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Characterizing chromatin protein dynamics in HIV-1 latency with a CASPEX approach

使用 CASPEX 方法表征 HIV-1 潜伏期染色质蛋白动态

基本信息

批准号：
10547359
负责人：
Jeffrey R Johnson
金额：
$ 21.13万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-08 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10547359
关键词：
Address Antibodies Biology Biotin Biotinylation CD4 Positive T Lymphocytes Cell Line Cell model Cells Chimeric Proteins Chromatin Chronic Communities Complement Complex DNA DNA-Protein Interaction Development Engineering Enzymes Genetic Genome Genomic Segment Guide RNA HIV HIV-1 Hydrogen Peroxide Infection Inflammation Lead Long Terminal Repeats Mass Spectrum Analysis Measures Methods Modeling Molecular Molecular Biology Nucleic Acids Peroxidases Persons Phenols Physiologic pulse Process Protein Dynamics Proteins Proteomics Recombinants Research Research Personnel Ribonucleoproteins Sampling Shock System T-Lymphocyte Technology Therapeutic Virus Virus Latency Western Blotting Work Yellow fever virus antiretroviral therapy ascorbate chromatin immunoprecipitation chromatin protein comorbidity endonuclease genomic locus improved in vivo mutant new technology next generation novel strategies novel therapeutics phenoxy radical prevent reactivation from latency recruit response screening small molecule therapeutic development therapy development viral rebound virus host interaction vpr Gene Products

项目摘要

PROJECT SUMMARY This project aims to develop and apply CASPEX technologies to measure protein changes in cells latently infected with HIV-1. CASPEX refers to the combination of a catalytically-dead Cas9 (dCas9) protein that can localize to a specific genomic locus fused to an engineered ascorbate peroxidase enzyme (APEX2) that is capable of biotinylating proteins within a sphere of proximity to the fusion protein. CASPEX enables the capture of proteins and nucleic acids in proximity to a genomic locus defined by guide RNAs complexed with dCas9. This powerful approach can be combined with mass spectrometry analysis to identify and quantify changes in proteins associated with genomic loci in an unbiased manner. HIV-1 latency represents the major barrier to developing new therapies for HIV-1 treatment and cure. The molecular processes governing HIV-1 latency have been investigated using high-throughput genetics, small molecule screening, and focused molecular biology studies. CASPEX has the potential to provide complementary information regarding HIV-1 latency processes that can confirm prior work or open new avenues of research. In this project, we will develop two CASPEX approaches and apply them to models of HIV-1 latency. In Specific Aim 1, we will engineer cell line models of HIV-1 latency for CASPEX analysis and characterize protein changes at integrated HIV-1 chromatin loci in response to latency reversal. Findings made in cell line models of HIV-1 latency are not always recapitulated in vivo, so in Specific Aim 2 we will develop a novel approach to apply CASPEX in primary CD4+ T cells. Successful completion of this project will advance the molecular understanding of HIV-1 latency and latency reversal, potentially informing on the development of next- generation therapies to treat or cure HIV-1 infection.

项目总结