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

(1)项目目标： - 确定最佳HIV-1感染所需的细胞基因，或 参与HIV-1发病机制。 - 将获得的知识应用于抗病毒治疗的开发 针对病毒和细胞基因。 (2)实验方法： 一组突变体来源于人T细胞系克隆CEM， 显示出对HIV-1感染的不同易感性。 开发了多种测定来分析这些克隆： 表面受体的流式细胞术分析和可溶性GP 120的结合。 (b)定量PCR测量病毒进入和前病毒DNA形成。（c）第（1）款 基于vaccinia-gp 160-载体的合胞体测定。(d)凝胶阻滞测定， 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）第（1）款 氯霉素乙酰转移酶（CAT）测定，以测量 HIV-1 LTR。 (3)主要发现： - 表面CD 4受体的数量与细胞增殖之间不存在直接的相关性。 和HIV-1感染的动力学。 - 两个CEM亚克隆被分离，具有显著降低的敏感性 感染多种HIV-1病毒。这些突变体被发现具有 属于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。还原 NF-kB蛋白的水平不影响细胞生长，只有HIV-1 易感性这些基因可能是反义抑制的靶点， 维持患者PBL处于静息状态，防止 整合病毒

(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.