Late Replication Functions of Retroviruses

描述(由申请人提供)：这项计划的长期目标 建议阐明晚期逆转录病毒复制的机制，涉及 病毒从细胞中萌发。建议进行实验，以跟进我们的 共同发现病毒萌发所需的逆转录病毒L结构域 细胞和我们随后鉴定的两种潜在的细胞蛋白 (Nedd4和TSG1O1)，都与泛素化有关，与 RSV和HIV1的L结构域。有人提议用实验来证明 这些细胞蛋白是病毒萌发所需的生物伙伴。我们 将寻找细胞蛋白与细胞内GAG的共同定位， 显性负表达对病毒在细胞内萌发的干扰 细胞蛋白片段，以及与GAG共免疫沉淀 从依赖于L结构域的提取物中提取各自的细胞蛋白。 已经在体内建立了两种细胞蛋白相互作用的证据 GAG、含有遗传基因或基因的细胞系的各自的L结构域序列 将构建候选细胞蛋白的表型敲除并 用来检验它们在萌芽过程中的作用。此外，免疫 使用具有和不具有L结构域的GAG构建物的沉淀技术 序列将被用于恢复额外的细胞蛋白质，这些蛋白质可能是 参与萌芽过程。这些蛋白质的特性将是 利用免疫和生化技术建立的植物及其在发芽中的作用 将如上所述建立。这将开始定义一条 从细胞中萌发病毒所需的相互作用。在不同的方向上，我们将 对相互作用的序列要求进行新的遗传分析 一种新的体内诱变作用对L结构域及其细胞伙伴的影响 程序。这些研究将为观察萌芽状态奠定基础 详细阐述了其作用机理。此外，发现RSV L结构域的PY基序是 发现于广泛类别的包膜病毒中(包括Rhabdo-、Filo-和 疱疹病毒)对抗病毒药物的发展具有广泛的意义 针对与病毒从细胞中释放有关的细胞蛋白的药物。

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.