IDENTIFICATION OF CD8+ CELL ANTI HIV FACTOR

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。感染艾滋病毒并保持健康超过10年的人被认为是长期幸存者。这些无症状的个体具有强烈的细胞介导的免疫反应，抑制HIV复制。这种活性随着感染者的疾病进展而丧失。抗病毒应答由分泌的CD 8+细胞抗病毒因子（CAF）介导，其通过HIV启动子阻断转录。CAF由来自这些健康感染个体的CD 8+细胞以低水平产生。这种蛋白质似乎与任何已知的细胞因子、趋化因子和人类生长因子都不同。CAF鉴定将最有可能通过蛋白质纯化和质谱法实现。 方法将来自健康HIV感染个体的CD 8+细胞在缺乏血清和白蛋白（如果可能）的培养基中生长。在实验室中通过标准程序测定液体的抗病毒活性。然后通过各种生物化学技术（包括离子交换、色谱法和通过尺寸确定柱）浓缩和分级活性流体。通过2-D凝胶分析具有富集的CAF活性的级分并通过质谱法评估。 结果到目前为止，我们已经能够表明CAF的分子大小约为50 kD。通过差异2D凝胶分析，似乎有至少20个不同的点与CAF活性相关。通过质谱法进行分析的研究正在进行中。将进一步评价含CAF液体特有的蛋白质斑点。 通过质谱分析，我们希望能够鉴定介导这种抗HIV活性的蛋白质（CAF）。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction Individuals who have been infected with HIV and remain healthy for over 10 years are considered long-term survivors. These asymptomatic individuals have a strong cell-mediated immune response that suppresses HIV replication. This activity is lost as the infected person advances to disease. The antiviral response is mediated by a secreted CD8+ cell antiviral factor (CAF) which blocks transcription via the HIV promoter. CAF is produced at low levels by CD8+ cells from these healthy infected individuals. The protein appears to be unlike any of the known cytokines, chemokines and human growth factors. CAF identification will most likely be achieved through protein purification and mass spectrometry. Methods CD8+ cells from healthy HIV-infected individuals are grown in culture medium lacking serum and where possible, albumin. Fluids are assayed for antiviral activity by standard procedures in the laboratory. Active fluids are then concentrated and fractionated by various biochemical techniques including ion exchange, chromatography, and by sizing columns Fractions with enriched CAF activity are analyzed by 2-D gel and evaluated by mass spectrometry. Results Thus far, we have been able to show that CAF has a molecular size of about 50 kD. By differential 2D gel analysis, there appears to be at least 20 different spots that are associated with CAF activity. Studies are in progress to analyzed by mass spectrometry. Those protein spots that are unique to CAF-containing fluids will be further evaluated. Discussion By mass spectrometry, we hope to be able to identify the protein (CAF) that mediates this anti-HIV activity.