Late Replication Functions of Retroviruses

逆转录病毒的晚期复制功能

• 批准号: 6633031
• 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/6633031
• 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（申请人提供）：这是此的长期目标 建议是阐明涉及晚期逆转录病毒复制的机制 细胞病毒的萌芽。提议实验跟进我们 从 细胞和我们随后对两个潜在细胞蛋白的鉴定 （NEDD4和TSG1O1），均与泛素化有关，与 RSV和HIV-1 L领域。提出了实验以证明 这些细胞蛋白是病毒萌芽所需的生物伴侣。我们 将寻找与细胞内部插孔的细胞蛋白共定位， 通过主要的负面表达来破坏细胞中病毒的萌芽 细胞蛋白的片段，以及与GAG的comumune降水 来自提取物的各自的细胞蛋白取决于L结构域。 建立了体内证据，表明两种细胞蛋白与 相应的L域序列的GAG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。另外，免疫 使用有或没有L域的插孔构建的降水技术 序列将用于恢复可能是的其他细胞蛋白 参与了萌芽过程。这些蛋白质的身份将是 使用免疫和生化技术及其在萌芽中的作用建立 将如上所述建立。这将开始定义 来自细胞的芽病毒所需的相互作用。在一个不同的方向上，我们将 对相互作用的序列要求进行新的遗传分析 通过新颖的体内诱变与其细胞伴侣的L领域 程序。这些研究将为研究萌芽奠定基础 详细的机制。此外，发现RSV L域Py图案为 在广泛的包裹病毒中发现（包括rhabdo-，filo-和 疱疹病毒）对抗病毒的发展具有广泛的影响 针对细胞蛋白参与细胞释放病毒的药物。