised (Fig. 6B). These results are consistent with the inability of the Tat TH64mut transfectants to achieve the TNF transcriptional activity and protein levels in control cells and indicated that interference of the recruitment of P/CAF to the TNF enhanceosome by Tat TH64 might be the functional explanation for its repression of TNF gene expression.

Overexpression of P/CAF overcomes the repression of Tat E TH64

To examine this hypothesis, we next tested whether overexpression of P/CAF could overcome the repression of TNF gene expression by Tat TH64 wt. Because we already demonstrated that the TNF promoter (–200 to +87 relative to the mRNA cap site) alone was sufficient for inhibition by Tat E (Fig. 2A) in Jurkat cells, and demonstrated using primers to the promoter that there was differential recruitment of P/CAF in Tat E stable transfectants, we cotransfected the TNF reporter gene in the presence of the pCDNA3.1 control vector, Tat LAI, or Tat TH64wt in the presence or absence of P/CAF and measured TNF reporter activity. There was a significant increase in TNF reporter activity in those cells cotransfected with TH64wt and P/CAF as compared with TH64wt alone, whereas transfection of P/CAF at a 1:1 ratio had no effect upon the cells cotransfected with pCDNA3.1, and had a nonsignificant effect upon the cells transfected with Tat LAI (Fig. 7). Furthermore, not only was repression of TNF reporter activity relieved by cotransfection of P/CAF with Tat Th64wt, but the levels of TNF gene expression were significantly elevated and equivalent to those observed in the cells cotransfected with Tat LAI and P/CAF expression vectors (Fig. 7). Thus, these results were consistent with the hypothesis that Tat TH64 was sequestering P/CAF, because when the P/CAF concentration was increased to match the concentration of Tat TH64 wt, the repressive effect of Tat TH64 upon TNF reporter activity was overcome.


Figure 7
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  		FIGURE 7. Overexpression of P/CAF overcomes the repression of TH64 Tat. Jurkat cells were cotransfected with pCDNA3.1 control vector, Tat LAI, or TH64wt and an equal amount (0.4 µg/ml) of the P/CAF expression vector with the TNF luciferase reporter gene and a control Renilla-luciferase plasmid using the Effectene Transfection kit (Qiagen). We first tested the effect of cotransfecting control vector or P/CAF at 50, 100, and 150% of the concentration of the cotransfected LAI or TH64 Tats (data not shown) upon TNF reporter activity and determined that a 1:1 ratio of P/CAF gave a maximal effect (data not shown). Sixteen hours after transfection, cells were mock or PHA (4 µg/ml) plus PMA (20 ng/ml)-stimulated for 8 h, and luciferase assays were performed. Results are shown as fold induction of TNF promoter activity. Overexpression of P/CAF significantly (p < 0.02) relieves TNF suppression by Tat TH64 wt. Results are shown as mean + SEM. *, p < 0.05.

 
Changes at residue 32 impact the structure of Tat in models of Tat of E and B

To further pursue the possibility that Tat TH64 wt had a distinct interaction with P/CAF, we modeled the structure of Tat LAI, TH64wt, and the TH64mut. We first screened the PDB using the PDB viewer software (Swiss-Pdb Viewer version 3.7b1) (26, 27, 28, 29) and constructed a three-dimensional model of the 86-aa HIV-Tat protein using the Swiss-Model protein modeling server (<http://www.expasy.org/spdbv/>) to find a sequence homologous to each Tat protein. Using this method, we determined the sequence homology between the Tat proteins used in this study and a Tat protein whose three-dimensional structure was reported in the PDB. We selected the Tat protein from the HIV-1 B isolate BRU (PDB code, 1JFW) (40, 41) as a template for modeling the TH64wt, TH64 mut, and LAI Tats, because it has 97% sequence homology with residues 1–86 of Tat LAI (the only changes are at positions 24 and 77), and Tat BRU has a Phe at position 32 as does Tat LAI. In addition, Tat BRU shares 62.8% homology with Tat TH64wt, making it a suitable template for modeling this protein as well. As shown in Fig. 8, the three Tat structural models have a similar three dimensional structure, and all superimpose well upon the Tat BRU template (data not shown).


Figure 8
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  		FIGURE 8. Ribbon representations of three-dimensional models of Tat LAI (A), Tat TH64 wt (B), and TH64mut W>G (C) showing amino acid residues potentially important in the Tat-P/CAF interaction. Amino acid residues are shown in stick and dots. Lys28 (green), which is acetylated by P/CAF, is conserved in all subtypes. Position 32, which we speculate affects the Tat-P/CAF interaction, is Phe32 in LAI (red), Trp32 in TH64 wt (violet), and Gly32 in TH64 mut (yellow). A putative preferential plane of interaction with P/CAF is comprised of residues at positions 33, 36, 63, and 65. His33 and His65 (gray) are conserved between subtypes. Positions 36 and 63 (gold in LAI, and pink in TH64 wt) show changes between subtypes. The sequence alignment of aas 25–66 of the Tat proteins is shown. Residues involved in the putative P/CAF-Tat binding site are colored and shown in boxes. The molecular views were drawn using PyMOL software (<www.PyMOL.org>).

 
The C-rich region of Tat (aas 25–36, and boxed in the sequence alignment at the bottom of Fig. 8) is formed by two beta turns and is on an exposed surface of the protein (Fig. 8 and Refs.40 and 41). Phe and Trp are hydrophobic amino acids, and Gly is an amino acid that lacks hydrophobic side chains. We therefore speculated that the presence or absence of these specific amino acids at position 32 could theoretically influence protein-protein interactions of Tat with other proteins such as P/CAF. In this regard, it is important to note that P/CAF binds to aas 20–40 of Tat in the absence of Tat Lys28 acetylation. Moreover, acetylation of Tat at Lys28 interferes with the Tat-P/CAF interaction (42). Finally, P/CAF itself acetylates Lys28 LAI (36).

Previously, it has been demonstrated that position +4 C-terminal to the reactive lysine has a particularly important role in the substrate selectivity of P/CAF as shown in the analysis of the crystal structure of the P/CAF/H3 complex (43). Strikingly, position 32 of Tat is +4 C-terminal to the reactive lysine at residue 28 (Lys28). As shown in Fig. 8, the presence of a Phe, Trp, or Gly at residue 32 impacts the conformation of Tat in the region, and it could be expected that the presence of the bulky Trp side chains present in TH64 wt Tat would influence the accessibility of Lys28 to acetylation. By contrast, the presence of Gly at position 32, which lacks hydrophobic side chains, would allow easier access of P/CAF to the binding groove as compared with the bulky Trp, and thus could be anticipated to influence the accessibility of Lys28 to acetylation and the release of P/CAF. In conclusion, these models support the hypothesis that the change at residue 32 from Phe to Trp or Gly would affect the ability of P/CAF to acetylate and to bind to Tat in the region of the protein between aas 20–40. Furthermore, these models provide a mechanistic explanation of the impact of the W to G change at residue 32 and its effect upon recruitment of p/CAF and the accessibility of Lys28 to acetylation by P/CAF.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, in an in vitro CD4+ T cell model, we have shown that Tat from HIV-1 subtype E isolates differentially repress TNF production in a CD4+ T cell line by interference with the recruitment of P/CAF to the TNF promoter and with chromatin remodeling of the TNF locus. These effects result in impaired TNF gene activation and protein production in stimulated Jurkat T cells. Thus, this study demonstrates both the role of chromatin remodeling of the TNF promoter in TNF gene regulation in activated T cells and that the Tat E proteins tested differentially influence the expression of TNF, a gene whose downstream signaling pathway is critical in driving HIV-1 replication.

HIV-1 subtypes have spread unevenly, such that the HIV-1 B subtype is predominantly associated with the infection in North America and Europe. By contrast, subtypes C and E have spread in Africa and Asia (6) and are currently the most prevalent subtypes globally, spreading faster than other subtypes (6, 44). Notably, the C and E viral subtypes differ from the B subtype in the sequences of several important viral genes, including Tat.

The relationship between virus subtype and pathogenicity is not well defined, in part because virus replication and virus-host interaction studies have been performed predominantly with subtype B viruses. Sequence analysis of HIV-1 has shown distinct sequences between the subtypes. Molecular differences between distinct subtypes and their impact upon viral-host interactions and viral replication, transmission, and dissemination remains to be determined.

Several laboratories have shown that LTRs from different subtypes have different activation profiles in response to a variety of stimuli. For example, E subtype LTRs are relatively less responsive to TNF than a B subtype LTR (45), whereas subtype C LTRs are relatively more responsive to p65 and Tat from a B subtype than subtype E LTR representatives (46). It has also been shown that E subtype LTRs were less responsive to T cell activation in the absence of Tat (47), similar to our findings presented in Fig. 2C. These differences were attributed to relatively decreased binding of NF-{kappa}B to the E subtype LTR and increased binding of NF-{kappa}B to the C LTR (45, 46, 47). Interestingly, the impaired binding of NF-{kappa}B to the E subtype LTR was correlated with enhanced binding of another transcription factor, GA binding protein (GABP). Furthermore, the presence of this GABP binding site increases LTR activity in the SupT-1 T cell line, which has low levels of NF-{kappa}B (48).

Our studies presented here, using a model Jurkat T cell system, allowed us to correlate TNF gene transcription, protein production, chromatin remodeling, and coactivator recruitment in vivo. Our experiments demonstrate a novel mechanism by which HIV-1 manipulates TNF, a host immune response gene that is important in its own replication, in certain circumstances via inhibition of chromatin remodeling and the recruitment of P/CAF. Thus, this model system allowed us to determine a mechanistic explanation for our findings, demonstrating the specific repressive action of Tat from subtype E HIV-1 isolates upon TNF transcription and protein levels. In support of these findings is the observation that TNF is regulated in a cell and inducer-specific fashion (17, 21, 22), and, furthermore, that there is differential HAT recruitment to the TNF enhanceosome in activated T cells (23). We also demonstrated a difference in TNF activation in a monocytic cell line U937 by Tat E, which had no impact upon TNF gene expression in contrast to Tat B and Tat C, which significantly increased TNF gene expression when cotransfected. Although out of the scope of the present study, it will be important to investigate TNF levels in T cells and monocytes from subtype E HIV-1-infected individuals or to infect T cell and monocytes with subtype E viruses and compare TNF levels to subtype C or B HIV-1-infected cells. Such experiments will allow the determination of the specific effects of clade E Tat upon TNF and potentially other host immune response genes in vivo in the human host.

In the analysis presented in this study, we analyzed Tat genetic sequences from four different HIV-1 subtype E isolates and found a Trp (W) at position 32 along with infrequent naturally occurring variations, at position 32 where the W was replaced with either a Gly (G) or an Arg (R). To date, W at position 32 has been only detected in subtype E and in the phylogenetically close (49) subtype G and AG recombinant HIV-1 isolates (Los Alamos Database) (7). Sequence analysis of the Tat and LTR genes from the four HIV-1 isolates examined demonstrate that they are both from subtype E (Fig. 1 and data not shown). Thus, W at residue 32 appears to be specific for E Tats.

Intriguingly, the Tat E inhibitory effect upon TNF transcription can be mapped to a single amino acid change at residue 32 of Tat E, because naturally occurring Tat E variants with a change at position 32 from Trp to Gly significantly increases TNF transcription and protein production and P/CAF recruitment to the TNF promoter. Strikingly, our structural modeling studies are consistent with the hypothesis that the bulky Trp at position 32 in Tat E wt interferes with the acetylation of Tat at Lys28 by P/CAF. Because acetylation of Lys28 is necessary for P/CAF to be released from Tat, it could be expected that P/CAF would be sequestered and thus not be available to interact with the TNF promoter. These speculations are supported by the fact that we can overcome TH64 wt’s repressive effect upon TNF gene expression by overexpression of P/CAF. Thus, Tat E has an ability to modulate TNF gene expression and thus protein production via its interaction with P/CAF.

Given the key role that TNF has in activating the NF-{kappa}B pathway and the nuclear translocation of NF-{kappa}B, it is interesting that the LTR of subtype E HIV-1 isolates have relatively weak NF-{kappa}B binding ability as compared with subtypes B and C. In this regard, it is intriguing to speculate that Tat E’s inhibitory effect upon TNF leading to relatively lower levels of nuclear NF-{kappa}B might preferentially allow the superior LTR E activator, GABP (48), to be recruited to the E subtype LTR GABP/NF-{kappa}B binding site. We note that viral manipulation of TNF levels has precedent. In the case of pox viruses, an immunoevasion strategy involves viral production of an analog of TNF receptors, which downmodulates TNF protein production and thus counteracts the negative impact of TNF upon the viral life cycle (50).

These studies also underscore the importance of considering subtype-specific factors in the design of immunotherapeutic strategies and effective vaccines for AIDS that will have the greatest global relevance and safety. For example, Tat from subtype B has been studied as a potential vaccine by a number of groups (51, 52, 53, 54) and has shown excellent safety profiles in clinical phase I and II trials (55, 56). Results obtained in monkey experiments using Tat as a vaccine demonstrated some protection against infectious challenge with SHIV (52, 53, 54, 57). However, in our study, we have shown that Tat from subtype E isolates differentially manipulates at least one major host innate immune response gene, TNF. Therefore, further studies and characterization of Tat from different subtypes upon host genes is imperative if Tat-based therapeutic strategies are further developed. Finally, these results underscore the impact of subtype-specific HIV-1 Tat sequence differences in the virus-host interaction and their potential impact upon therapeutic strategies and vaccine development.


   Acknowledgments
 
We are grateful to H. Holmes and the Centralized Facility for AIDS Reagents, (National Institute for Biological Standards and Control, South Mimms, U.K.), S. Osmanov of World Health Organization-United National Programme on HIV/AIDS (Geneva, Switzerland) and the National Institutes of Health Research and Reference Reagents Program for critical reagents, and to D. Thanos for the p/CAF expression vector.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (AI60433 and GM056492; to A.E.G.) and the Center for AIDS Research of Harvard University Medical School and the Campbell Foundation (to S.R.). Back

2 Address correspondence and reprint requests to Dr. Anne E. Goldfeld, CBR Institute for Biomedical Research, Harvard Medical School, 800 Huntington Avenue, Boston, MA 02115. E-mail address: goldfeld{at}cbr.med.harvard.edu Back

3 Abbreviations used in this paper: HIV-1, HIV type 1; Tat, transactivator of transcription; LTR, long terminal repeat; HAT, histone acetyltransferase; P/CAF, p300/CBP-associating factor; wt, wild type; mut, mutant; DH, DNaseI hypersensitivity; ChIP, chromatin immunoprecipitation; GABP, GA binding protein. Back

Received for publication November 28, 2005. Accepted for publication January 12, 2006.


   References
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 Introduction
 Materials and Methods
 Results
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 Disclosures
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