Retrovirus Entry and Virus Evolution

逆转录病毒进入和病毒进化

• 批准号: 6575465
• 负责人: Mark J Federspiel
• 金额: $ 8.52万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6575465
• 关键词: Retroviridae; X ray crystallography; avian leukosis virus; conformation; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene targeting; glycoproteins; green fluorescent proteins; polymerase chain reaction; receptor binding; site directed mutagenesis; structural biology; tissue /cell culture; virus envelope; virus infection mechanism; western blottings

DESCRIPTION (provided by applicant): Retroviruses, which include many important pathogens of humans and animals, have become important research tools as benign gene delivery vectors, and have potential as therapeutic vectors for gene therapy. Whether pathogenic or therapeutic, the initial infection and subsequent dissemination of the virus depends on efficient entry of the retrovirus into host cells. While a detailed understanding of the mechanisms of viral entry has not been clearly defined for any retrovirus, all retroviruses share a common overall strategy for entry into cells. The initial step of retrovirus entry, the interaction between the viral surface glycoprotein (SU) and a cellular receptor, is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as a receptor, often a protein that has no obvious homology to the original receptor, and retain efficient entry functions. How do retroviruses do this? Understanding this ability will provide valuable information for antiviral intervention and targeting gene delivery. We hypothesize that: (1) the envelope glycoproteins are organized into functional domains that allow changes to occur in receptor choice by mutation and/or recombination while maintaining a critical level of both receptor binding affinity and the ability to trigger glycoprotein conformational changes to initiate the fusion process; (2) multiple, noncontiguous receptor interaction determinants located in the SU hypervariable domains are required for binding affinity and to restrict or broaden receptor usage; (3) regions outside of the SU hypervariable domains will function to connect receptor binding to triggering the glycoprotein lock mechanism and will be conserved as the virus changes receptor usage. The homologous group of retroviruses, the subgroups A through E (A-E) avian leukosis viruses (ALV), provide a powerful model system to test these hypotheses by supplying highly related viruses that have evolved from a common ancestor to utilize different receptors. Specifically, we aim to:1. Genetically define functional regions/residues of the subgroup A-E ALV envelope glycoproteins important for receptor binding affinity. 2. Genetically define functional regions/residues of the subgroup A-E ALV glycoproteins important for the specificity of receptor usage. 3. Identify and characterize regions/residues in the ALV glycoproteins important for connecting receptor binding to triggering the conformational changes that initiate the fusion process. 4. Genetically define functional regions/residues of the ALV receptors necessary for binding affinity and triggering a conformational change in the ALV glycoproteins. 5. Test soluble forms of the ALV glycoproteins for the ability to produce crystals suitable for structural studies.

描述（申请人提供）：逆转录病毒包括人类和动物的许多重要病原体，作为良性基因递送载体已成为重要的研究工具，并具有作为基因治疗的治疗载体的潜力。无论是致病性的还是治疗性的，病毒的初始感染和随后的传播取决于逆转录病毒有效进入宿主细胞。虽然对任何逆转录病毒的病毒进入机制的详细理解尚未明确定义，但所有逆转录病毒都有一个共同的进入细胞的总体策略。逆转录病毒进入的初始步骤，病毒表面糖蛋白（SU）和细胞受体之间的相互作用是复杂的，涉及两种蛋白质中指定受体选择、结合亲和力和触发病毒糖蛋白构象变化的能力的多个非连续决定簇。尽管这种相互作用很复杂，但逆转录病毒有能力进化其包膜糖蛋白的结构，以使用不同的细胞蛋白作为受体，通常是与原始受体没有明显同源性的蛋白质，并保留有效的进入功能。逆转录病毒是如何做到这一点的？了解这种能力将为抗病毒干预和靶向基因递送提供有价值的信息。 我们假设：（1）包膜糖蛋白被组织成功能结构域，其允许通过突变和/或重组在受体选择中发生变化，同时保持受体结合亲和力和触发糖蛋白构象变化以启动融合过程的能力的临界水平;（2）多个，位于SU高变结构域的非连续受体相互作用决定簇是结合亲和力和限制或拓宽受体用途所必需的;（3）SU高变结构域之外的区域将起到连接受体结合以触发糖蛋白锁定机制的作用，并且将随着病毒改变受体用途而保守。逆转录病毒的同源组，亚组A至E（A-E）禽白血病病毒（ALV），提供了一个强大的模型系统，以测试这些假设，通过提供高度相关的病毒，已经从一个共同的祖先进化到利用不同的受体。具体而言，我们的目标是：1。从遗传学上确定对受体结合亲和力重要的亚组A-E ALV包膜糖蛋白的功能区/残基。2.从遗传学上确定对受体使用特异性重要的亚组A-E ALV糖蛋白的功能区/残基。3.识别和表征ALV糖蛋白中对于连接受体结合与触发启动融合过程的构象变化至关重要的区域/残基。4.从遗传学上定义ALV受体的功能区域/残基，这些区域/残基是结合亲和力和触发ALV糖蛋白构象变化所必需的。5.测试ALV糖蛋白的可溶形式产生适合结构研究的晶体的能力。