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

• 批准号: 7755150
• 负责人: Phalguni GUPTA
• 金额: $ 21.85万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7755150
• 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

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）：已证明CD 8 + T细胞以MHC不受限的非细胞溶解方式抑制人类免疫缺陷病毒1型（HIV-1）的转录。然而，T细胞介导的抗病毒因子（CAF）的身份在过去的20年里仍然难以捉摸。先前在CAF纯化中的研究主要基于他们的努力，假设可溶性因子，如细胞因子或趋化因子，介导抗病毒活性。我们考虑了另一种新的假设，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，我们已经证明了介导HIV-1转录抑制的膜定位活性，其通过被称为外泌体的30-100纳米大小的囊泡的细胞外分泌以膜结合形式伴随分泌。我们已经成功地从纯化的外泌体中提取了这种抗病毒活性的可溶形式，使用了一种消除血清和细胞膜的新方法。蛋白质质谱分析表明，这种无血清的蛋白质馏分具有抗病毒活性，只有有限数量的蛋白质在it. The本研究的目的是最终确定的HIV抑制蛋白的水溶性提取物的外来体。本项目的具体目标是：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抗病毒活性部分纯化的一系列生化程序将被串联使用。报告基因介导的HIV-1转录抑制试验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法鉴定HIV-1 LTR抑制因子的最可能候选蛋白质/基因序列。将使用差分同位素标记结合多维液相色谱（MDLC）的定量蛋白质质谱分析应用于含有抗HIV因子的无血清蛋白质级分; 3）作为LTR启动子抑制性HIV-1抑制因子的候选蛋白质/基因序列的结论性确认。RNA干扰、重组蛋白生产和/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为HIV-1抑制因子。.由于其对具有不同共受体性质的多种HIV-1的非细胞溶解作用模式和活性，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。公共卫生相关性：来自HIV-1感染受试者的CD 8 + T细胞可以抑制人类免疫缺陷病毒1型（HIV-1）复制。在过去的20年里，这种抗病毒因子的身份仍然难以捉摸。我们最近已经鉴定了一种有效的膜结合HIV-1抑制活性，其从转化的CD 8 + T细胞分泌为30-100 nm大小的内体衍生的囊泡，称为外泌体。我们已经成功地从纯化的外泌体中提取了这种抗病毒活性的可溶形式，使用了一种消除血清和细胞膜的新方法。本研究的目的是利用生物化学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确定HIV-1抑制因子。