HIGH RESOLUTION FUNCTIONAL MAPPING OF RETROVIRAL GENES

逆转录病毒基因的高分辨率功能图谱

• 批准号: 2885499
• 负责人: ILA R SINGH
• 金额: $ 9.09万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2885499
• 关键词: DNA footprinting; Retroviridae; gag protein; gene mutation; genetic library; polymerase chain reaction; tissue /cell culture; virus DNA; virus genetics; virus infection mechanism

Acquired Immunodeficiency Syndrome (AIDS) and cancer are major world health problems today. Despite recent advances in anti- retroviral therapy, it is clear that current measures are insufficient to eradicate AIDS. In order to develop better anti- retroviral strategies, a more complete understanding of the biology of retroviruses is essential. This proposal outlines a novel approach designed to understand the process of retroviral replication and to elucidate the roles played by the different viral proteins in the various steps of infection. A detailed knowledge of these processes is also important for understanding basic biological processes such as cellular growth control, and for advances in biotechnology such as in creating better retroviral vectors for gene therapy. Aim 1 consists of creating several comprehensive libraries of mutants in a retroviral sequence using the new technique of genetic footprinting. Genetic footprinting is an efficient method for construction and parallel analysis of a very large set of mutations in a sequence. Aim 2 consists of identifying regions of the retroviral genome that are essential for viral replication. Since genetic footprinting allows the identification of functionally essential regions in a gene without isolating or sequencing individual mutants, it will be applied to retroviral sequences, starting with gag, the region coding for structural proteins that form the viral capsid. This will define all regions in gag that are essential for viral replication. Aim 3 consists of identifying regions in the retroviral genome that are essential for individual steps of viral entry and replication. Aim 4 consists of performing a detailed analysis of some of the mutants identified in the above screens using standard biochemical and cell-biological methods of studying viral entry and replication. This will help identify the specific roles of different regions of a gene in greater detail. The experiments are designed to provide extensive data on the process of retroviral replication and infection.

获得性免疫缺陷综合症（艾滋病）和癌症是当今世界的主要健康问题。尽管最近在抗逆转录病毒疗法方面取得了进展，但显然目前的措施不足以根除艾滋病。为了开发更好的抗逆转录病毒策略，对逆转录病毒生物学的更完整的理解是必不可少的。该提案概述了一种新的方法，旨在了解逆转录病毒复制的过程，并阐明不同的病毒蛋白在感染的各个步骤中所发挥的作用。对这些过程的详细了解对于理解基本的生物学过程（如细胞生长控制）和生物技术的进步（如创造更好的逆转录病毒载体用于基因治疗）也很重要。目标1包括使用新的遗传足迹技术在逆转录病毒序列中创建几个综合的突变体库。遗传足迹法是一种构建和并行分析序列中大量突变的有效方法。目标2包括确定逆转录病毒基因组中对病毒复制至关重要的区域。由于遗传足迹法可以在不分离或测序单个突变体的情况下鉴定基因中的功能性必需区域，因此它将被应用于逆转录病毒序列，从gag开始，该区域编码形成病毒衣壳的结构蛋白。这将定义gag中对病毒复制至关重要的所有区域。目标3包括确定逆转录病毒基因组中对病毒进入和复制的各个步骤至关重要的区域。目的4包括使用研究病毒进入和复制的标准生物化学和细胞生物学方法对上述筛选中鉴定的一些突变体进行详细分析。这将有助于更详细地确定基因不同区域的具体作用。这些实验旨在提供有关逆转录病毒复制和感染过程的广泛数据。