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（由申请人提供）：逆转录病毒（包括许多重要的人类和动物病原体）已成为重要的研究工具作为良性基因递送载体，并且具有作为基因治疗的治疗媒介的潜力。无论是致病性还是治疗性，初始感染和随后的病毒传播取决于逆转录病毒的有效进入宿主细胞。尽管尚未明确定义对病毒进入机制的详细理解，但所有逆转录病毒都具有进入细胞的共同总体策略。逆转录病毒进入的第一步，即病毒表面糖蛋白（SU）和细胞受体之间的相互作用是复杂的，涉及两种蛋白质中多种，无连续的决定因素，这些蛋白质指定受体选择，结合亲和力以及触发病毒性糖蛋白中构象变化的能力。尽管这种相互作用的复杂性，但逆转录病毒具有进化其包膜糖蛋白使用不同细胞蛋白作为受体的结构，通常是一种与原始受体具有明显同源性并保留有效进入功能的蛋白质。逆转录病毒如何做到这一点？了解此能力将为抗病毒干预和靶向基因输送提供有价值的信息。 我们假设：（1）将包膜糖蛋白组织成功能结构域，从而使受体结合亲和力的临界水平和触发糖蛋白构型构象变化的能力保持临界水平，以通过突变和/或重组在受体选择中发生变化； （2）在SU高变量域中的多个非连续受体相互作用决定因素是结合亲和力并限制或扩大受体用法所必需的； （3）SU高变量域外的区域将起作用以连接受体结合以触发糖蛋白锁定机制，并随着病毒的受体使用而被保存。同源逆转录病毒的同源群，亚组A到E（A-E）禽白血病病毒（ALV），通过提供从共同祖先进化的高度相关病毒来利用不同受体的高度相关病毒，为这些假设提供了强大的模型系统。具体来说，我们的目标是：1。基因定义了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域/残基。 2。在遗传上定义了亚组A-E ALV糖蛋白的功能区域/残基，对受体使用的特异性很重要。 3。识别和表征ALV糖蛋白中的区域/残基，对于连接受体结合以触发启动融合过程的构象变化很重要。 4。在遗传上定义了与结合亲和力和触发ALV糖蛋白发生构象变化所必需的ALV受体的功能区域/残基。 5。测试ALV糖蛋白的可溶性形式，以产生适合结构研究的晶体。