Understanding the Mechanism of Retrovirus Budding

描述（由申请人提供）：逆转录病毒的出芽依赖于其Gag多蛋白编码的小l结构域序列，该序列作为参与内体分选的细胞蛋白的结合位点（ESCRT-I, -II, -III）。这些细胞蛋白为病毒样颗粒从质膜上释放提供了机械手段。根据逆转录病毒的不同，有三个不同的L结构域被单独或联合使用。为了了解这些蛋白复合物的性质，将在天然条件下从细胞中分离出来自禽肉瘤（ASV）、人类免疫缺陷、I型、（HIV-1）和Moloney小鼠白血病（MuLV）病毒的Gag多蛋白，通过二维色谱分离相关细胞蛋白，并通过质谱技术鉴定以l结构域依赖方式结合Gag的蛋白。这将定义哪些细胞蛋白是共享的，哪些不是。通过分析野生型和Gag衍生的复合物，这些复合物含有l结构域缺失，但与特异性ESCRT-I， -II， -III蛋白融合，从而挽救或不挽救出芽缺陷，从而阐明每种病毒进入出芽途径的切入点。ASV和MuLV Gag都使用相关但不同的PPxY l结构域，它们是E3泛素连接酶的特定结合位点。这些基序将在ASV和MuLV Gag多蛋白之间交换，以证明这改变了出芽过程中使用的E3蛋白的特异性。针对不同E3蛋白的细胞内相关PY基序和siRNA表型耗竭的残基的位点定向诱变也将进行，以确定它们在出芽过程中的机制作用。E3泛素连接酶Nedd4的C2转运结构域的可能功能将在ASV Gag转运到细胞表面的过程中进行研究。最后，我们将分析一系列已知的泛素突变体对Gag释放的影响。总之，这些实验将提供有关出芽机制的重要新信息，并为开发破坏病毒出芽过程的抗病毒药物提供细胞靶点。与针对病毒编码酶的药物相比，逆转录病毒对这些药物产生耐药性的可能性更小。公共卫生相关性：我们将利用生化蛋白质组学和定点诱变技术比较三种不同逆转录病毒细胞出芽的机制。这些研究将为了解病毒复制的基本过程奠定基础，并为开发抗病毒药物提供靶点，使逆转录病毒不太可能产生耐药变异。

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.