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Mechanisms of transcriptional regulation of ccr5 and host genetic control of HIV

ccr5转录调控机制与HIV宿主遗传控制

基本信息

批准号：
9926037
负责人：
Richard Sutton
金额：
$ 76.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-17 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9926037
关键词：
ATAC-seq Africa African African American American Asian Americans Authorization documentation Bioinformatics Biological Assay CCR5 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cell surface Cis-Acting Sequence Clinic Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Complementary DNA Coupled DNA sequencing Data Defect Disease remission Distal Down-Regulation Enhancers Enterochromaffin Cells Ethnic Origin European Family member Freezing Genes Genetic Genetic Transcription Genetic study Genomics Grant Guide RNA HIV HIV Infections HIV Long Terminal Repeat HLA-B Antigens Health Hispanic Americans Homologous Gene Household Human In Vitro Individual Inheritance Patterns Kinetics Laboratories Lentivirus Vector Link Master&apos s Degree Medical Messenger RNA Methods Molecular Nature Paper Patients Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Phenotype Population Production Protein Isoforms Proteins Publishing RNA Race Recording of previous events Reporter Reproducibility Resistance Sampling Ships Slave T memory cell T-Cell Activation T-Lymphocyte T-Lymphocyte Subsets Techniques Technology Testing Training Transcriptional Regulation Transfection Uganda Universities Variant Vesicular stomatitis Indiana virus Virus Virus Replication antiretroviral therapy base causal variant chromosome conformation capture cohort exome sequencing genetic pedigree genome wide association study immune function indexing interest macrophage novel overexpression pandemic disease receptor recruit transcription factor transcriptome sequencing vector viral resistance virology

项目摘要

Replication-competent human immunodeficiency virus (HIV) present in latently infected memory T cells in HIV+ individuals is a major barrier to virus eradication, and in the history of the pandemic only two individuals are thought to have been truly cured. There is, however, a small subset of HIV+ individuals who are able to suppress viral replication to undetectable levels for years, in the absence of antiretroviral therapy. These individuals, termed 'elite controllers' or ECs have been intensively studied, and genome-wide association studies indicate that virologic control is due to coding variants in the HLA-B class I molecule, but that can explain only ~20% of the effect. This suggests that other, perhaps non-immune based mechanisms, are responsible for the EC phenotype. We were interested in linking the EC phenotype to genetic or transcriptional changes, and by studying nearly 200 ECs (and viremic controllers or VCs) we were able to demonstrate that a subset of them have CD4+ T cells that are relatively resistant to R5-tropic viruses in single cycle infectivity assays. This in vitro phenotype, seen in ~20% of all EC/VCs, was highly reproducible, depended upon the method of T cell activation, not observed in macrophages, and reversed by the introduction of CCR5, the R5 co-receptor. This phenotype of in vitro R5 virus resistance inversely correlated with both mRNA and protein levels of CCR5 and CCR2, the latter being the closest homolog to CCR5 and just 10 kb upstream. The effect, however, extended for hundreds of kb surrounding ccr5/ccr2. Family members of Index ECs with this phenotype had similar decreases in both ccr5 and ccr2 RNA levels, suggesting an autosomal dominant inheritance pattern. ccr5 and ccr2 RNA half-lives were identical to those of non-resistant ECs, suggesting that the effect was not post-transcriptional in nature, and ChIP data were consistent with a transcriptional effect. Here we wish to further explore the mechanism(s) underpinning this phenotype. In the first aim we will determine how both ccr5 and ccr2 are transcriptionally regulated in primary CD4+ T cells. We will perform an unbiased CRISPR KO screen in primary T cells to identify genes which regulate ccr5/ccr2. Chromosome conformation capture methods will be used to identify putative enhancers for the ccr5/ccr2 loci, confirmed by functional studies, including use of advanced CRISPR/dCas9 techniques. We will also examine the molecular basis of CD4 T cell resistance to replication-competent virus, since we have observed profound inhibition of X4 virus in CD4+ T cells of these ECs. This will include KO of up-regulated restriction factors and other genes, as seen by RNA-Seq. Finally, in an ongoing collaboration with Makerere University in Kampala, Uganda we wish to extend these studies to East Africans, who are genetically distinct from our cohorts. CD4+ T cells from EC/VCs will be analyzed as described above to identify potentially novel mechanisms of host genetic control. At the conclusion of these studies we hope to have a more complete understanding as to how these EC/VCs are able to control viral replication and achieve functional cure while retaining immune function.

人类免疫缺陷病毒（HIV）存在于潜伏感染的记忆T细胞中