THE IDENTIFICATION OF AN ANTI-HIV MECHANISM: A PROTEOMIC BASED APPROACH

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 现在人们普遍认为，仅使用高效抗逆转录病毒疗法（HAART）来恢复免疫功能是不完全的。由于HIV-1耐药株的出现，需要治疗性免疫策略来加强HAART治疗HIV疾病。 我们以前已经观察到一种多抗原免疫调节剂，称为派，它由灭活流感和细菌疫苗的混合物组成，诱导MHC非限制性非细胞溶解性抗HIV-1活性。 基于我们的初步数据记录病毒抑制，我们建议测试的假设，派诱导的抗病毒活性可以通过蛋白质组学方法的差异确定。 为了解决这一假设，我们的具体目标是： 具体目标1：通过进行二维凝胶电泳（2D-GE）和图像分析，确定用效应细胞（PBMC、CD 8+和CD 8+耗竭的T细胞）上清液处理的靶T细胞的细胞内裂解物中的差异蛋白表达。 具体目标#2：通过使用质谱（MS）进行肽质量指纹图谱、细胞培养物中氨基酸稳定同位素标记（SILAC）分析和数据库检索，鉴定和比较派处理的靶T细胞差异表达的蛋白质。 具体目标#3：通过蛋白质印迹和/或qRT-PCR验证质谱法鉴定的差异表达蛋白的同一性。 初步发现鉴定了响应派治疗的PBMC蛋白。比较主图谱以评估蛋白质表达的差异。这揭示了派处理的PBMC中的47个差异表达点。通过串联MS（MS/MS）分析改变的蛋白质用于蛋白质鉴定。在对NCBInr蛋白质数据库查询MS/MS数据后，我们鉴定了11个差异表达的蛋白质点。我们相信，所确定的蛋白质将产生PAI-抗HIV-1反应的蛋白质组特征。我们将在不同的细胞类型中制作派治疗的蛋白质组参考图谱，并将其各自相关的HIV机制提供给其他人用于比较研究。

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. It is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease. We have previously observed that a polyantigenic immunomodulator, known as PAI, which consists of a mixture of inactivated influenza and bacterial vaccine, induces MHC non restricted non-cytolytic anti-HIV-1 activity. Based on our preliminary data documenting viral suppression, we propose to test the hypothesis that PAI induced antiviral activity can be differentially determined by a proteomic approach. To address this hypothesis, our specific aims are: Specific Aim #1: To determine differential protein expression in intracellular lysates from target T cells treated with supernatant from effector cells (PBMC, CD8+and CD8+ depleted T cells) by performing two-dimensional gel electrophoresis (2D-GE) and image analysis. Specific Aim #2: To identify and compare differentially expressed proteins from target T cells treated with PAI by performing peptide mass finger printing using mass spectrometry (MS), stable-isotope labeling by amino acids in cell culture (SILAC) analysis and database search. Specific Aim #3: To validate by Western blots and/or qRT-PCR the identity of differentially expressed proteins identified by mass spectrometry. Preliminary findings identified PBMC proteins responsive to PAI treatment. Master maps were compared to assess differences in protein expression. This revealed 47 differentially expressed spots in PAI treated PBMC. The altered proteins were analyzed by tandem MS (MS/MS) for protein identification. After querying the MS/MS data against the NCBInr protein database, we have identified 11 differentially expressed protein spots. We believe that the identified proteins will generate a proteomic signature of the PAI-anti HIV-1 response. We will make the proteome reference map of PAI treatment in different cell type with their respective related HIV mechanisms available for others to use for comparative studies.