Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection.

The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.

New pyridinone derivatives as potent HIV-1 nonnucleoside reverse transcriptase inhibitors.

Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized and evaluated for their anti HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (10) with methyl substituents at positions 3 and 5 of the cyclohexyloxy moiety had potent antiviral activity against wild-type HIV-1. Subsequent stereoselective synthesis of a stereoisomer displaying both methyl groups in equatorial position was found to have the best EC(50). Further modulations focused on position 3 of the pyridinone ring improved the antiviral activity against mutant viral strains. Compounds bearing a 3-ethyl (22) or 3-isopropyl group (23) had the highest activity against wild-type HIV-1 and displayed low-nanomolar potency against several clinically relevant mutant strains.

Expression, purification, and structural prediction of the Ets transcription factor ERM.

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors. Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an alpha-helix. In contrast, the 250-residue central domain seems to have very little structure.

Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity.

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481-4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632-2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.

Administration of HDAC inhibitors to reactivate HIV-1 expression in latent cellular reservoirs: implications for the development of therapeutic strategies.

The discovery of powerful antiviral compounds in the 90's raised the hope that the human immunodeficiency virus type 1 (HIV-1) might be eradicated. However, if these drugs succeed in decreasing and controlling viral replication, complete eradication of the virus is nowadays impossible. The persistence of virus even after long periods of highly active antiretroviral therapy (HAART) mainly results from the presence of cellular reservoirs that contain transcriptionally competent latent viruses capable of producing infectious particles after cellular activation. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of HAART. Activation of HIV gene expression in these cells combined with an effective HAART has been proposed as an adjuvant therapy that could lead to the elimination of the latently infected cells and then to the eradication of the infection. In this context, we have previously demonstrated that deacetylase inhibitors (HDACi) synergize with TNF-induced NF-kappaB to activate the HIV-1 promoter. The physiological relevance of the TNF/HDACi synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Based on these results, we propose the administration of deacetylase inhibitor(s) together with continuous HAART as a new potential therapeutic perspective to decrease the pool of latent HIV reservoirs by forcing viral expression.

Gene activation and gene silencing: a subtle equilibrium.

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of several genomes has been determined, the major challenging problem is to understand how cell differentiation, proliferation or death are controlled. Major steps include analysis of the determinants of the cell cycle, the unravelling of RNAs and proteins involved in the control of gene expression and the dissection of the protein-destruction machinery. The successive steps to be considered are transcription of RNA on the DNA template, mRNA stabilization or degradation, and mRNA translation and protein localization in the right cell compartment. Gene expression or gene silencing is the result of many DNA-RNA-protein interactions and chromatin is among the key regulators of gene expression. Open chromatin (euchromatin) allows expression of the DNA message. This chromatin structure is generally characterized by the presence on the gene promoters of transcription complexes associated with histone acetyltransferases (HATs). On the contrary, closed chromatin (heterochromatin) is poorly acetylated and more condensed. It contains histone deacetylases (HDACs), potentially associated with DNA methyltransferases (DNMTs). DNMT activity leads to methylation and silencing of the DNA. Thus, a major problem in the field of gene regulation resides in understanding chromatin structure at each promoter, a formidable task for the years to come.

A defect in nucleosome remodeling prevents IL-12(p35) gene transcription in neonatal dendritic cells.

To gain insight into the inability of newborns to mount efficient Th1 responses, we analyzed the molecular basis of defective IL-12(p35) expression in human neonatal monocyte-derived dendritic cells (DCs). Determination of IL-12(p35) pre-mRNA levels by real-time RT-PCR revealed that transcriptional activation of the gene in lipopolysaccharide-stimulated neonatal DCs was strongly impaired compared with adult DCs. We next showed that p50/p65 and p65/p65 dimers interact with kB#1 site, a critical cis-acting element of the IL-12(p35) promoter. We found that LPS-induced p65 activation was similar in adult and newborn DCs. Likewise, in vitro binding activity to the Sp1#1 site, previously shown to be critical for IL-12(p35) gene activation, did not differ in adults and newborns. Since the accessibility to this Sp1#1 site was found to depend on nucleosome remodeling, we used a chromatin accessibility assay to compare remodeling of the relevant nucleosome (nuc-2) in adult and neonatal DCs. We observed that nuc-2 remodeling in neonatal DCs was profoundly impaired in response to lipopolysaccharide. Both nuc-2 remodeling and IL-12(p35) gene transcription were restored upon addition of recombinant interferon-gamma. We conclude that IL-12(p35) transcriptional repression in neonatal DCs takes place at the chromatin level.

Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites.

To get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region. First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line. On stimulation with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) -310 to -160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2). Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the -396/-241 region contained critical cis-acting elements. Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-gamma-inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line. Moreover, we found that in DCs, LPS and IFN-gamma synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis. Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.

Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-kappaB and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies.

The transcription factor NF-kappaB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-kappaB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of kappaB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IkappaBalpha. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-kappaB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.

Bovine leukemia virus SU protein interacts with zinc, and mutations within two interacting regions differently affect viral fusion and infectivity in vivo.

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.