Molecular mechanisms involved in HIV-1 transcriptional latency and reactivation: implications for the development of therapeutic strategies.

The persistence of latently HIV-infected cellular reservoirs, despite prolonged treatment with ART (antiretroviral therapy), represents the major hurdle to virus eradication. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of ART. Therefore, a greater understanding of the molecular mechanisms regulating viral latency and reactivation should lead to rational strategies aimed at purging the latent HIV reservoirs. Our laboratory is studying elements critical for the mechanisms of viral transcriptional reactivation including: 1) the transcription factor NF-kB, which is induced by proinflammatory cytokines (such as TNFalpha) and binds to two sites kB in the HIV-1 promoter region; 2) the specific remodeling of a single nucleosome (called nuc-1 and located immediately downstream of the HIV transcription start site under latency conditions) upon activation of the HIV-1 promoter; 3) post-translational acetylation of histones and of non-histone proteins (following treatment with deacetylase inhibitors [HDACi]), which induces viral transcription and nuc-1 remodeling. Recently, we have identified a new regulatory link between the first (NF-kB) and the third (protein acetylation) element by demonstrating a strong synergistic activation of HIV-1 promoter activity by TNFalpha (an inducer of NF-kB) and HDACi. In addition to the prototypical subtype B promoter, we have observed the TNFalpha/HDACi synergism with viral promoters from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of kB sites present in the respective promoters and the amplitude of the TNFalpha/HDACi synergism. Importantly, the physiological relevance of this 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 administrating deacetylase inhibitor(s) together with continuous ART in order to force viral expression and decrease the pool of latently HIV-infected cellular reservoirs.

Interaction between C/EBPbeta and Tax down-regulates human T-cell leukemia virus type I transcription.

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein beta (C/EBPbeta) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPbeta has also been found to interact with Tax, we analyzed the effects of C/EBPbeta on viral Tax-dependent transcription. We show here that C/EBPbeta represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPbeta. We also analyzed the physical interactions between Tax and C/EBPbeta and found that the central region of C/EBPbeta, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPbeta would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPbeta was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPbeta may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response.

The cAMP response element binding protein-2 (CREB-2) can interact with the C/EBP-homologous protein (CHOP).

cAMP response element binding protein-2 (CREB-2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE-dependent transcription but that can also act as a transcriptional activator. Moreover, CREB-2 is able to function in association with the viral Tax protein as an activator of the human T-cell leukemia virus type I (HTLV-I) promoter. Here we show that CREB-2 is able to interact with C/EBP-homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer-dependent transcription. Cotransfection of CHOP with CREB-2 results in decreased activation driven by the cellular CRE motif or the HTLV-I proximal Tax-responsive element, confirming that CREB-2 and CHOP can interact with each other in vivo.