CHEMOTHERAPY OF HIV INFECTION--ULTRASTRUCTURAL APPROACH

HIV感染的化疗——超微结构方法

• 批准号: 3142602
• 负责人: GUEH-DJEN HSIUNG
• 金额: $ 13.04万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3142602
• 关键词: AIDS therapy; Betaherpesvirinae; HIV infections; RNA directed DNA polymerase; antiAIDS agent; antiviral agents; cell components; cellular pathology; electron microscopy; enzyme linked immunosorbent assay; human immunodeficiency virus 1; human immunodeficiency virus 2; human subject; immunoelectron microscopy; immunofluorescence technique; lymphocyte; macrophage; monocyte; pathologic process; tissue /cell culture; virus antigen; virus infection mechanism; virus morphology; virus replication; zidovudine

The specific aims of this proposal are: 1) to compare the pathogenesis of HIV in lymphocytes and monocytes/macrophages (mo/m0) with emphasis on virus attachment, penetration, maturation and release; and 2) to study the effects of antiviral agents, either synthetic or natural products, on HIV replication in lymphocytes and/or mo/mo/, especially in persistently -infected macrophages. The experimental design will rely mainly on ultrastructural approaches. 1) Pathogenesis of HIV in lymphocytes and mo/mo/: HIV-infected primary cultures of lymphocytes and mo/mo/will be monitored for HIV attachment during the first 3-4 hours after infection and for viral replication on days 7, 10, 14 (acute) and 40 (chronic) post-infection by: a) assaying supernatant for reverse transcriptase activity and HIV antigen levels (ELISA); and b) processing cell pellets of lymphocytes and mo/mo/monolayers for EM and IEM. Dually-infected mo/mo/or H9 cells with HIV and HCMV will also be monitored as in b). A detailed search for pseudotype virions will be made using immunogold-labelling EM and/or immunoferritin EM techniques. 2) Antiviral drug assays: Primary cultures of lymphocytes and mo/mo obtained form the same donors will be treated with polybrene and then infected with HIV-1 or HIV-2, after which various concentrations of antiviral drugs will be added to the culture medium. Drug inhibition of HIV replication in infected cultures will be determined as describe in 1). In EM studies, special attention will be given to the effects of drugs on HIV attachment and penetration during the early states of infection, i.e. the first 4 hours after infection; and on inhibition of HIV release on days 7, 10 and 14 post-infection. The ultrastructural information obtained from these basic studies should expand our knowledge of the pathogenesis of HIV infection and the mechanisms of antiviral drug action. This information may lead to improved strategies for the treatment of AIDS and related disorders.

该提案的具体目的是：1）比较发病机制 淋巴细胞和单核细胞/巨噬细胞中 HIV 的含量 (mo/m0)，重点是 病毒附着、渗透、成熟和释放； 2）学习 抗病毒药物（无论是合成产品还是天然产品）对 HIV 在淋巴细胞和/或 mo/mo/ 中复制，特别是在 持续感染的巨噬细胞。 实验设计将主要依靠超微结构方法。 1) HIV在淋巴细胞和mo/mo/中的发病机制：HIV感染的原发性 淋巴细胞培养物和 mo/mo/will 将监测 HIV 附着情况 在感染后的前 3-4 小时内以及病毒复制期间 感染后第 7、10、14 天（急性）和 40 天（慢性）：a) 检测 上清液用于检测逆转录酶活性和 HIV 抗原水平 （酶联免疫吸附试验）； b) 处理淋巴细胞的细胞沉淀物和 用于 EM 和 IEM 的 mo/mo/monolayers。 双重感染mo/mo/或H9细胞 HIV 和 HCMV 也将按照 b) 进行监测。 详细搜索 假型病毒粒子将使用免疫金标记 EM 和/或 免疫铁蛋白电镜技术。 2) 抗病毒药物测定：主要 从同一供体获得的淋巴细胞和 mo/mo 培养物将 用聚凝胺处理，然后感染 HIV-1 或 HIV-2，之后 培养物中将添加不同浓度的抗病毒药物 中等的。 药物抑制感染培养物中的 HIV 复制将是 如 1) 中所述确定。 在 EM 研究中，将特别关注 考虑到药物对艾滋病病毒附着和渗透的影响 感染的早期状态，即感染后的前 4 小时； 以及感染后第 7 天、第 10 天和第 14 天抑制 HIV 释放。 从这些基础研究中获得的超微结构信息应该 扩大我们对艾滋病毒感染发病机制的认识 抗病毒药物的作用机制。 此信息可能会导致 改善艾滋病及相关疾病的治疗策略。