SIGNALS FOR TRANSACTIVATION OF LATENT HIV-1 GENOME

潜伏 HIV-1 基因组反激活信号

• 批准号: 3756016
• 负责人: WILLIAM M O BOTO
• 金额: --
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3756016
• 关键词: HIV infections; T lymphocyte; biological signal transduction; cellular pathology; gene expression; human immunodeficiency virus 1; immunofluorescence technique; immunologic assay /test; interleukin 2; interleukin 6; latent virus infection; molecular pathology; northern blottings; nucleic acid probes; pathologic process; polymerase chain reaction; protein kinase C; scintillation counter; transfection; tumor necrosis factor alpha; virus envelope; virus replication

The signal which triggers replication of the HIV genome in T4 cells is an attractive target for the design of new promising anti-AIDS chemotherapy. A number of recent investigations have shown that polyclonal mitogens, tumor promoters, certain cytokines, and other stimuli promote the infection of T4 cells by HIV, and stimulate marked increases in the expression of previously latent HIV genome in these lymphocytes. Nevertheless, it is still uncertain whether these structurally diverse classes of agonists stimulate replication of the virus via a common or distinct molecular mechanisms. This study will examine the role of the signal transducing enzyme. PKC, in the infection of T4 cells by HIV-1, and in trans-activation of latent viral genome in the lymphocytes. Mitogen-responsive T4 cells lines will be infected with a standard preparation of HIV-1 in the presence of PKC-activating agents, or PKC inhibitors. Time-dependent internalization of the HIV-1 nucleocapsid, and RNA template-directed reverse transcription of the first copies of proviral DNA intermediates, as well as virus replication in T4 cells will be analyzed by means of the following techniques: a) immunofluorescence; b) Taq DNA polymerase-dependent chain reaction (PCR) using oligonucleotide primer pairs complementary to conserved sequences in the LTR, GAG, and ENV genes; c) MTT chromogenic test for virus-induced lysis of target cells; and d) p55 antigen capture immunoassay. To assess the role of PKC in trans-activation and replication of the latent HIV-1 genome, T4 cells will be stably transfected with HIV-pBR322 plasmid constructs which express the TAT, ENV as well as heterologous CAT reporter gene ligated at positions down stream from the LTR. DNA- transfected cells will be incubated with the modulators of PKC. The trans-activating effects of the ligands on the expression of the HIV-1 genome will be quantified by means of several techniques: by a) liquid scintillation assay for the LTR-directed expression of the CAT reporter gene; and b) northern blot hybridization using the ENV and TAT cDNA probes. In addition, a potential role of PKC-dependent secretion of specific cytokines in the regulation of the HIV-1 replication will be examined. MRNA isolated from normal peripheral blood mononuclear cells following in vitro infection with HIV-1 will be analyzed by means of northern blot hybridization to quantify the levels of expression of the genes encoding IL 2, IL 6, INF-alpha, INF-gamma, and TNF-alpha. The goal of this investigation is to elucidate the cellular and molecular mechanisms which determine the latent and lytic course of HIV-1 infection.

触发T4细胞中HIV基因组复制的信号是 设计新的有前途的反援助的有吸引力的目标 化学疗法。 最近的许多调查表明 多克隆有丝分裂原，肿瘤启动子，某些细胞因子和其他 刺激通过HIV促进T4细胞的感染，并刺激标记 在这些中的表达增加 淋巴细胞。 然而，仍然不确定这些是否 结构上多样化的激动剂刺激复制 通过常见或不同的分子机制病毒。 这项研究会 检查信号转导酶的作用。 PKC，在感染中 HIV-1的T4细胞以及潜在病毒基因组的反式激活 淋巴细胞。 有丝分裂原反应T4细胞系将被感染 在存在PKC激活剂的情况下，HIV-1的标准制备， 或PKC抑制剂。 HIV-1的时间依赖性内在化 Nucleocapsid和RNA模板定向的逆转录 病毒DNA中间体的第一份以及病毒复制 在T4中，将通过以下技术进行分析：a） 免疫荧光； b）TAQ DNA聚合酶依赖性链反应（PCR） 使用与保守序列互补的寡核苷酸底漆对 在LTR，插科打and和Env基因中； c）病毒诱导的MTT发色测试 靶细胞的裂解； d）p55抗原捕获免疫测定。 评估 PKC在潜在HIV-1的反式激活和复制中的作用 基因组，T4细胞将被HIV-PBR322质粒稳定转染 表达TAT，ENV和异源猫的结构 记者基因在LTR的位置下方的位置连接。 脱氧核糖核酸- 转染的细胞将与PKC的调节剂一起孵育。 这 配体对HIV-1表达的反式激活作用 基因组将通过几种技术进行量化：a）液体 猫记者的LTR指导表达的闪烁测定法 基因; b）使用ENV和TAT cDNA北部印迹杂交 探针。 另外，PKC依赖性分泌的潜在作用 HIV-1复制调节中的特定细胞因子将是 检查。 从正常的外周血单核细胞中分离出的mRNA 通过在体外感染HIV-1后将通过 北印迹杂交以量化 编码IL 2，IL 6，Inf-Alpha，Inf-Gamma和TNF-Alpha的基因。 目标 这项研究是为了阐明细胞和分子 确定HIV-1的潜在和裂解过程的机制 感染。