A Novel Approach to Identify HIV Suppression Factor from CD8 Cells

描述(申请人提供)：CD8+T细胞已被证明以一种MHC不受限制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)的特性在过去20年中仍然难以捉摸。以前对CAF纯化的研究主要是基于他们的努力，假设一种可溶性因子，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抑制是一种膜依赖现象。为了证明这一假设，我们证明了一种膜定位的活性，它介导了HIV-1转录抑制，并通过30-100纳米大小的囊泡(称为外小体)的细胞外分泌，以膜结合的形式伴随着分泌。我们已经成功地从纯化的外切体中提取了这种抗病毒活性的可溶性形式，使用了一种新的方法，消除了血清和细胞膜。对这种具有抗病毒活性的无血清蛋白质组分的蛋白质质谱分析表明，其中只有有限数量的蛋白质。本研究的目的是确证外切体水提物中的HIV抑制蛋白。该项目的具体目标是：1)生化分离抗HIV因子，使其接近纯度。在我们的初步研究中显示部分纯化的抗病毒活性的一系列生化程序将被串联使用。报告基因介导的HIV-1转录抑制实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学方法鉴定HIV-1 LTR抑制因子最可能的候选蛋白质/基因序列。差示同位素标记结合多维液相色谱(MDLC)的蛋白质定量分析将应用于含有抗HIV因子的无血清蛋白质组分；3)确定候选蛋白质/基因序列为LTR启动子抑制HIV-1抑制因子。将使用RNA干扰、重组蛋白生产和/或抗体中和蛋白质因子的组合来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特性的多种HIV-1病毒的非细胞溶解作用和活性，抑制因子的识别对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。在过去的20年里，这种抗病毒因子的身份仍然难以捉摸。我们最近发现了一种有效的膜结合的HIV-1抑制活性，它是由转化的CD8+T细胞分泌的30-100 nm大小的内小体衍生的囊泡，称为外小体。我们已经成功地从纯化的外切体中提取了这种抗病毒活性的可溶性形式，使用了一种新的方法，消除了血清和细胞膜。这项拟议研究的目的是使用生化和蛋白质组学相结合的方法，从外体衍生的可溶性因子中确定HIV-1抑制因子。

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.