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

• 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
• 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' 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

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细胞中存在可复制的人类免疫缺陷病毒(HIV) 艾滋病毒+个体感染是根除病毒的主要障碍，而在大流行的历史上只有两个 个人被认为是真正被治愈的。然而，有一小部分HIV+患者是 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多年检测不到的水平。 这些被称为精英控制者或ECs的个体已经被深入研究，并在全基因组范围内 关联研究表明，病毒学控制是由于人类白细胞抗原B类分子中的编码变异，但 这只能解释大约20%的影响。这表明，其他可能不基于免疫的机制， 是导致EC表型的主要原因。我们感兴趣的是将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 证明它们中的一个亚群具有相对耐受R5嗜性病毒的单个 循环感染性分析。这种体外表型在~20%的EC/VC中可见，具有高度的重复性， 依赖于T细胞激活的方法，在巨噬细胞中没有观察到，并被引入逆转 CCR5，R5共受体。体外R5病毒耐药性的这种表型与两者呈负相关 CCR5和CCR2的mRNA和蛋白水平，后者是与CCR5最接近的同源物，仅为10kb 在上游。然而，这种影响延伸到了CCR5/CCR2周围的数百kb。索引的家庭成员 具有这种表型的ECS在CCR5和CCR2 RNA水平上都有类似的下降，表明是常染色体 显性遗传模式。CCR5和CCR2 RNA半衰期与非耐药内皮细胞相同， 这表明这种效应本质上不是转录后效应，芯片数据与 转录效应。在这里，我们希望进一步探讨支持这种表型的机制(S)。在 首先，我们将确定CCR5和CCR2在原代CD4+T细胞中的转录调控方式。我们 将在原代T细胞中进行无偏见的CRISPR KO筛查，以识别调节CCR5/CCR2的基因。 染色体构象捕获方法将用于鉴定CCR5/CCR2基因座的可能增强子， 经功能研究证实，包括使用先进的CRISPR/dCas9技术。我们还将检查 CD4T细胞抵抗复制能力病毒的分子基础，自从我们观察到深刻的 X4病毒对这些内皮细胞中CD4+T细胞的抑制作用这将包括上调限制因子的KO和 其他基因，如RNA-Seq.最后，在与坎帕拉的Makerere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同龄人截然不同。CD+4+ 将如上所述对EC/VC中的T细胞进行分析，以确定潜在的宿主新机制 基因控制。在这些研究结束后，我们希望对如何 这些EC/VC能够控制病毒复制，在保留免疫功能的同时实现功能性治愈。