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ABSTRACTPackaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only ~2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

ABSTRACTRepression of viral expression is a major strategy developed by retroviruses to escape from the host immune response. The absence of viral proteins (or derived peptides) at the surface of an infected cell does not permit the establishment of an efficient immune attack. Such a strategy appears to have been adopted by animal oncoviruses such as bovine leukemia virus (BLV) and human T-cell leukemia virus (HTLV). In BLV-infected animals, only a small fraction of the infected lymphocytes (between 1 in 5,000 and 1 in 50,000) express large amounts of viral proteins; the vast majority of the proviruses are repressed at the transcriptional level. Induction of BLV transcription involves the interaction of the virus-encoded Tax protein with the CREB/ATF factors; the resulting complex is able to interact with three 21-bp Tax-responsive elements (TxRE) located in the 5' long terminal repeat (5' LTR). These TxRE contain cyclic AMP-responsive elements (CRE), but, remarkably, the “TGACGTCA” consensus is never strictly conserved in any viral strain (e.g.,AGACGTCA, TGACGGCA, TGACCTCA). To assess the role of these suboptimal CREs, we introduced a perfect consensus sequence within the TxRE and showed by gel retardation assays that the binding efficiency of the CREB/ATF proteins was increased. However,trans-activation of a luciferase-based reporter by Tax was not affected in transient transfection assays. Still, in the absence of Tax, the basal promoter activity of the mutated LTR was increased as much as 20-fold. In contrast, mutation of other regulatory elements within the LTR (the E box, NF-?B, and glucocorticoid- or interferon-responsive sites [GRE or IRF]) did not induce a similar alteration of the basal transcription levels. To evaluate the biological relevance of these observations made in vitro, the mutations were introduced into an infectious BLV molecular clone. After injection into sheep, it appeared that all the recombinants were infectious in vivo and did not revert into a wild-type virus. All of them, except one, propagated at wild-type levels, indicating that viral spread was not affected by the mutation. The sole exception was the CRE mutant; proviral loads were drastically reduced in sheep infected with this type of virus. We conclude that a series of sites (NF-?B, IRF, GRE, and the E box) are not required for efficient viral spread in the sheep model, although mutation of some of these motifs might induce a minor phenotype during transient transfection assays in vitro. Remarkably, a provirus (pBLV-?21-bp) harboring only two TxRE was infectious and propagated at wild-type levels. And, most importantly, reconstitution of a consensus CRE, within the 21-bp enhancers increases binding of CREB/ATF proteins but abrogates basal repression of LTR-directed transcription in vitro. Suboptimal CREs are, however, essential for efficient viral spread within infected sheep, although these sites are dispensable for infectivity. These results suggest an evolutionary selection of suboptimal CREs that repress viral expression with escape from the host immune response. These observations, which were obtained in an animal model for HTLV-1, are of interest for oncovirus-induced pathogenesis in humans.

Using the OM-10.1 promyelocytic model of inducible human immunodeficiency virus type 1 (HIV-1) infection, we tested a panel of known protein kinase inhibitors for an ability to block tumour necrosis factor-α-induced HIV-1 expression. Among the compounds tested, the broad-spectrum protein kinase inhibitor H-7 uniquely blocked HIV-1 expression at the level of viral transcription, but did not inhibit nuclear factor κB activation or function. In structure—activity analysis this inhibitory activity of H-7 on HIV-1 expression corresponded with the known structural requirements for the interaction of H-7 with the ATP-binding region of protein kinase C, suggesting that it was indeed related to the kinase inhibitory properties of H-7. The mechanism of H-7 transcriptional inhibition did not involve chromatin remodelling at the HIV-1 long terminal repeat promoter, as shown by nuc-1 disruption, and appeared to involve HIV-1 RNA elongation but not initiation. Therefore, H-7 and related isoquinoline-sulphonamide analogues are most likely inhibiting a kinase target essential for HIV-1 transcriptional elongation whose identity may provide new therapeutic targets for intervention.

After integration in the host cell genome, the HIV‐1 provirus is packaged into chromatin. A specific chromatin disruption occurs in the HIV‐1 promoter during transcriptional activation in response to TNF‐alpha, suggesting that chromatin plays a repressive role in HIV‐1 transcription and that chromatin modification(s) might result in transcriptional activation. We have treated several cell lines latently infected with HIV‐1 with two new specific inhibitors of histone deacetylase, trapoxin (TPX) and trichostatin A (TSA), to cause a global hyperacetylation of cellular histones. Treatment with both drugs results in the transcriptional activation of the HIV‐1 promoter and in a marked increase in virus production. Dose‐response curves and kinetic analysis show a close correlation between the level of histone acetylation and HIV‐1 gene expression. In contrast, both TPX and TSA have little or no effect on HIV‐1 promoter activity following transient transfection of an HIV‐1 promoter‐reporter plasmid. Activation of HIV‐1 transcription by TSA and TPX treatment occurs in the absence of NF‐kappa B induction. Chromatin analysis of the HIV‐1 genome shows that a single nucleosome (nuc‐1) located at the transcription start and known to be disrupted following TNF‐alpha treatment, is also disrupted following TPX or TSA treatment. This disruption is independent of transcription as it is resistant to alpha‐amanitin. These observations further support the crucial role played by nuc‐1 in the suppression of HIV‐1 transcription during latency and demonstrate that transcriptional activation of HIV‐1 can proceed through a chromatin modification.

Chromatin organization of eukaryotic promoters is increasingly recognized as an important factor in the regulation of transcription in vivo. To determine the role of chromatin in HIV‐1 expression, we have examined the nucleosome organization of the promoter of HIV‐1 under low and high transcription rates. Independently of the cell line examined, nucleosomes are precisely positioned in the viral 5′ long terminal repeat (5′ LTR) and define two large nucleosome‐free regions encompassing nt 200–450 and 610–720. A nucleosome positioned between these two regions, immediately after the transcription initiation site (nuc‐1), is disrupted following TPA or TNF‐alpha treatment. The disruption of nuc‐1 from DNA is independent of DNA replication since it is completed in 20 min and independent of transcription as it is alpha‐amanitin insensitive. A model is proposed in which nuc‐1 plays an organizing role in the HIV‐1 promoter to bring in close proximity factors bound to DNA in the two nucleosome‐free regions, upstream and downstream of the site of transcription initiation. These results define chromatin as an integral component of the HIV‐1 transcriptional regulatory machinery and identify a chromatin transition associated with activation of viral gene expression.

After integration in the host cell genome, the HIV‐1 provirus is packaged into chromatin. A specific chromatin disruption occurs in the HIV‐1 promoter during transcriptional activation in response to TNF‐alpha, suggesting that chromatin plays a repressive role in HIV‐1 transcription and that chromatin modification(s) might result in transcriptional activation. We have treated several cell lines latently infected with HIV‐1 with two new specific inhibitors of histone deacetylase, trapoxin (TPX) and trichostatin A (TSA), to cause a global hyperacetylation of cellular histones. Treatment with both drugs results in the transcriptional activation of the HIV‐1 promoter and in a marked increase in virus production. Dose‐response curves and kinetic analysis show a close correlation between the level of histone acetylation and HIV‐1 gene expression. In contrast, both TPX and TSA have little or no effect on HIV‐1 promoter activity following transient transfection of an HIV‐1 promoter‐reporter plasmid. Activation of HIV‐1 transcription by TSA and TPX treatment occurs in the absence of NF‐kappa B induction. Chromatin analysis of the HIV‐1 genome shows that a single nucleosome (nuc‐1) located at the transcription start and known to be disrupted following TNF‐alpha treatment, is also disrupted following TPX or TSA treatment. This disruption is independent of transcription as it is resistant to alpha‐amanitin. These observations further support the crucial role played by nuc‐1 in the suppression of HIV‐1 transcription during latency and demonstrate that transcriptional activation of HIV‐1 can proceed through a chromatin modification.

Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements.

Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13-->L). Both tat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13-->L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.

When transcriptionally active, the human immunodeficiency virus (HIV) promoter contains a nucleosome-free region encompassing both the promoter/enhancer region and a large region (255 nucleotides [nt]) downstream of the transcription start site. We have previously identified new binding sites for transcription factors downstream of the transcription start site (nt 465 to 720): three AP-1 sites (I, II, and III), an AP3-like motif (AP3-L), a downstream binding factor (DBF) site, and juxtaposed Sp1 sites. Here, we show that the DBF site is an interferon-responsive factor (IRF) binding site and that the AP3-L motif binds the T-cell-specific factor NF-AT. Mutations that abolish the binding of each factor to its cognate site are introduced in an infectious HIV-1 molecular clone to study their effect on HIV-1 transcription and replication. Individual mutation of the DBF or AP3-L site as well as the double mutation AP-1(III)/AP3-L did not affect HIV-1 replication compared to that of the wild-type virus. In contrast, proviruses carrying mutations in the Sp1 sites were totally defective in terms of replication. Virus production occurred with slightly delayed kinetics for viruses containing combined mutations in the AP-1(III), AP3-L, and DBF sites and in the AP3-L and DBF-sites, whereas viruses mutated in the AP-1(I,II,III) and AP3-L sites and in the AP-1(I,II,III), AP3-L, and DBF sites exhibited a severely defective replicative phenotype. No RNA-packaging defect could be measured for any of the mutant viruses as determined by quantification of their HIV genomic RNA. Measurement of the transcriptional activity of the HIV-1 promoter after transient transfection of the HIV-1 provirus DNA or of long terminal repeat-luciferase constructs showed a positive correlation between the transcriptional and the replication defects for most mutants.

Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.

An intragenic enhancer in the pol gene of human immunodeficiency virus type 1 has previously been identified (Verdin et al., Proc. Natl. Acad. Sci. USA 87:4874-4878, 1990). This element is composed of two subdomains both exhibiting phorbol ester-inducible enhancing activity on the viral thymidine kinase promoter in HeLa cells. Examination of the nucleotide sequence of one of these domains (nucleotides 4079 to 4342, HXB2 isolate) revealed the presence of three short DNA regions highly homologous to the recognition site for cellular transcription factor AP-1. Two short oligonucleotides containing these AP-1 sites each functioned as a phorbol ester-inducible enhancer when cloned upstream of the thymidine kinase promoter and transfected into HeLa cells. Gel mobility shift assays and competition experiments using the same two oligonucleotides demonstrated that they bound affinity-purified AP-1 or AP-1 present in uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced HeLa nuclear extracts. Footprinting experiments confirmed that all three predicted sites bound purified AP-1. These results suggest that the AP-1 factor could play a role in the transcriptional regulation of human immunodeficiency virus type 1 gene expression.

Chromatin organization of eukaryotic promoters is increasingly recognized as an important factor in the regulation of transcription in vivo. To determine the role of chromatin in HIV‐1 expression, we have examined the nucleosome organization of the promoter of HIV‐1 under low and high transcription rates. Independently of the cell line examined, nucleosomes are precisely positioned in the viral 5′ long terminal repeat (5′ LTR) and define two large nucleosome‐free regions encompassing nt 200–450 and 610–720. A nucleosome positioned between these two regions, immediately after the transcription initiation site (nuc‐1), is disrupted following TPA or TNF‐alpha treatment. The disruption of nuc‐1 from DNA is independent of DNA replication since it is completed in 20 min and independent of transcription as it is alpha‐amanitin insensitive. A model is proposed in which nuc‐1 plays an organizing role in the HIV‐1 promoter to bring in close proximity factors bound to DNA in the two nucleosome‐free regions, upstream and downstream of the site of transcription initiation. These results define chromatin as an integral component of the HIV‐1 transcriptional regulatory machinery and identify a chromatin transition associated with activation of viral gene expression.