CELLULAR GENES REQUIRED FOR HIV-1 INFECTION AS TARGETS FOR THERAPY

(1)项目目标： -确定最佳艾滋病毒-1感染所需的细胞基因，或 参与了HIV-1的发病机制。 -将所获得的知识应用于抗病毒疗法的发展 这些基因同时针对病毒和细胞基因。 (2)实验方法： 一组突变体来自人类T细胞克隆CEM， 表现出对艾滋病毒-1感染的不同易感性。 开发了多种分析方法来分析这些克隆：(A)定量 表面受体和可溶性gp120结合的流式细胞术分析。 (B)定量聚合酶链式反应以测量病毒进入和前病毒DNA的形成。(C) 以痘苗病毒gp160载体为基础的合胞体检测。(D)凝胶延迟分析 研究最佳HIV-1前病毒所需的DNA结合蛋白水平 重新激活。(E)蛋白激酶C酶活性测定。(F) 氯霉素乙酰转移酶(CAT)法检测细胞活性 HIV-1 LTR. (三)主要发现： -表面CD4受体的数量之间不存在直接关系 以及HIV-1感染的动力学。 -分离到两个CEM亚克隆，敏感度显著降低 被多种HIV-1毒株感染。这些突变体被发现具有 NFK-B的DNA结合蛋白水平显著降低 家庭(P50)。其他DNA结合蛋白正常表达。约简 核因子-kB蛋白水平仅影响HIV-1细胞生长 敏感度。这些基因可能成为反义抑制的靶点。 将患者的PBL维持在静息状态，防止再次激活 集成病毒。

(1) Goals of project: - To identify cellular genes required for optimal HIV-1 infection, or involved in HIV-1 pathogenesis. - To apply the knowledge gained towards development of anti-viral therapy which target both viral and cellular genes. (2) Experimental approach: A panel of mutants were derived from the human T cell line clone CEM, demonstrating different susceptibilities to HIV-1 infection. Multiple assays were developed to analyze these clones: (a) Quantitative flow cytometry analysis of surface receptors and binding of soluble gp120. (b) Quantitative PCR to measure viral entry and proviral DNA formation. (c) Vaccinia-gp160-vector based syncytia assay. (d) Gel retardation assays to study the levels of DNA binding proteins required for optimal HIV-1 provira reactivation. (e) Protein Kinase C enzymatic activity assay. (f) Chloramphenicol acetyl transferase (CAT) assay to measure activation of the HIV-1 LTR. (3) Major Findings: - No direct correlation exist between the number of surface CD4 receptors and the kinetics of HIV-1 infection. - Two CEM subclones were isolated with dramatically reduced susceptibility to infection by multiple strains of HIV-1. These mutants were found to have a markedly reduced level of DNA binding protein belonging to the NFk-B family (p50). Other DNA binding proteins are normally expressed. The reduce levels of NF-kB proteins does not effect cell growth only HIV-1 susceptibility. These genes could be the target of anti-sense suppression t maintain patients' PBL at a resting state, preventing reactivation of integrated virus.