Harnessing Genetic Interactions to Uncover HIV-1 Rev Host Factors

利用遗传相互作用揭示 HIV-1 Rev 宿主因素

基本信息

批准号：
9042832
负责人：
Jason Wojcechowskyj
金额：
$ 5.8万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9042832
关键词：
Acquired Immunodeficiency Syndrome Affect Affinity Chromatography Animal Model Antiviral Therapy Bioinformatics Biological Assay CD4 Positive T Lymphocytes Cell Line Cell Separation Cell physiology Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Code Cyan Fluorescent Protein Data Disease Flow Cytometry Gene Deletion Genes Genetic Genetic Epistasis Genome Genome engineering Goals HIV-1 Hela Cells Human Individual Infection Integration Host Factors Link Location Mass Spectrum Analysis Measurement Measures Messenger RNA Monitor Morbidity - disease rate Nature Neurodegenerative Disorders Nuclear Nuclear Export Nuclear Import Orthologous Gene Pathway interactions Patients Phenotype Play Primary Infection Process Proteins Quantitative Genetics RNA RNA Interference Reporter Research Resolution Response Elements Role Saccharomyces cerevisiae Small Interfering RNA Surveys Technology Tertiary Protein Structure Testing Therapeutic Transcript Vaccines Viral Viral Proteins Virus Replication Yeasts base cell type cellular targeting deletion library enzyme substrate genetic variant genome-wide global health homologous recombination insight mortality mutant novel novel therapeutics pathogen pointed protein protein complex public health relevance research study rev Protein screening small molecule therapeutic target tool yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, remains a global health crisis with millions of new infections each year. While modern antiviral therapies can manage HIV-1 disease in many patients, a cure or vaccine remains out of reach. HIV-1 requires a multitude of host factors in order to replicate and the targeting of cellular factors with small molecules or genome engineering has reinvigorated the search for novel HIV-1 therapeutics. Yet despite decades of research, the full armament of host factors hijacked by HIV-1 remain uncharacterized. The use of yeast as a model organism has been pivotal for our current understanding of basic cell biology and disease mechanisms, from human neurodegenerative disorders to human pathogens. I have recently demonstrated that an epistasis mini-array profile (E-MAP), a genetic interaction (GI) analysis technology, in yeast is well suited to uncover host factors of HIV-1 Rev, an essential viral protein. The hypothesis of this proposal is that a quantitative, genome-wide survey of Rev domain-specific cellular GIs will uncover novel HIV-1 host factors and provide key mechanistic insights into their role in Rev-dependent processes. The goal of Aim 1 is to generate a genome-wide E-MAP of Rev in the context of well defined Rev point mutants (a pE-MAP). A genome-wide pE-MAP of Rev mutants that are defective in nuclear location, multimerization, and nuclear export will be generated in S. cerevisiae. With the aid of bioinformatics, these experiments will uncover novel functional interactions between individual Rev domains and cellular genes. Bridging these findings into human CD4+ T cells with functional HIV-1 infectivity assays will further our understanding of how HIV-1 hijacks cellular processes. Aim 2 will establish the role of putative Rev host factors during HIV-1 replication in human cells with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Depleting cells of the expression of putative Rev host factors with CRISPRs will allow the interrogation of their role in HIV-1 infection in various human cell types. Experiments with a Rev-dependent reporter CD4+ T cell line and various Rev functional assays will clarify the mechanism(s) for how the identified host factors affect specific Rev functions. These studies will culminate in determining the role of Rev host factors during HIV-1 infection of primary human CD4+ T cells. Results from this proposal will greatly expand our understanding of HIV-1 host factors and may identify potential anti-HIV-1 therapeutic targets.

描述（由申请人提供）：人类免疫缺陷病毒 1 (HIV-1) 是艾滋病的病原体，它仍然是一个全球性的健康危机，每年有数百万新感染者，而现代抗病毒疗法可以控制许多患者的 HIV-1 疾病。然而，HIV-1 的治疗或疫苗仍然遥不可及，需要多种宿主因子才能复制，而小分子或基因组工程靶向细胞因子重新激发了对新型 HIV-1 疗法的探索。研究表明，被 HIV-1 劫持的宿主因子的全套装备仍未得到表征。使用酵母作为模型生物对于我们目前了解基本细胞生物学和疾病机制（从人类神经退行性疾病到人类病原体）至关重要。我最近证明了上位性微型阵列图谱（E-MAP），这是一种遗传基因。酵母中的相互作用 (GI) 分析技术非常适合揭示 HIV-1 Rev 的宿主因素，该提案的假设是，对 Rev 域特异性细胞 GI 进行定量、全基因组调查将发现新的 HIV-1 宿主因子，并为它们在 Rev 依赖性过程中的作用提供关键的机制见解。目标 1 是在明确定义的 Rev 点突变体（pE-MAP）的背景下生成 Rev 的全基因组 E-MAP。Rev 突变体的全基因组 pE-MAP 在核定位、多聚化、核出口将借助生物信息学，这些实验将揭示个体 Rev 结构域和细胞基因之间的新功能相互作用，通过功能性 HIV-1 感染性检测将这些发现桥接到人类 CD4+ T 细胞中，这将进一步加深我们对 HIV 的了解。 -1 劫持细胞过程，目标 2 将通过成簇规则间隔短回文重复 (CRISPR) 确定假定的 Rev 宿主因子在人类细胞中 HIV-1 复制过程中的作用。使用 CRISPR 消除细胞中推定的 Rev 宿主因子的表达将有助于探究它们在各种人类细胞类型中的 HIV-1 感染中的作用，使用 Rev 依赖性报告 CD4+ T 细胞系和各种 Rev 功能测定进行的实验将阐明这一点。已确定的宿主因子如何影响特定 Rev 功能的机制将最终确定 Rev 宿主因子在人类原代 CD4+ T 细胞感染过程中的作用。 HIV-1 宿主因素并可能确定潜在的抗 HIV-1 治疗靶点。