HIV-1 Tat Second Exon limits the Extent of Tat-mediated Modulation of Interferon-Stimulated Genes in Antigen Presenting cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE42926 | GEO | 2013/06/01
SECONDARY ACCESSION(S): PRJNA183878
REPOSITORIES: GEO
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Project description:BackgroundWe have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and macrophages.ResultsWe evaluated how Tat alleles and mutants differ in cellular gene modulation of immature dendritic cells and monocyte-derived macrophages and what similarities this modulation has with that induced by interferons. The tested alleles and mutants modulated to different degrees ISG, without concomitant induction of interferons. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all modulated genes to a significantly greater extent than full-length wild type, two-exon Tat, indicating that Tat second exon is critical in reducing the innate response triggered by HIV-1 in these cells. Mutants with reduced LTR transactivation had a substantially reduced effect on host gene expression modulation than wild type TatSF2. However, the more potent LTR transactivator TatSF2A58T modulated ISG expression to a lower degree compared to TatSF2. A cellular gene modulation similar to that induced by Tat and Tat mutants in immature dendritic cells could be observed in monocyte-derived macrophages, with the most significant pathways affected by Tat being the same in both cell types. Tat expression in cells deleted of the type I IFN locus or receptor resulted in a gene modulation pattern similar to that induced in primary immature dendritic cells and monocyte-derived macrophages, excluding the involvement of type I IFNs in Tat-mediated gene modulation. ISG activation depends on Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters and a single exon Tat protein more strongly modulated the luciferase activity mediated by MAP2K3, MAP2K6, and IRF7 promoter sequences located 5' of the RNA start site than the wild type two-exon Tat, while a cysteine and lysine Tat mutants, reduced in LTR transactivation, had negligible effects on these promoters. Chemical inhibition of CDK9 or Sp1 decreased Tat activation of MAP2K3-, MAP2K6-, and IRF7-mediated luciferase transcription.ConclusionsTaken together, these data indicate that the second exon of Tat is critical to the containment of the innate response stimulated by Tat in antigen presenting cells and support a role for Tat in stimulating cellular transcription via its interaction with transcription factors present at promoters.
| S-EPMC4036831 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-06-01 | E-GEOD-42926 | biostudies-arrayexpress
Project description:HIV-1 Tat induces the expression of interferon (IFN)-inducible genes in immature dendritic cells (iDC) in the absence of IFN production. We evaluated how three alleles of Tat and some Tat mutants differ in cellular gene modulation and whether a similar gene induction pattern could be detected by treating cells with IFN’s. The three alleles and mutants, with the exception of mutants TatSF21-47 and TatSF2G48-R57A that do not localize in the nucleous, modulated to different degrees IFN-inducible genes without concomitant induction of IFN’s. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all induced genes to a significantly greater extent than full-length Tat. The 2nd exon appears to diminish the gene modulation that can be observed when the first exon alone is expressed. To investigate what domain of Tat are critical to the host-pathogen interactions that are Tat-dependent during HIV infection, we evaluated a variety of Tat-mutants and found that in antigen presenting cells, blood-derived myeloid iDC and macrophages, the second exon of Tat reduces innate immunological responses which are maximal when a Single exon Tat is expressed.
2013-06-01 | GSE42925 | GEO
Project description:HIV-1 Tat induces the expression of interferon (IFN)-inducible genes in immature dendritic cells (iDC) in the absence of IFN production. We evaluated how three alleles of Tat and some Tat mutants differ in cellular gene modulation and whether a similar gene induction pattern could be detected by treating cells with IFNM-bM-^@M-^Ys. The three alleles and mutants, with the exception of mutants TatSF21-47 and TatSF2G48-R57A that do not localize in the nucleous, modulated to different degrees IFN-inducible genes without concomitant induction of IFNM-bM-^@M-^Ys. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all induced genes to a significantly greater extent than full-length Tat. The 2nd exon appears to diminish the gene modulation that can be observed when the first exon alone is expressed. To investigate what domain of Tat are critical to the host-pathogen interactions that are Tat-dependent during HIV infection, we evaluated a variety of Tat-mutants and found that in antigen presenting cells, blood-derived myeloid iDC and macrophages, the second exon of Tat reduces innate immunological responses which are maximal when a Single exon Tat is expressed. 1X10e6 macrophages were infected with HIVBal (20 ng of p24/ 106 cells) for 10 days and with adenoviruses expressing TatHXB2 or LacZ control (5 pfu per cell). Cells infected with Ad-Tat or Ad-LacZ were collected at 24 hours and cells infected with HIV or treated with medium were harvested at 7d and 10d post-infection. THP-Mac were infected with Ad-Tat or Ad-tTA control for 24 hours. RNA was isolated and mRNA expression levels were analyzed by microarrays. adenovirus expressing HIV Tat for 30 hours. and then RNA was isolated, labeled, and prepared for hybridization on Affymetrix microarrays.
2013-06-01 | E-GEOD-42925 | biostudies-arrayexpress
Project description:As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.
| S-EPMC3656449 | biostudies-literature
Project description:The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.
| S-EPMC2879518 | biostudies-literature
Project description:Human immunodeficiency virus type 1 (HIV-1) Tat is a mediator of viral transcription and is involved in the control of virus replication. However, associations between HIV-1 Tat diversity and functional effects during primary HIV-1 infection are still unclear. We estimated selection pressures in tat exon 1 using the mixed-effects model of evolution with 672 viral sequences generated from 20 patients infected with HIV-1 subtype C (HIV-1C) over 500 days postseroconversion. tat exon 1 residues 3, 4, 21, 24, 29, 39, and 68 were under positive selection, and we established that specific amino acid signature patterns were apparent in primary HIV-1C infection compared with chronic infection. We assessed the impact of these mutations on long terminal repeat (LTR) activity and found that Tat activity was negatively affected by the Ala(21) substitution identified in 13/20 (65%) of patients, which reduced LTR activity by 88% (± 1%) (P < 0.001). The greatest increase in Tat activity was seen with the Gln(35)/Lys(39) double mutant that resulted in an additional 49% (± 14%) production of LTR-driven luciferase (P = 0.012). There was a moderate positive correlation between Tat-mediated LTR activity and HIV-1 RNA in plasma (P = 0.026; r = 0.400) after 180 days postseroconversion that was reduced by 500 days postseroconversion (P = 0.043; r = 0.266). Although Tat activation of the LTR is not a strong predictor of these clinical variables, there are significant linear relationships between Tat transactivation and patients' plasma viral loads and CD4 counts, highlighting the complex interplay between Tat mutations in early HIV-1C infection.
| S-EPMC3648179 | biostudies-literature
Project description:HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%-100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA.
| S-EPMC4974531 | biostudies-literature
Project description:BackgroundSince HIV-1 Tat and Vpr genes are involved in promoter transactivation, apoptosis, etc, we carried out studies to find out nature and extent of natural variation in the two genes from seropositive patients from Northern India and determined their functional implications.MethodsHIV-1 tat exon 1 and vpr were amplified from the genomic DNA isolated from the blood of HIV-1 infected individuals using specific primers by Polymerase Chain reaction (PCR) and subjected to extensive genetic analysis (CLUSTAL W, Simplot etc). Their expression was monitored by generating myc fusion clones. Tat exon 1 and Vpr variants were co-transfected with the reporter gene construct (LTR-luc) and their transactivation potential was monitored by measuring luciferase activity. Apoptosis and cell cycle analysis was done by Propidium Iodide (PI) staining followed by FACS.ResultsExon 1 of tat was amplified from 21 samples and vpr was amplified from 16 samples. One of the Tat exon 1 variants showed phylogenetic relatedness to subtype B & C and turned out to be a unique recombinant. Two of the Vpr variants were B/C/D recombinants. These natural variations were found to have no impact on the stability of Tat and Vpr. These variants differed in their ability to transactivate B LTR and C LTR promoters. B/C recombinant Tat showed better co-operative interaction with Vpr. B/C/D recombination in Vpr was found to have no effect on its co-operativity with Tat. Recombinant Tat (B/C) induced more apoptosis than wild type B and C Tat. The B/C/D recombination in Vpr did not affect its G2 arrest induction potential but reduced its apoptosis induction ability.ConclusionsExtensive sequence and region-specific variations were observed in Tat and Vpr genes from HIV-1 infected individuals from Northern India. These variations have functional implications & therefore important for the pathogenicity of virus.
| S-EPMC3868622 | biostudies-literature
Project description:BACKGROUND: During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. RESULTS: To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20 degrees C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37 degrees C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. CONCLUSION: Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.
| S-EPMC2483996 | biostudies-literature