Rapamycin-induced inhibition of HTLV-I LTR activity is rescued by c-Myb.

BACKGROUND: Rapamycin is an immunosuppressive which represses translation of transcripts harbouring a polypyrimidine motif downstream of the mRNA cap site through the mammalian target of rapamycin complex. It inhibits the abnormal autologous proliferation of T-cell clones containing a transcriptionally active human T-lymphotropic virus, type I (HTLV-I) provirus, generated from infected subjects. We showed previously that this effect is independent of the polypyrimidine motifs in the viral long terminal repeat (LTR) R region suggesting that HTLV-I transcription, and not translation, is being affected. Here we studied whether rapamycin is having an effect on a specific transcription factor pathway. Further, we investigated whether mRNAs encoding transcription factors involved in HTLV-I transcriptional activation, specifically CREB, Ets and c-Myb, are implicated in the rapamycin-sensitivity of the HTLV-I LTR. RESULTS: An in vitro analysis of the role of SRE- and NF-kappaB-mediated transcription highlighted the latter as rapamycin sensitive. Over-expression of c-Myb reversed the rapamycin effect. CONCLUSION: The sensitivity of HTLV-I transcription to rapamycin may be effected through an NF-kappaB-pathway associated with the rapamycin-sensitive mTORC1 cellular signalling network.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Integrating p300 functions in HTLV-1 transcription initiation

2014 Summer.Includes bibliographical references.The HTLV-1 provirus overcomes a repressive chromatin environment for efficient transcription of its genome. This is accomplished by the robust recruitment of the coactivator protein, p300, to the viral enhancer sites through interactions with DNA bound pCREB and the viral transactivating protein, Tax. Recruitment of p300 to the HTLV-1 promoter results in histone acetylation and nucleosome depletion from the promoter region in the presence of the histone chaperone, Nap1. To study the histone acetylation requirements for Nap1-dependent nucleosome disassembly, we utilized immobilized in vitro assembled chromatin templates containing site specific K→R mutations within the N-terminal tails of the histones. Through these studies, we identified histone H3, lysine 14 as the functionally relevant acetylation site for Nap1-dependent nucleosome disassembly. Additionally, we found a significant correlation between nucleosome disassembly from the HTLV-1 promoter and acetylation-dependent transcription activation. These studies suggest that nucleosome disassembly is a prerequisite for transcription activation, as nucleosome disassembly creates a nucleosome free region within the HTLV-1 promoter, allowing for the subsequent recruitment of Pol II and general transcription machinery for activation of transcription. The identification of a single and specific acetyl-lysine residue led us to the hypothesis that the p300 acetyl-lysine binding domain (bromodomain) was involved in HTLV-1 transcription activation through recognition of H3K14ac. To test this hypothesis, we utilized a p300 bromodomain deletion mutant and a CBP/p300 specific bromodomain inhibitor, (SGC-CBP30), to investigate the involvement of the p300 bromodomain in HTLV-1 transcription activation. Importantly, we found that the p300 bromodomain is not involved in the initial recruitment of the coactivator to the chromatin template as previously proposed, rather the bromodomain functions after recruitment to the promoter and following acetylation of the histone tails. These findings are consistent with a role for the p300 bromodomain in nucleosome disassembly and uncover a novel function for the bromodomain in gene activation

Converging Strategies in Expression of Human Complex Retroviruses

The discovery of human retroviruses in the early 1980s revealed the existence of viral-encoded non-structural genes that were not evident in previously described animal retroviruses. Based on the absence or presence of these additional genes retroviruses were classified as ‘simple’ and ‘complex’, respectively. Expression of most of these extra genes is achieved through the generation of alternatively spliced mRNAs. The present review summarizes the genetic organization and expression strategies of human complex retroviruses and highlights the converging mechanisms controlling their life cycles

HTLV Tax, HBZ and APH-2 regulatory protein interaction with host cell factors: implications for NF-\u3baB pathway deregulation and tumorigenesis.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects approximately 20 million people worldwide and 5% of them develop adult T-cell leukemia (ATL), a fatal T-cell malignancy with a poor prognosis. The HTLV-1 genome encodes two proteins that play a pivotal role in the oncogenic process, the regulatory protein Tax and the accessory protein HBZ. The expression of HTLV-1 oncoprotein Tax plays a key role in CD4+ T-cell transformation, interacting with host factors and deregulating several cell pathways implicated in the regulation of cell cycle and cell survival. Among them, Tax activates contitutively the NF-\u3baB pathway, which play a primary role in inflammation, immunity, cell proliferation, apoptosis and cancer. HTLV-1 basic zipper protein (HBZ), encoded by the antisense viral genome strand, is essential for viral persistence and promotion of T-cell proliferation, acting in concert with Tax and contributing to ATL development. The purpose of my PhD research is to expand the knowledge of the molecular mechanism of NF-\u3baB pathway deregulation mediated by the interactions of Tax and HBZ regulatory proteins with selected host factors. We studied the effect of Tax-1 and HBZ on NF-\u3baB promoter activation by comparative analyses with the homologous regulatory proteins expressed by the genetically related HTLV type 2, which is not associated with ATL disease. We focused the experimental analyses on two relevant aspects of the NF-\u3baB deregulation: the interactions of Tax and the antisense proteins with key factors of the NF-\u3baB pathway mediating p65 activation, and their role on the alternative NF-\u3baB pathway modulation. Our data demonstrated for the first time that HBZ and APH-2 differ in the inhibitory mechanism of Tax-dependent NF-\u3baB activation. By confocal microscopy, we observed that APH-2, unlike HBZ, was recruited in Tax-2-cytoplasmic structures containing the NF-\u3baB factors that are essential for the activation of the pathway, the adaptor protein TAB2 and the NF-\u3baB modulator NEMO. The formation of these complexes results in the impairment of p65 transcription factor translocation into the nucleus. The analyses of these complexes showed that TRAF3, a key factor of the alternative NF-\u3baB pathway, interacts with Tax and APH-2. Applying the CRISPR/Cas9 system, we generated a cell model that allowed us to define the contribution of TRAF3 in the inhibition of NF-\u3baB. The results obtained revealed for the first time that the absence of TRAF3 dramatically reduced the Tax-1 transactivating activity of NF-\u3baB. In conclusion, the results of my PhD thesis identify a new cellular factor essential for the action of HTLV Tax protein in the deregulation of cellular pathways and support the hypothesis that the different molecular mechanism of HBZ and APH-2 in the NF-\u3baB inhibition may reflect divergent effects on HTLV-infected cells survival and probably on leukemogenesis induced by HTLV

Host restriction factors against human retrovirus: the role of CIITA and TRIM22.

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Detection, quantification and genetic analyses of human immunodeficiency virus type 2 (HIV-2) infections in the Gambia, West Africa

Infection with Human Immunodeficiency Virus type 2 (HIV-2) has been investigated in collaborative studies with the MRC unit, Fajara, in The Gambia, West Africa using virological, serological and molecular techniques. Serum antibody levels of anti-HIV-2 were measured with competitive serological assays using culture-derived viral antigens which were applied in field studies in West Africa. These were subsequently replaced with a recombinant β-galactosidase fusion protein of an immunodominant region of the transmembrane glycoprotein (gp36) of HIV-2. Competitive EIAs were demonstrated to be a specific and sensitive means of detecting anti-HIV-2 and, in most cases, for speciation of HIV-1 and HIV-2 infections and for the detection of related non-human primate lentiviruses. A recombinant HIV-2 p26 antigen was produced as a glutathione-S-transferase fusion protein by PCR amplification and cloning into the pGEX-3X expression vector. These studies yielded a soluble recombinant antigen which was used as an immunogen to raise a polyclonal anti-p26- specific reagent. An assay for the detection of p26 antigens was also developed. Partial nucleotide sequence of the p26 gene of four Gambian HIV-2 strains (CBL.20-23) indicated these to be subtype A strains. Nested PCR assays were applied to HIV-2 pro viral DNA detection using oligonucleotide sequences in the Long Terminal Repeat (LTR), pol, and vpx regions of the HIV-2 genome. In a cohort of Gambian seropositive individuals, HIV-2 provirus was detected in DNA extracted from PBMCs in 84/86 (97%) of individuals with LTR primers and 39/41 (95%) in a similarly conserved region of pol (integrase). LTR sequences and recombinant Pfu DNA polymerase, rather than Taq polymerase, were applied in a radiometric incorporation assay for quantification of HIV-2 proviral DNA and evaluated by an end-point limiting dilution method. A wide range of proviral DNA levels were found in HIV-2-infected individuals where increased proviral loads, expressed as copies per 105 CD4-positive lymphocytes, were strongly associated with CD4 depletion. Nested HIV-1 and HIV-2 PCR primers were also used to identify dual infections. Amplification and direct sequence analysis of the U3 region of the HIV-2 LTR, from either proviral DNA or viral RNA, indicated a high level of sequence conservation of four cis-acting elements in vivo. A PCR-based approach to genotyping HIV-2 strains was also developed, capable of differentiating HIV-2 subtype A strains from subtype B or SIV-like viruses based on U3 sequence-specificity. Subtype A viruses were found to predominate in the Gambian samples analysed

Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.Comparative StudyJournal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe