Allan Guiguen

CpG methylation controls reactivation of HIV from latency.

DNA methylation of retroviral promoters and enhancers localized in the provirus 5′ long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5′ LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5′ LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5′ LTR in viremic patients. However, even dense methylation of the HIV-1 5′LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-α, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.

The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line POTENTIAL INVOLVEMENT OF DIRECT INHIBITION OF cAMP-RESPONSIVE ELEMENT (CRE)-BINDING PROTEIN/CRE MODULATOR/ACTIVATION TRANSCRIPTION FACTOR BINDING

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2′-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5′-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator TaxBLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267LTaxSN 5′-LTR compared with the L267 5′-LTR. Interestingly, DNA methylation inhibitors and TaxBLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the −154 or −129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at −129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5′-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Synthesis, biological evaluation and molecular modeling studies of quinolonyl diketo acid derivatives: New structural insight into the HIV-1 integrase inhibition

New quinolonyl diketo acid compounds bearing various substituents at position 6 of the quinolone scaffold were designed and synthesized as potential HIV-1 integrase inhibitors. These new compounds were evaluated for their antiviral and anti-integrase activity and showed inhibitory potency similar to that of 6-bromide analog 2. Molecular modeling and docking studies were performed to rationalize these data and to provide a detailed understanding of the mechanism of inhibition for this class of compounds.

Structural and theoretical studies of [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid as HIV-1 integrase inhibitor.

Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid 2 were synthesized as potential HIV-1 integrase inhibitors and evaluated for their enzymatic and antiviral activity, acidic compound 2 being more potent than ester compound 1. X-ray diffraction analyses and theoretical calculations show that the diketoacid chain of compound 2 is preferentially coplanar with the quinolinone ring (dihedral angle of 0-30 degrees ). Docking studies suggest binding modes in agreement with structure-activity relationships.

Role of the cofactor CTIP2 (COUP-TF Interacting Protein 2) in the transcriptional repression of HTLV-1 (Human T-lymphotropic Virus 1)

Background Following entry and reverse transcription, HTLV-1 integrates into the host cell genome. The viral promoter activity is directly governed by its chromatin environment. Epigenetic modifications, such as DNA methylation and histone deacetylation, are crucial for transcriptional silencing of the virus. We have previously reported that the cofactor CTIP2 recruits a multienzymatic chromatin-modifying complex, including histone deacetylases and a methyltransferase to the HIV-1 promoter, thereby establishing a heterochromatic environment at the promoter. CTIP2 is recruited to the HIV-1 promoter via its association with the transcription factor Sp1. Here, we investigated the potential role of CTIP2 in transcriptional regulation of the HTLV-1 promoter. Methods Electrophoretic mobility shift assays (EMSAs), transient transfection and chromatin immunoprecipitation (ChIP) assays were performed. Results Analysis of the nucleotide sequence of the HTLV-1 promoter (LTRHTLV-1) revealed two Sp1 binding sites within the R region. We demonstrated that Sp1 and Sp3 bind in vitro to these sites by EMSAs. We showed, by cotransfection assays in epithelial HEK293T cells and T-lymphoid Jurkat cells, that the cofactor CTIP2 inhibited Tax-mediated transactivation of the HTLV-1 promoter. We demonstrated by ChIP assays the in vivo recruitment of CTIP2 to the LTRHTLV-1 in a latently HTLV-1 infected cell line (TLom1). Conversely, CTIP2 was absent from the proviral genome in a HTLV-1 productive cell line (SLB1). The identification of the multienzymatic complex recruited by CTIP2 to the viral promoter under latent conditions is currently under investigation.

The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

We have previously identified three AP-1 binding sites in the pol gene of human immunodeficiency virus type 1 (HIV-1) and shown that short oligonucleotides containing these sites functioned as phorbol ester-inducible enhancers (Van Lint et al., 1991, J. Virol., 65:7066-7072). These sites are located in a region, called fragment 5103, exhibiting a phorbol ester-inducible enhancing activity on the viral thymidine kinase promoter in HeLa cells. In this study, we have further characterized each of the AP-1 binding sites and have shown that transcription factors cFos, JunB and JunD interacted in vitro with these motifs. For each site, we have identified mutations abolishing AP1 factor binding without altering the underlying amino acid sequence of the HIV-1 reverse transcriptase. By transient transfection assays, we have demonstrated that the intragenic AP-1 binding sites were entirely responsible for the PMA-dependent transcriptional activity of fragment 5103. Moreover, this PMA-stimulated activity of fragment 5103 was inhibited by a dominant-negative A-Fos mutant provided the AP-1 sites were not mutated. Finally, we have investigated the biological significance of the intragenic AP-1 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity. from Frontiers of Retrovirology: Complex retroviruses, retroelements and their hosts Montpellier, France. 21-23 September 2009