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Principal Investigator: Sonia Mediouni Jablonski
Organization: UNIVERSITY OF FLORIDA
Fiscal Year: 2024
Award: $289,800
Funding agency: National Institute of Allergy and Infectious Diseases
ABSTRACT
The persistence of latently infected memory CD4+ T cells releasing a low-level trickling of viral RNAs and
proteins in people living with HIV (PLWH) on antiretroviral therapy (ART) provides a strong rationale for the
development of cure strategies. HIV functional cure approaches are being explored, at the transcriptional level,
to achieve sustained ART-free viral remission that would result from epigenetic changes over time. These
approaches are supported by evidence in PLWH who spontaneously control their viral load below the limit of
detection, in the absence of ART (elite controllers, ECs). These ECs were shown to have a large proportion of
their proviruses in condensed chromatin (heterochromatin) loci, with virus refractory to reactivation.
The chromatin environment surrounding the HIV promoter is believed to be regulated by host transcription
factors, host chromatin remodelers and the viral Tat protein. Our hypothesis is that the pioneer factor (PF) family
may also remodel the HIV locus chromatin, via their unique epigenetic modulator properties, resulting in the
regulation of HIV transcription. PFs play critical roles in chromatin remodeling, mostly during development and
disease phenotypes. Unique properties of PFs include 1) trigger assembly of regulatory factors on target DNA
in heterochromatin; 2) bookmark genes for rapid reactivation/repression and 3) induce persistent epigenetic
modulation of the cellular chromatin. A few PFs were reported to modulate HIV latency and persistence in primary
CD4+ T cells but were poorly studied mechanistically. Preliminary data using shRNAs targeting 13 PFs in the
HIV latently infected CD4+ T cells, revealed 3 novel hits that hindered HIV transcription. This data further
highlights the significance of uncovering the role played by the PFs on HIV transcription in CD4+ T cells, with the
long-term goal to harness them for HIV cure approaches and/or diagnostic purposes. Here, we propose to:
Aim 1. Screen a shRNAmir library targeting 31 PFs (and controls) by flow cytometry, taking advantage of
the HIV reporter pMorpheus-V5 vector in Jurkat T cells. This vector allows the distinction of productively infected,
latently infected, and uninfected cells. The shRNAmir relative abundance in these populations will reflect their
activity on HIV transcription. The follow-up of the candidates will be based namely on reproducibility and novelty.
Aim 2. Validate the hits identified in preliminary data and Aim 1. Their activity will be confirmed in cell line
models of latency with different HIV transcriptional environments, and in primary blood CD4+ T cells from
uninfected and virally suppressed ART treated female and male donors since estrogen modulates certain PFs’
activity. Their impact on CD4+ T cell proliferation, activation and cytotoxicity will also be measured.
Aim 3. Characterize the 3 hits identified in preliminary data. Their recruitment to the host/HIV promoters
(ChIP-Seq analysis), their modulation of the HIV chromatin (MNase nucleosomal mapping, enChIP-MS) and
transcriptional regulation (RNA Seq analysis) in cell models of HIV latency will be studied for their comprehensive
transcriptional regulatory profiling. Cell based- and in vitro- assays will define their dependence to HIV Tat.
Terms: <AIDS Virus><Acceleration><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Active Follow-up><Acute><Basal Transcription Factor><Basal transcription factor genes><Binding><Biological Markers><Blood><Blood Reticuloendothelial System><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><Cell Body><Cell Isolation><Cell Line><Cell Segregation><Cell Separation><Cell Separation Technology><Cell model><CellLine><Cells><Cellular model><ChIP Sequencing><ChIP-seq><ChIPseq><Characteristics><Chromatin><Circulatory Collapse><Cytolysis Induction><DNA><DNA-Dependent RNA Polymerase II><Data><Deoxyribonucleic Acid><Dependence><Development><Diagnostic><Disease remission><Down-Regulation><Drug resistance><Ensure><Environment><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Estrogens><Family><Family Study><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gene Inactivation><Gene Silencing><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Goals><HIV><HIV Infections><HTLV-III Infections><HTLV-III-LAV Infections><Heterochromatin><Homeo Domain Proteins><Homeobox Family Protein><Homeobox Proteins><Homeodomain Family Protein><Homeodomain Proteins><Homeoproteins><Homeotic Proteins><Host Factor><Host Factor Protein><Human><Human Immunodeficiency Viruses><Human T-Lymphotropic Virus Type III Infections><Immune><Immunes><In Vitro><Integration Host Factors><Interruption><LAV-HTLV-III><Laboratories><Libraries><Life><Literature><Lymphadenopathy-Associated Virus><Lytotoxicity><Maps><Measures><Mediating><Modeling><Modern Man><Molecular Interaction><NIAID><NIH><National Institute of Allergy and Infectious Disease><National Institutes of Health><Nucleosomes><Persons><Physical condensation><Play><Population><Production><Productivity><Property><Proteins><Proviruses><RNA Expression><RNA Polymerase B><RNA Polymerase II><RNA Seq><RNA sequencing><RNAseq><Refractory><Regulation><Remission><Reporter><Reporting><Repression><Reproducibility><Research Proposals><Resistance development><Resistant development><Role><Shock><Stigmatization><Strains Cell Lines><T-Cell Proliferation><T-Cells><T-Lymphocyte><T4 Cells><T4 Lymphocytes><Therapeutic><Therapeutic Estrogen><Time><Toxic effect><Toxicities><Trans-Activation of Transcription Protein><Trans-Activator of Transcription of HIV><Transactivating Regulatory Protein><Transcription><Transcription Activation><Transcription Factor Proto-Oncogene><Transcription Regulation><Transcription factor genes><Transcriptional Activation><Transcriptional Control><Transcriptional Regulation><United States National Institutes of Health><Viral><Viral Burden><Viral Gene Products><Viral Gene Proteins><Viral Load><Viral Load result><Viral Proteins><Virus><Virus-HIV><accelerated aging><accelerated biological age><accelerated biological aging><active followup><age acceleration><antiretroviral therapy><antiretroviral treatment><bio-markers><biologic marker><biomarker><cell sorting><chromatin immunoprecipitation-sequencing><chromatin remodeling><circulatory shock><condensation><cultured cell line><cytotoxicity><detection limit><developing resistance><developmental><disease phenotype><drug resistant><epigenetically><female treatment><flow cytophotometry><follow up><follow-up><followed up><followup><improved><in vitro Assay><in vivo Model><inhibitor><knock-down><knockdown><latency/reactivation><latent infection><male><member><memory CD4 T cell><memory CD4 T lymphocyte><novel><promoter><promotor><reactivation from latency><recruit><resistance to Drug><resistant to Drug><shRNA><shocks><short hairpin RNA><side effect><small hairpin RNA><social role><tat Protein><thymus derived lymphocyte><tool><transcription factor><transcriptional silencing><transcriptome sequencing><transcriptomic sequencing><treat females><treat women><treatment among females><treatment among women><treatment in females><treatment in women><vector><viral RNA><viral rebound><virus RNA><virus protein><virus rebound><women's treatment>