Understanding the Mechanism of Retrovirus Budding

描述(申请人提供)：逆转录病毒的萌发依赖于其Gag多蛋白中编码的小L结构域序列，这些序列充当参与内体分选的细胞蛋白(ESCRT-I、-II、-III)的结合位点。这些细胞蛋白为病毒样颗粒从质膜中释放提供了机械手段。根据逆转录病毒的不同，使用了三个不同的L结构域，单独或组合使用。为了了解这些蛋白质复合体的性质，来自禽肉瘤病毒、人类免疫缺陷病毒、I型病毒、人类免疫缺陷病毒和莫洛尼小鼠白血病病毒的Gag多聚蛋白将在自然条件下从细胞中分离出来，相关细胞蛋白通过二维层析进行分离，那些与Gag结合的蛋白将通过质谱学技术进行鉴定。这将定义那些共享或不共享的细胞蛋白质。通过分析野生型和含有L结构域缺失的Gag与特定的ESCRT-I、-II、-III蛋白融合形成的复合体，这些蛋白可以挽救或不挽救萌发中的缺陷，从而阐明每种病毒进入萌发途径的切入点。ASV和MuLV Gag都使用相关但不同的PPxY L结构域基序，这些基序是E3泛素连接酶的特异性结合位点。这些基序将在ASV和MuLV Gag多蛋白之间交换，以证明这改变了E3蛋白在出芽过程中使用的特异性。还将对相关PY基序之间不同的残基进行定点突变，并针对不同的E3蛋白进行细胞内siRNA表型耗尽，以确定它们在出芽过程中的机制作用。将研究E3泛素连接酶C2转运域Nedd4在ASV Gag到细胞表面运输中的可能作用。最后，将分析一系列已知的泛素突变体对GAG释放的影响。综上所述，这些实验将提供关于出芽机制的重要新信息，并为开发干扰病毒出芽过程的抗病毒药物提供细胞靶点。与靶向病毒编码酶的药物相比，逆转录病毒对此类药物产生抗药性的可能性可能较小。公共卫生相关性：将使用生化蛋白质组学和定点突变技术比较三种不同逆转录病毒的细胞萌发机制。这些研究将为理解病毒复制的基本过程奠定基础，并为抗病毒药物的开发提供目标，逆转录病毒可能不太可能对其产生抗药性变异。

DESCRIPTION (provided by applicant): The budding of retroviruses is dependent upon small L-domain sequences encoded in their Gag polyproteins that serve as binding sites for cellular proteins involved in endosomal sorting (ESCRT-I, -II, -III). These cell proteins provide the mechanical means for virus-like particles to release from the plasma membrane. Depending upon the retrovirus, three different L domains are used, individually or in combination. To understand the nature of these protein complexes, Gag polyproteins from avian sarcoma (ASV), human immunodeficiency, type I, (HIV-1) and Moloney murine leukemia (MuLV) viruses will be isolated from cells under native conditions, associated cell proteins fractionated by two-dimensional chromatography, and those that bind to Gag in an L-domain dependent fashion will be identified by mass spectrographic techniques. This will define those cellular proteins that are shared or not. By analysis of complexes derived from wild type and Gag containing L-domain deletions but fused to specific ESCRT-I, -II, -III proteins that rescue or not the budding defect, the entry points for each virus into the budding pathway will be elucidated. Both ASV and MuLV Gag use related but different PPxY L-domain motifs, which are specific binding sites for E3 ubiquitin ligases. These motifs will be exchanged between ASV and MuLV Gag polyproteins to demonstrate that this changes the specificity for the E3 protein used in the budding process. Site directed mutagenesis of residues that differ between related PY motifs and siRNA phenotypic depletion inside of cells targeting different E3 proteins will also be carried out to establish their mechanistic role in the budding process. The possible function of the C2 transport domain of the E3 ubiquitin ligase, Nedd4, will be examined for a role in transport of ASV Gag to the cell surface. Finally, a series of known ubiquitin mutants will be analyzed for their effect on Gag release. Taken together, these experiments will provide important new information about the mechanism of budding and suggest cellular targets for the development of antiviral agents that disrupt the viral budding process. Retroviruses may be less likely to develop drug resistance to such agents than to drugs that target viral encoded enzymes. PUBLIC HEALTH RELEVANCE: The mechanism of budding from cells of three different retroviruses will be compared using biochemical proteomics and site directed mutagenesis techniques. These studies will lay the basic foundation for understanding an essential process in viral replication and suggest targets for the development of antiviral agents for which retroviruses may be less likely to develop drug resistance variants.