CONTROL OF HIV REPLICATION BY CYCLIN L2

细胞周期蛋白 L2 对 HIV 复制的控制

基本信息

批准号：
8993271
负责人：
George Kyei
金额：
$ 16.45万
依托单位：
WASHINGTON UNIVERSITY
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：
2015-06-15 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8993271
关键词：
Acquired Immunodeficiency Syndrome Adverse drug effect Affect Alternative Splicing Amino Acids Anti-Retroviral Agents Arginine Attention Biochemical Boxing C-terminal Cell Cycle Cell Cycle Regulation Cells Cellular biology Complex Cyclins Data Dendritic Cells Development Fatigue Genes Genome Genomics Goals HIV HIV-1 HIV-2 Hybrids Immune Immunoprecipitation Infection Interphase Cell Interruption K-Series Research Career Programs Knowledge Lead Life Light Literature Maps Mass Spectrum Analysis Mentors Mentorship Messenger RNA Methods Molecular Molecular Biology Molecular Genetics Molecular Target N-terminal Patients Phosphorylation Phosphotransferases Physicians Polymerase Chain Reaction Positioning Attribute Process Proteins Proteomics Quality of life Quantitative Reverse Transcriptase PCR RNA Sequences RNA Splicing Resources Rest Role SIV Scientist Serine Source T-Lymphocyte Testing Training Transcript United States Universities Viral Viral Load result Virus Virus Replication Washington Work Yeasts antiretroviral therapy attenuation base cell type cost experience improved innovation kinase inhibitor mRNA Precursor macrophage molecular domain multicatalytic endopeptidase complex new therapeutic target novel novel therapeutics pandemic disease public health relevance research study skills therapeutic target transcriptome sequencing ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): The goal of this Mentored Career Development Award is to facilitate Dr. George Kyei's transition to independence as a physician-scientist studying how the human immunodeficiency virus (HIV) is able to survive in latently infected cell like resting T cells and macrophages. The proposed experiments will be conducted under the mentorship of Dr. Lee Ratner, and they will examine the role of the protein cyclin L2 and how it controls HIV replication in the aforementioned cell types. HIV continues to be a global pandemic with over 35 million people living with the virus. In the United States, over 50,000 new infections happen every year. Although current anti-retroviral therapy (ART) can suppress the virus and improve the quality of life for patients, these treatments do not lead to cure. Within weeks of patients stopping therapy, the viral load begins to rise again. Lifelong treatment has the attendant problems of patient fatigue, drug side effects and unsustainable costs. Hence viral elimination or cure is a vital goal. The main reason for viral resurgence upon treatment interruption is the presence of viral reservoirs like resting T cell and macrophages, which have the virus integrated in their genomes in a resting state. As soon as treatment is interrupted, these cells serve as sources of virus. Despite their crucial role in sustaining HIV persistence, current knowledge of viral replication requirements in macrophages and resting T cells is very limited. Recently, we identified cyclin L2 as critical for HIV replication in macrophages. The goal of this project is to test the hypothesis that cyclin L2 controls HIV replication in macrophages and resting T cells by modulating viral alternative splicing in cooperation with specific cellular kinases. In order to test this hypothesis, we will use biochemical, molecular genetics, genomics and proteomics approaches to test the following specific aims: 1. to characterize the mechanisms by which cyclin L2 controls HIV pre-mRNA alternative splicing. We will use gene knockdown, polymerase chain reaction, whole genome RNA sequencing and biochemical methods to define how cyclin L2 controls the splicing of HIV. 2. To determine the effect of phosphorylation on cyclin L2 function. Cyclin L2 is known to be heavily phosphorylated. We will use proteomics, gene knockdown approaches and specific kinase inhibitors to investigate the importance of Dyrk1A and CDK11B as potential kinases for cyclin L2. 3. To identify and characterize the molecular domains of cyclin L2 required supporting HIV replication in non- cycling cells. The rationale for this aim is to identify the exact motifs of cyclin L2 required forHIV replication, in order to map out a potential therapeutic target. . The proposed experiments will shed new light in our understanding of HIV replication requirements in these critical cell types and potentially reveal novel molecular targets for HIV elimination.

描述（由申请人提供）：该指导性职业发展奖的目标是促进 George Kyei 博士作为一名医师科学家独立过渡，研究人类免疫缺陷病毒 (HIV) 如何能够在潜伏感染的细胞（如静息 T）中存活。拟议的实验将在 Lee Ratner 博士的指导下进行，他们将研究细胞周期蛋白 L2 的作用以及它如何控制上述细胞中的 HIV 复制。 HIV 仍然是一种全球流行病，在美国有超过 3500 万人感染该病毒，新增感染人数超过 50,000 人。尽管目前的抗逆转录病毒治疗（ART）可以抑制病毒并改善患者的生活质量，但这些治疗并不能在患者停止治疗后几周内治愈，病毒载量会再次开始上升。治疗会带来患者疲劳、药物副作用和不可持续的费用等问题，因此消除或治愈病毒是一个重要目标，治疗中断后病毒复发的主要原因是存在静息 T 细胞和巨噬细胞等病毒库。病毒整合了一旦治疗中断，这些细胞就会成为病毒的来源，尽管它们在维持 HIV 持久性方面发挥着至关重要的作用，但目前对巨噬细胞和静息 T 细胞的病毒复制要求的了解非常有限。我们确定细胞周期蛋白 L2 对于巨噬细胞中的 HIV 复制至关重要。该项目的目的是检验细胞周期蛋白 L2 通过与特定细胞激酶配合调节病毒选择性剪接来控制巨噬细胞和静息 T 细胞中 HIV 复制的假设。为了检验这一假设，我们将使用生化、遗传分子、基因组学和蛋白质组学。方法来测试以下具体目标： 1. 表征细胞周期蛋白 L2 控制 HIV 前体 mRNA 选择性剪接的机制我们将使用基因敲低、聚合酶链式反应、全基因组 RNA 测序。和生化方法来确定细胞周期蛋白 L2 如何控制 HIV 剪接 2. 为了确定磷酸化对细胞周期蛋白 L2 功能的影响，我们将使用蛋白质组学、基因敲低方法和特定激酶抑制剂进行研究。 Dyrk1A 和 CDK11B 作为细胞周期蛋白 L2 潜在激酶的重要性 3. 识别和表征细胞周期蛋白 L2 的分子结构域。这一目标的基本原理是确定 HIV 复制所需的细胞周期蛋白 L2 的确切基序，以便找出潜在的治疗靶标。这些关键细胞类型中艾滋病毒复制的要求，并有可能揭示消除艾滋病毒的新分子靶点。