SIGNALS FOR TRANSACTIVATION OF LATENT HIV-1 GENOME

触发HIV基因组在T4细胞中复制的信号是 一个有吸引力的目标，设计新的有前途的抗艾滋病 化疗 最近的一些调查显示， 多克隆有丝分裂原、肿瘤促进剂、某些细胞因子和其他 刺激促进HIV感染T4细胞，并刺激显著的 在这些人中，以前潜伏的HIV基因组的表达增加， 淋巴细胞 然而，目前还不确定这些 结构上不同的激动剂刺激复制的 病毒通过共同或不同的分子机制。 本研究将 研究信号转导酶的作用。 蛋白激酶C在感染中 HIV-1对T4细胞的作用，以及HIV-1对潜伏病毒基因组的反式激活作用。 淋巴细胞 有丝分裂原反应性T4细胞系将被感染 在PKC活化剂存在下的HIV-1标准制剂， 或PKC抑制剂。 HIV-1的时间依赖性内化 核衣壳和RNA模板指导的逆转录。 前病毒DNA中间体的第一个拷贝，以及病毒复制 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引物对 c）MTT显色试验，用于病毒诱导的LTR、GAG和ENV基因的表达; 靶细胞裂解;和d）p55抗原捕获免疫测定。 评估 PKC在潜伏HIV-1转录激活和复制中的作用 T4细胞将用HIV-pBR 322质粒稳定转染 表达达特、ENV以及异源CAT的构建体 报告基因连接在LTR下游的位置。 DNA- 将转染的细胞与PKC调节剂一起孵育。 的 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定量：a）液体 用于CAT报告基因的LTR定向表达的闪烁测定 B）使用ENV和达特cDNA的北方印迹杂交 probes. 此外，PKC依赖性分泌的一个潜在的作用， 在调节HIV-1复制的特定细胞因子将是 考察 从正常外周血单个核细胞中分离的mRNA 体外感染HIV-1后，将通过 北方印迹杂交以定量表达的水平， 编码IL 2、IL 6、INF-α、INF-γ和TNF-α的基因。 目标 这项研究的目的是阐明细胞和分子 决定HIV-1潜伏和裂解过程的机制 感染

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.