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细胞中存在有复制能力的人类免疫缺陷病毒（HIV） 在HIV+个体中的感染是根除病毒的主要障碍，在大流行的历史上， 被认为是真正治愈的人。然而，有一小部分艾滋病毒阳性个体 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，并在全基因组范围内 相关研究表明，病毒学控制是由于HLA-B I类分子的编码变体， 这只能解释20%的影响。这表明其他的，也许是非免疫机制， 负责EC表型。我们感兴趣的是将EC表型与遗传或 通过研究近200个EC（和病毒血症控制器或VC），我们能够 证明其中的一个亚群具有CD 4 + T细胞，这些细胞在单个细胞中对R5嗜性病毒具有相对抗性。 循环感染性测定。在所有EC/VC的约20%中观察到的这种体外表型是高度可重复的， 依赖于T细胞活化的方法，在巨噬细胞中未观察到，并且通过引入 CCR 5，R5共同受体。这种体外R5病毒抗性的表型与两种 CCR 5和CCR 2的mRNA和蛋白质水平，后者是CCR 5最接近的同源物，仅10 kb 上游然而，这种效应延伸到ccr 5/ccr 2周围的数百kb。Index家族成员 具有这种表型的EC在ccr 5和ccr 2 RNA水平上都有类似的降低，这表明常染色体 显性遗传模式ccr 5和ccr 2 RNA半衰期与非抗性EC的半衰期相同， 这表明这种效应在本质上不是转录后的，ChIP数据与一个实验结果一致。 转录效应在这里，我们希望进一步探索这种表型的机制。在 第一个目标是确定ccr 5和ccr 2在原代CD 4 + T细胞中是如何转录调节的。我们 将在原代T细胞中进行无偏倚的CRISPR KO筛选，以鉴定调节ccr 5/ccr 2的基因。 染色体构象捕获方法将用于鉴定ccr 5/ccr 2基因座的推定增强子， 通过功能研究证实，包括使用先进的CRISPR/dCas 9技术。我们亦会研究 CD 4 T细胞抵抗复制能力病毒的分子基础，因为我们已经观察到深刻的 抑制这些EC的CD 4 + T细胞中的X4病毒。这将包括上调限制因子的KO， 其他基因，如RNA-Seq所见。最后，在与坎帕拉的马凯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，他们在遗传上与我们的同龄人不同。CD4+ 将如上所述分析来自EC/VC的T细胞，以鉴定宿主免疫应答的潜在新机制。 基因控制在这些研究的结论中，我们希望能够更全面地了解 这些EC/VC能够控制病毒复制并在保持免疫功能的同时实现功能性治愈。