ABSTRACT
Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial.
Subject(s)
Antiretroviral Therapy, Highly Active , Genetic Therapy , Genetic Vectors/genetics , Hematopoietic Stem Cells/metabolism , Lentivirus/genetics , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Transduction, Genetic , Animals , Gene Expression , Hematopoietic Stem Cell Transplantation , Immunophenotyping , Lymphocyte Count , Macaca nemestrina , Simian Acquired Immunodeficiency Syndrome/therapy , Simian Acquired Immunodeficiency Syndrome/virology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/radiation effects , T-Lymphocyte Subsets/virology , Transgenes , Transplantation Conditioning , Viral LoadABSTRACT
The largest transcription factor IID (TFIID) subunit, TBP-associated factor 1 (TAF1), possesses protein kinase and histone acetyltransferase (HAT) activities. Both enzymatic activities are essential for transcription from a subset of genes and G(1) progression in mammalian cells. TAF7, another TFIID subunit, binds TAF1 and inhibits TAF1 HAT activity. Here we present data demonstrating that disruption of the TAF1/TAF7 interaction within TFIID by protein phosphorylation leads to activation of TAF1 HAT activity and stimulation of cyclin D1 and cyclin A gene transcription. Overexpression and small interfering RNA knockdown experiments confirmed that TAF7 functions as a transcriptional repressor at these promoters. Release of TAF7 from TFIID by TAF1 phosphorylation of TAF7 increased TAF1 HAT activity and elevated histone H3 acetylation levels at the cyclin D1 and cyclin A promoters. Serine-264 of TAF7 was identified as a substrate for TAF1 kinase activity. Using TAF7 S264A and S264D phosphomutants, we determined that the phosphorylation state of TAF7 at S264 influences the levels of cyclin D1 and cyclin A gene transcription and promoter histone H3 acetylation. Our studies have uncovered a novel function for the TFIID subunit TAF7 as a phosphorylation-dependent regulator of TAF1-catalyzed histone H3 acetylation at the cyclin D1 and cyclin A promoters.
Subject(s)
Cyclin A/metabolism , Cyclin D1/metabolism , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/chemistry , Animals , Cell Cycle , Cell Nucleus/metabolism , HeLa Cells , Histone Acetyltransferases , Histones/metabolism , Humans , Insecta/cytology , Phosphorylation , Protein Interaction Mapping/methods , Serine/chemistry , Transcription Factor TFIID/metabolism , TransfectionABSTRACT
The open-source Computational Proteomics Analysis System (CPAS) contains an entire data analysis and management pipeline for Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) proteomics, including experiment annotation, protein database searching and sequence management, and mining LC-MS/MS peptide and protein identifications. CPAS architecture and features, such as a general experiment annotation component, installation software, and data security management, make it useful for collaborative projects across geographical locations and for proteomics laboratories without substantial computational support.
