Inducible Cellular Resistance to HIV-1 infection.

诱导细胞对 HIV-1 感染的抵抗力。

• 批准号: 6511545
• 负责人: Malgorzata Simm
• 金额: $ 27.13万
• 依托单位: ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511545
• 关键词: HIV infections; T lymphocyte; cellular immunity; clinical research; cytokine; human immunodeficiency virus 1; human subject; immunologic assay /test; molecular cloning; protein biosynthesis; protein purification; protein sequence

DESCRIPTION: (Adapted from Applicant's Abstract) We have induced defensive cellular responses by attenuated infection with HIV-1 mutated in the vif gene. Resistance was induced in transformed human T cells by co-cultivation with HIV-1 resistant PBL, which were latently infected with vif-negative HIV-1. The resulting latently infected transformed T cell clones were found to secrete a soluble HIV-1 Resistance Factor (HRF), a new factor that confers resistance to HIV-1 upon uninfected cells by blocking HIV-1 transcription. HRF activity in HRF(+) clones was shown initially through their resistance to productive HIV-1 infection and their block to TAT activated transcription of a marker gene. More detailed studies indicate that HRF prevents formation of NF-kB/DNA complex in nuclear extracts of cells that have been exposed to supernatants of HRF(+) cells. This observation prompted the development of Rapid Suppression Assay (RSA) that measures the activity of HRF by inhibition of phorbol myristate induction of HIV-1 in ACH-2 cells. In this application we propose to (1) isolate and purify HRF active in RSA from cell supernatants through sequential gel exclusion chromatographic steps. Purified HRF will be subjected to N'-terminal sequencing, yielding sequences for primer design. (2) We shall construct a cDNA library from HRF(+) cells, identify a full-length cDNA using HRF sequence based primers, and clone HRF into a Baculovirus expression vector or a mammalian expression vector for high level expression. Anti-HRF antibodies will be raised against recombinant HRF. (3) In parallel we will employ state of the art technologies to identify up-regulated genes in HRF(+) clones using cDNA expression arrays. In this approach we will search known genes involved in hematology, immunology and transcription regulation to complement biochemical isolation and identification. (4) The activity of recombinant HRF will be tested in the optimal dose for inhibition of a panel of primary isolates in different cell types. (5) HRF expression in Iymphocytes from HIV-1 infected persons will be tested by RT-PCR and the levels of expression will be correlated to the disease state of the donor. Having T cell clones producing HRF creates a unique opportunity to isolate, characterize and apply this protein for treatment of HIV-1 infection in culture, and in long term goals to design new therapeutics against AIDS and to understand cellular protective responses to infection by attenuated HIV-1.

描述：（改编自申请人的摘要）我们已经引起了防御 通过VIF基因中的HIV-1突变的HIV-1感染通过减弱的细胞反应。 通过与 HIV-1抗性PBL，该PBL被VIF阴性HIV-1潜在地感染。这 结果发现被视为被视为转化的T细胞克隆分泌A 可溶性HIV-1抗性因子（HRF），这是一种赋予对抗性的新因素 通过阻断HIV-1转录，在未感染细胞上的HIV-1。人力资源管理活动 HRF（+）克隆最初是通过对生产性HIV-1的抵抗而显示的 感染及其对标记基因的TAT激活转录的阻滞。更多的 详细的研究表明，HRF阻止了NF-KB/DNA复合物的形成 暴露于HRF上清液的细胞的核提取物（+） 细胞。该观察结果促使发展快速抑制测定法 （RSA）通过抑制佛波尔肉豆蔻酸酯来测量HRF的活性 ACH-2细胞中HIV-1的诱导。在此应用中，我们建议（1） 分离并纯化RSA中的HRF通过顺序从细胞上清液中活跃起来 凝胶排除色谱步骤。纯化的HRF将受到 n'-末端测序，产生引物设计的序列。 （2）我们将 从HRF（+）细胞构建cDNA库，使用使用的全长cDNA 基于HRF序列的引物，并克隆HRF到杆状病毒表达载体 或用于高水平表达的哺乳动物表达载体。抗HRF抗体 将针对重组HRF提出。 （3）并行运用 使用cDNA鉴定HRF（+）克隆中上调基因上调基因的艺术技术 表达阵列。在这种方法中，我们将搜索涉及的已知基因 血液学，免疫学和转录调节以补充生化 隔离和识别。 （4）重组HRF的活性将是 在最佳剂量中测试以抑制一组主要分离株 不同的细胞类型。 （5）来自HIV-1感染的HIMPHOCYTES中的HRF表达 人员将通过RT-PCR进行测试，表达水平将为 与供体的疾病状态相关。有T细胞克隆产生 人力资源管理创造了一个独特的机会来隔离，表征和应用此 用于治疗培养中HIV-1感染的蛋白质，在长期目标中 设计针对艾滋病的新疗法并了解细胞保护性 HIV-1对感染的反应。