A Novel Approach to Identify HIV Suppression Factor from CD8 Cells

从 CD8 细胞中鉴定 HIV 抑制因子的新方法

基本信息

批准号：
7871341
负责人：
Phalguni GUPTA
金额：
$ 18.75万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-12 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7871341
关键词：
Antibodies Antigens Antiviral Agents Binding Biochemical Biological Assay CD8-Positive T-Lymphocytes CD8B1 gene Candidate Disease Gene Cell Line Cell membrane Cells Cleaved cell Code Complementary DNA Coupled Data Endosomes Fractionation Gene Proteins Genes Genetic Transcription Goals HIV HIV-1 Infection Isotopes Liquid Chromatography Manuscripts Mass Spectrum Analysis Mediating Membrane Methods Molecular Monitor Monoclonal Antibodies Natural History Nature Neutralization Tests Peptides Performance Procedures Production Property Protein Fragment Protein Sequence Analysis Proteins Proteomics RNA Interference Recombinant Proteins Recombinants Reporter Reporter Genes Research Personnel Sampling Sequence Analysis Series Serum Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization T-Lymphocyte Testing Trypsin Vesicle aqueous base cDNA Expression chemokine cytokine cytotoxic extracellular factor A nano nanoscale novel novel strategies numb protein promoter public health relevance receptor

项目摘要

DESCRIPTION (provided by applicant): CD8+ T cells have been shown to suppress transcription of human immunodeficiency virus type 1 (HIV-1) in a MHC-unrestricted non-cytolytic manner. Yet the identity of the T cell mediated antiviral factor (CAF) remained elusive for last 20 years. Previous studies in the purification of the CAF were largely based on their efforts on an assumption that a soluble factor, such as a cytokine or chemokine, mediates the antiviral activity. We considered an alternative and novel hypothesis that non-cytotoxic HIV-1 suppression is a membrane dependent phenomenon. To prove this hypothesis we have demonstrated a membrane-localized activity mediating HIV-1 transcription suppression that is concomitantly secreted in membrane-bound form through extracellular secretion of 30-100 nanometer sized vesicles known as exosomes. We have been successful in extracting a soluble form of this antiviral activity from purified exosomes using a novel procedure that eliminates serum and cell membrane. Protein mass spectrometric analysis of this serum-free protein fraction with antiviral activity indicated only a limited number of proteins in it. The objective of this study is to conclusively identify the HIV suppressive protein in aqueous soluble extracts of exosomes. Specific aims of the project are: 1) Biochemical isolation of the anti-HIV factor to near purity. A series of biochemical procedures that showed partial purification of antiviral activity in our Preliminary studies will be used in tandem. A reporter gene mediated HIV-1 transcription suppression assay will be used to monitor antiviral activity during purification; 2) Identification of a most probable candidate protein/gene sequence for the HIV-1 LTR suppressing factor by a quantitative protein mass spectrometry approach. Quantitative protein mass spectrometric analysis using differential isotope-tagging coupled with multidimensional liquid chromatography (MDLC) will be applied to serum-free protein fraction containing anti-HIV factor; 3) Conclusive confirmation of a candidate protein/gene sequence as the LTR promoter suppressive HIV-1 suppressive factor. A combination of RNA-interference, recombinant protein production and/or antibody neutralization of the protein factor will be used to confirm whether a candidate gene sequence is the HIV-1 suppressive factor. . Because of its non-cytolytic mode of action and activity against a diverse range of HIV-1 with different co-receptor properties, the identification of the suppressive factor has a profound effect on therapy and natural history studies of HIV-1 infection. PUBLIC HEALTH RELEVANCE: CD8+ T cells from HIV-1-infected subjects can suppress Human Immunodeficiency Virus type 1 (HIV-1) replication. Identity of this antiviral factor remained elusive for last 20 years. We have recently identified a potent membrane-bound HIV-1 suppressing activity that is secreted from transformed CD8+ T cells as 30-100 nm sized endosome-derived vesicles termed exosomes. We have been successful in extracting a soluble form of this antiviral activity from purified exosomes using a novel procedure that eliminates serum and cell membrane. The objective of the proposed study is to use a combined biochemical and proteomic approach to conclusively identify the HIV-1 suppressive factor from the exosome-derived soluble factor.

描述（由申请人提供）：CD8+T细胞已被证明能够以MHC不受限制的非溶细胞方式抑制人类免疫缺陷病毒1型（HIV-1）的转录。然而，在过去 20 年里，T 细胞介导的抗病毒因子 (CAF) 的身份仍然难以捉摸。先前对 CAF 纯化的研究主要基于这样的假设：细胞因子或趋化因子等可溶性因子介导抗病毒活性。我们考虑了另一种新颖的假设，即非细胞毒性 HIV-1 抑制是一种膜依赖性现象。为了证明这一假设，我们证明了介导 HIV-1 转录抑制的膜定位活性，该活性通过称为外泌体的 30-100 纳米大小的囊泡以膜结合形式同时分泌到细胞外。我们已经成功地使用一种消除血清和细胞膜的新方法从纯化的外泌体中提取了这种抗病毒活性的可溶形式。对这种具有抗病毒活性的无血清蛋白质级分进行蛋白质质谱分析表明，其中仅含有有限数量的蛋白质。本研究的目的是最终鉴定外泌体水溶性提取物中的 HIV 抑制蛋白。该项目的具体目标是： 1) 将抗 HIV 因子生化分离至接近纯度。在我们的初步研究中显示出部分纯化抗病毒活性的一系列生化程序将串联使用。报告基因介导的 HIV-1 转录抑制测定将用于监测纯化过程中的抗病毒活性； 2) 通过定量蛋白质质谱方法鉴定最可能的 HIV-1 LTR 抑制因子候选蛋白质/基因序列。使用差异同位素标记与多维液相色谱 (MDLC) 相结合的定量蛋白质质谱分析将应用于含有抗 HIV 因子的无血清蛋白质级分； 3) 最终确认候选蛋白质/基因序列作为LTR启动子抑制HIV-1抑制因子。 RNA干扰、重组蛋白产生和/或蛋白因子的抗体中和的组合将用于确认候选基因序列是否是HIV-1抑制因子。。由于其非溶细胞作用模式和针对具有不同共受体特性的多种 HIV-1 的活性，抑制因子的鉴定对 HIV-1 感染的治疗和自然史研究具有深远的影响。公共健康相关性：来自 HIV-1 感染者的 CD8+ T 细胞可以抑制人类免疫缺陷病毒 1 型 (HIV-1) 的复制。过去 20 年来，这种抗病毒因子的身份仍然难以捉摸。我们最近发现了一种有效的膜结合 HIV-1 抑制活性，它是由转化的 CD8+ T 细胞分泌的，大小为 30-100 nm 的内体衍生囊泡，称为外泌体。我们已经成功地使用一种消除血清和细胞膜的新方法从纯化的外泌体中提取了这种抗病毒活性的可溶形式。拟议研究的目的是利用生化和蛋白质组学相结合的方法从外泌体衍生的可溶性因子中最终鉴定出 HIV-1 抑制因子。