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：
10542351
负责人：
Richard Sutton
金额：
$ 63.55万
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/10542351
关键词：
ATAC-seq Africa African African American population 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 Macrophage Master of Science Medical Messenger RNA Methods Molecular Nature Paper Patients Peripheral Blood Mononuclear Cell Persons Pharmaceutical Preparations Phenotype Population Production Protein Isoforms Proteins Publishing RNA Race Recording of previous events Reporter Reproducibility Resistance Sampling 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 autosome causal variant chromosome conformation capture cohort exome sequencing genetic pedigree genome wide association study immune function indexing interest novel overexpression pandemic disease receptor recruit transcription factor transcriptome sequencing vector viral resistance

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.

在潜在感染的记忆T细胞中存在于复制能力的人免疫缺陷病毒（HIV）在艾滋病毒+中，个体是消除病毒的主要障碍，在大流行的历史中，人们认为个人已经得到了真正的治愈。但是，有一小部分艾滋病毒+个人在没有抗逆转录病毒疗法的情况下，能够抑制多年来无法检测到的病毒复制水平。这些人被深入研究，称为“精英控制器”或EC 关联研究表明，病毒学控制是由于HLA-B I类分子中的编码变体引起的，但是这只能解释约20％的效果。这表明其他也许是基于非免疫的机制，负责EC表型。我们有兴趣将EC表型与遗传或转录变化，通过研究近200个EC（和病毒控制器或VC），我们能够证明其中的一个子集的CD4+ T细胞相对抗单个R5循环病毒具有相对抗性的CD4+ T细胞循环感染性测定。在所有EC/VC的20％中，这种体外表型是高度可重复的，取决于T细胞激活的方法，在巨噬细胞中未观察到，并被引入逆转 CCR5，R5共受体。这种体外R5病毒抗性的表型与两者均与 CCR5和CCR2的mRNA和蛋白质水平，后者是最接近CCR5的同源物，仅为10 kb 上游。然而，该效果扩展了CCR5/CCR2周围数百个KB的效果。索引家庭成员具有这种表型的EC在CCR5和CCR2 RNA水平上均具有相似的降低，这表明常染色体主要的继承模式。 CCR5和CCR2 RNA的半衰期与非耐药EC相同，表明效果本质上不是转录后的，并且芯片数据与转录效应。在这里，我们希望进一步探讨该表型的基础的机制。在首先，我们将确定在初级CD4+ T细胞中如何在转录调控CCR5和CCR2。我们将在原代T细胞中执行无偏CRISPR KO筛选，以识别调节CCR5/CCR2的基因。染色体构象捕获方法将用于识别CCR5/CCR2基因座的推定增强剂，通过功能研究确认，包括使用晚期CRISPR/DCAS9技术。我们还将检查 CD4 T细胞对复制能力病毒的抗性的分子基础，因为我们观察到了深刻的在这些EC的CD4+ T细胞中抑制X4病毒。这将包括上调限制因素的KO和其他基因，如RNA-Seq所见。最后，在与坎帕拉的Makerere University进行的持续合作中乌干达，我们希望将这些研究扩展到与我们的同伙不同的东非人。 CD4+ 如上所述，将分析来自EC/VC的T细胞，以识别宿主的潜在新机制遗传控制。在这些研究结束时，我们希望对这些EC/VC能够控制病毒复制并实现功能性治疗，同时保持免疫功能。