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Control of HIV replication by interactions between SF3B1 and Tat

通过 SF3B1 和 Tat 之间的相互作用控制 HIV 复制

基本信息

批准号：
10327200
负责人：
George Kyei
金额：
$ 23.63万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-11 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10327200
关键词：
Affect Alanine Amino Acids Binding Binding Proteins Biological Assay C-terminal CD4 Positive T Lymphocytes Cells Chronic Disease Complement Complex Data Drug resistance Electrophoretic Mobility Shift Assay Engineering Environment Enzymes Future Genetic Transcription Goals HIV HIV Infections HIV therapy HIV-1 Histones Immunoprecipitation In Vitro Integrase Inhibitors Life Cycle Stages Luciferases Mammalian Cell Measures Mediating Methylation Modeling Mutagenesis Neural Tube Defects Nuclear Pathway interactions Physiological Play Proteins RNA RNA Polymerase II RNA Splicing Resistance Role Scanning Signal Transduction Surface System Technology Testing Therapeutic Therapeutic Intervention Viral Viral Proteins Weight Gain Work adverse drug reaction antiretroviral therapy base chromatin immunoprecipitation cyclin T1 experience fight against genetic manipulation in vivo inhibitor/antagonist interest knock-down luminescence macrophage molecular domain monocyte mutant nanoluciferase neuropsychiatric disorder new therapeutic target novel pathogen promoter protein protein interaction targeted treatment tat Protein therapeutic target

项目摘要

PROJECT SUMMARY Antiretroviral therapy (ART) has transformed HIV into a manageable non-fatal chronic disease. However, significant challenges remain with increasing drug resistance and adverse drug reactions. Therefore, there is an urgent need to identify novel therapeutic targets with unique mechanisms of action to sustain the gains of ART. HIV transcription represents a vital part of the viral life cycle that has not been successfully targeted for therapy. We recently discovered that an interaction between the essential viral protein Tat and splicing factor 3B subunit 1 (SF3B1) is required for HIV transcription. The primary goal of this project is to define the role played by the interaction between Tat and SF3B1 in HIV transcription. The long term objective is to identify compounds that can block this interaction and therefore HIV replication. We hypothesize that Tat-mediated HIV transcription requires Tat to interacts with SF3B1 and that this interaction, when blocked will inhibit HIV replication. Our specific aims are: Aim 1: Identify the molecular domains of Tat-SF3B1 interactions required for HIV-1 transcription. We have shown that Tat and SF3B1 interact, and that this interaction requires the C-terminal portion of Tat. However, the specific domains of Tat or SF3B1 required for this interaction are unknown. First, we will use mutagenesis, along with in-vitro and in-vivo protein binding studies to narrow down the residues of Tat and the domain of SF3B1 required for the interaction. Second, we will perform in-vivo alanine scanning to better define the interaction surfaces for the two binding domains. The effect of alanine substitutions will be quantified with the NanoLuc® Binary Technology (NanoBiT) split luciferase complementation protein: protein interaction system. We will also perform Tat-SF3B1 immunoprecipitations and transcription assays in primary CD4+ T cells and monocyte-derived macrophages (MDMs) to ascertain the physiological relevance of our findings. Aim 2: Define the specific role of SF3B1 in Tat-mediated HIV-1 transcription. We showed that depletion of SF3B1 abrogates Tat-mediated HIV transcription, but the exact mechanism for SF3B1 actions on HIV transcription is unknown. SF3B1 interacts with Tat, the nuclear inhibitor of PP1 (NIPP1), and is also known to methylate HIV transcription-activating histone H3K36me3. Therefore, we hypothesize that SF3B1 may control HIV transcription through one or more of these pathways in primary CD4+T cells, MDMs and HIV latency models. First, we will use RNA Immunoprecipitation (RNAIP) and fluorescent electrophoretic mobility shift assay (fEMSA) to quantify the effects of SF3B1 on Tat/TAR interactions. Second, we will determine how SF3B1 influences Tat methylation as well as the occupancy of the HIV promoter by H3K36me3. Finally we will determine if Tat-SF3B1 interactions plays a role in NIPP1-mediated inhibition of HIV transcription. This work will be highly significant to further the understanding of Tat-mediated HIV transcription and define a potential therapeutic target for HIV.

项目总结