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.

潜伏感染的记忆 T 细胞中存在具有复制能力的人类免疫缺陷病毒 (HIV) HIV+ 个体中的感染是根除病毒的主要障碍，在大流行历史上只有两个人们被认为已经真正治愈了。然而，有一小部分 HIV 感染者在没有抗逆转录病毒治疗的情况下，能够将病毒复制抑制到无法检测的水平多年。这些被称为“精英控制者”或 EC 的个体已被深入研究，并且在全基因组范围内关联研究表明病毒学控制是由于 HLA-B I 类分子的编码变异造成的，但是这只能解释约 20% 的效应。这表明其他可能是非免疫机制负责 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（以及病毒血症控制者或 VC），我们能够证明其中的一个子集具有 CD4+ T 细胞，在单次治疗中对 R5 嗜性病毒具有相对抵抗力循环感染性测定。这种体外表型在约 20% 的 EC/VC 中可见，具有高度可重复性，取决于 T 细胞激活的方法，在巨噬细胞中未观察到，并且通过引入逆转 CCR5，R5 共受体。体外 R5 病毒抗性的这种表型与两者呈负相关 CCR5 和 CCR2 的 mRNA 和蛋白质水平，后者是与 CCR5 最接近的同源物，仅 10 kb 上游。然而，该效应在 ccr5/ccr2 周围延伸了数百 kb。指数家族成员具有这种表型的 ECs ccr5 和 ccr2 RNA 水平都有类似的下降，表明常染色体显性遗传模式。 ccr5 和 ccr2 RNA 半衰期与非耐药 EC 相同，表明该效应本质上不是转录后的，并且 ChIP 数据与转录效应。在这里，我们希望进一步探讨支撑这种表型的机制。在第一个目标是确定 ccr5 和 ccr2 在原代 CD4+ T 细胞中如何进行转录调控。我们将在原代 T 细胞中进行无偏见的 CRISPR KO 筛选，以鉴定调节 ccr5/ccr2 的基因。染色体构象捕获方法将用于识别 ccr5/ccr2 位点的假定增强子，经功能研究证实，包括使用先进的 CRISPR/dCas9 技术。我们还将检查 CD4 T 细胞对具有复制能力的病毒具有抵抗力的分子基础，因为我们已经观察到了深刻的抑制这些 EC 的 CD4+ T 细胞中的 X4 病毒。这将包括上调限制因子的 KO 和其他基因，如 RNA-Seq 所示。最后，在与坎帕拉麦克雷雷大学的持续合作中，乌干达，我们希望将这些研究扩展到东非人，他们在基因上与我们的群体不同。 CD4+ 将如上所述分析来自 EC/VC 的 T 细胞，以确定宿主的潜在新机制遗传控制。在这些研究的结论中，我们希望能够更全面地了解如何这些 EC/VC 能够控制病毒复制并实现功能性治愈，同时保留免疫功能。