Late Replication Functions of Retroviruses

描述（由申请人提供）：本项目的长期目标 一项提案是阐明逆转录病毒复制晚期的机制， 病毒从细胞中出芽。实验建议跟进我们的 共同发现了逆转录病毒L结构域， 细胞和我们随后鉴定的两种潜在的细胞蛋白 （Nedd 4和TSG 1 O 1），两者都参与泛素化，与蛋白质相互作用。 RSV和HIV-1 L结构域。实验证明， 这些细胞蛋白质是病毒出芽所需的生物伴侣。我们 将寻找细胞蛋白与Gag在细胞内的共定位， 通过显性负表达破坏病毒在细胞中的出芽 细胞蛋白的片段，以及Gag与细胞蛋白的共免疫沉淀。 来自依赖于L结构域的提取物的相应细胞蛋白。 已经建立了体内证据，表明这两种细胞蛋白质与 Gag的相应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， 用来检查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构建体的沉淀技术 序列将被用来恢复额外的细胞蛋白， 参与了萌芽过程。这些蛋白质的身份将是 利用免疫和生物化学技术建立的， 将按上述方式建立。这将开始定义一条 相互作用所需的芽病毒从细胞。在另一个方向，我们将 对相互作用的序列要求进行新的遗传分析， 的L结构域与它们的细胞伴侣通过一种新的体内诱变 procedure.这些研究将为研究 机制细节。此外，发现RSV L结构域PY基序是 在广泛的一类包膜病毒（包括横纹肌病毒、丝状病毒和 疱疹病毒）对开发抗病毒药物具有广泛的意义 针对参与从细胞释放病毒的细胞蛋白质的药剂。

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.