A Novel Approach to Identify HIV Suppression Factor from CD8 Cells

描述（由申请人提供）：CD8+ T细胞已被证明以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，在过去的20年中，T细胞介导的抗病毒因子（CAF）的身份仍然是一个难以捉摸的问题。先前对CAF纯化的研究主要基于一种假设，即可溶性因子，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，我们已经证明了一种介导HIV-1转录抑制的膜定位活性，该活性通过30-100纳米大小的囊泡（称为外泌体）的细胞外分泌，以膜结合的形式同时分泌。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性形式，使用了一种新的方法，消除了血清和细胞膜。蛋白质质谱分析表明，该无血清蛋白片段中只有有限数量的蛋白质具有抗病毒活性。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)生化分离抗hiv因子至接近纯度。在我们的初步研究中显示部分纯化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转录抑制试验将用于在纯化过程中监测抗病毒活性；2)通过定量蛋白质谱法鉴定HIV-1 LTR抑制因子最可能的候选蛋白/基因序列。将差分同位素标记-多维液相色谱（MDLC）相结合的定量蛋白质谱分析方法应用于含抗hiv因子的无血清蛋白组分；3)最终确定LTR启动子抑制HIV-1抑制因子的候选蛋白/基因序列。rna干扰，重组蛋白生产和/或抗体中和蛋白因子的组合将用于确认候选基因序列是否是HIV-1抑制因子。由于其非细胞溶解的作用方式和对多种具有不同共受体特性的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。这种抗病毒因子的鉴定在过去的20年里一直是难以捉摸的。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，作为30-100 nm大小的内体衍生囊泡，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性形式，使用了一种新的方法，消除了血清和细胞膜。该研究的目的是使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.