Late Replication Functions of Retroviruses

逆转录病毒的晚期复制功能

• 批准号: 6724770
• 负责人: JONATHAN P LEIS
• 金额: $ 24.39万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724770
• 关键词: Rous sarcoma virus; aspartic endopeptidases; drug resistance; enzyme activity; gag protein; host organism interaction; human immunodeficiency virus 1; microorganism culture; virus assembly; virus protein; virus replication

DESCRIPTION (provided by the applicant): The long-term objective of this proposal is to elucidate mechanisms in late retrovirus replication that involve budding of virus from cells. Experiments are proposed to follow up our co-discovery of the retrovirus L domain required for budding of virus from cells and our subsequent identification of two potential cellular proteins (Nedd4 and TSG1O1), both involved with ubiquitination, that interact with the RSV and HIV-1 L domains, respectively. Experiments are proposed to prove that these cell proteins are the biological partners required for virus budding. We will look for co-localization of the cellular proteins with Gag inside cells, disruption of virus budding in cells by dominant negative expression of fragments of the cell proteins, and coimmune precipitation of Gag with the respective cell proteins from extracts that is dependent upon the L domain. Having established in vivo evidence that the two cell proteins interact with the respective L domain sequences of Gag, cells lines containing genetic or phenotypic knockouts of the candidate cellular proteins will be constructed and used to examine their roles in the budding process. In addition, immune precipitation techniques using Gag constructs with and without L domain sequences will be used to recover additional cellular proteins that may be involved in the budding process. The identity of these proteins will be established using immune and biochemical techniques and their role in budding will be established as described above. This will begin to define a pathway of interactions required to bud virus from cells. In a separate direction, we will carry out a novel genetic analysis of the sequence requirements for interaction of the L domains with their cell partners by a novel in vivo mutagenesis procedure. These studies will set the groundwork for looking at the budding mechanism in detail. Moreover, the finding that the RSV L domain PY motif is found in a broad class of enveloped viruses (including rhabdo-, filo-, and herpesviruses) has wide ranging implications for the development of antiviral agents directed at cell proteins involved in release of viruses from cells.

描述（由申请人提供）：本项目的长期目标 提议是阐明晚期逆转录病毒复制的机制，涉及 病毒从细胞中出芽。建议进行实验来跟进我们的 共同发现病毒出芽所需的逆转录病毒 L 结构域 细胞以及我们随后鉴定出的两种潜在细胞蛋白 （Nedd4 和 TSG1O1），两者都参与泛素化，与 分别是 RSV 和 HIV-1 L 结构域。建议进行实验来证明 这些细胞蛋白是病毒出芽所需的生物伙伴。我们 将寻找细胞蛋白与细胞内 Gag 的共定位， 通过显性负表达来破坏细胞中的病毒出芽 细胞蛋白片段，以及 Gag 与 提取物中的各个细胞蛋白依赖于 L 结构域。 已建立体内证据表明这两种细胞蛋白相互作用 Gag各自的L结构域序列，含有遗传或 将构建候选细胞蛋白的表型敲除并 用于检查它们在萌芽过程中的作用。此外，免疫 使用带有和不带有 L 结构域的 Gag 构建体的沉淀技术 序列将用于回收额外的细胞蛋白，这些蛋白可能是 参与萌芽过程。这些蛋白质的身份将是 使用免疫和生化技术建立及其在出芽中的作用 将如上所述建立。这将开始定义一条路径 病毒从细胞中萌芽所需的相互作用。我们将在一个单独的方向 对相互作用的序列要求进行新颖的遗传分析 通过新颖的体内诱变将 L 结构域与其细胞伴侣结合起来 程序。这些研究将为观察萌芽奠定基础 机制详细。此外，发现 RSV L 结构域 PY 基序是 存在于一大类有包膜病毒（包括弹状病毒、丝状病毒和 疱疹病毒）对于抗病毒药物的开发具有广泛的影响 针对参与从细胞释放病毒的细胞蛋白的药物。