Understanding the Mechanism of Retrovirus Budding

了解逆转录病毒出芽机制

• 批准号: 7505729
• 负责人: JONATHAN P LEIS
• 金额: $ 22.65万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-10 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7505729
• 关键词: Antiviral Agents; Avian Sarcoma; Avian Sarcoma Viruses; Binding; Binding Sites; Biochemical; Birds; Cell membrane; Cell surface; Cells; Chromatography; Complex; Condition; Cytolysis; Defect; Development; Drug Delivery Systems; Drug resistance; Enzymes; Foundations; Future; Gagging; HIV-1; Human; Immunologic Deficiency Syndromes; Infection; Mechanics; Murine leukemia virus; Mus; Nature; Pathway interactions; Process; Proline; Proteins; Proteomics; Public Health; Range; Recruitment Activity; Retroviridae; Role; Seminal; Series; Site-Directed Mutagenesis; Small Interfering RNA; Sorting - Cell Movement; Specificity; TSG101 gene; Techniques; Ubiquitin; Ubiquitination; Vacuolar Protein Sorting; Variant; Viral; Virus; Virus-like particle; base; cellular targeting; comparative; gag Gene Products; leukemia; mutant; research study; two-dimensional; ubiquitin-protein ligase

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.

描述（由申请方提供）：逆转录病毒的出芽依赖于其Gag多聚蛋白中编码的小L结构域序列，该序列作为参与内体分选的细胞蛋白（ESCRT-I、-II、-III）的结合位点。这些细胞蛋白为病毒样颗粒从质膜释放提供了机械手段。根据逆转录病毒，单独或组合使用三种不同的L结构域。为了了解这些蛋白质复合物的性质，将在天然条件下从细胞中分离来自鸟类肉瘤（ASV）、人类免疫缺陷病毒I型（HIV-1）和莫洛尼鼠白血病（MuLV）病毒的Gag多聚蛋白，相关细胞蛋白通过二维色谱法分离，并通过质谱技术鉴定以L结构域依赖方式与Gag结合的蛋白。这将定义那些共享或不共享的细胞蛋白质。通过分析衍生自野生型和含Gag的L-结构域缺失但融合至拯救或不拯救出芽缺陷的特异性ESCRT-I、-II、-III蛋白的复合物，将阐明每种病毒进入出芽途径的进入点。ASV和MuLV Gag都使用相关但不同的PPxY L结构域基序，其是E3泛素连接酶的特异性结合位点。这些基序将在ASV和MuLV Gag多聚蛋白之间交换，以证明这改变了出芽过程中使用的E3蛋白的特异性。还将进行相关PY基序之间不同的残基的定点诱变和靶向不同E3蛋白的细胞内的siRNA表型消耗，以确定它们在出芽过程中的机制作用。将检查E3泛素连接酶Nedd 4的C2转运结构域在ASV Gag转运至细胞表面中的作用。最后，将分析一系列已知的泛素突变体对Gag释放的影响。总之，这些实验将提供重要的新信息的机制，出芽，并建议细胞靶点的抗病毒药物的发展，破坏病毒出芽过程。逆转录病毒可能不太可能对这些药物产生耐药性，而不是靶向病毒编码酶的药物。公共卫生相关性：从三种不同的逆转录病毒的细胞出芽的机制将使用生化蛋白质组学和定点突变技术进行比较。这些研究将为理解病毒复制的基本过程奠定基础，并为开发逆转录病毒不太可能产生耐药性变体的抗病毒剂提供靶点。