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AVIAN RETROVIRUS STRUCTURE AND ASSEMBLY

禽逆转录病毒结构和组装

基本信息

批准号：
3481842
负责人：
Volker M. Vogt
金额：
$ 17.12万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-05-01 至 1993-07-31
项目状态：
已结题

项目摘要

The objectives of this proposal are to learn about the principles that govern the assembly of retrovirus particles at the plasma membrane of the infected cell. The formation of virus particles requires protein-membrane interactions, protein-protein interactions, and protein-RNA interactions. In addition, proteolytic cleavages take place in the maturation of virus particles. Using as a model system the avian sarcoma and leukemia viruses, we will continue to investigate each of these aspects of assembly. 1. Point mutations that render the viral protease defective will be introduced into the viral genome, and quail cell lines will be derived that produce the defective particles. The biochemical properties of these presumably immature virus particles will be characterized. Also, the regulation of protease activity will be studied in vitro, from partially purified protease fusion proteins obtained from an expression vector in E. coli. 2. The site of interaction of gag protein with lipids in the viral membrane will be studied, using different cross-linking agents. Both normal virus and protease-defective virus containing the uncleaved gag precursor protein Pr76 will be analyzed. Further, the interaction of Pr76 made in vitro with chicken membranes will be characterized to learn if this is a biologically relevant model. Studies will be continued to reconstitute phospholipids around delipidated immature virus cores. 3. Detailed deletion and linker scanning mutagenesis of defined 5' and 3' regions on the RNA will be carried out, in order to learn exactly what RNA sequences are required for an RNA to be recognized and efficiently packaged into virus particles. The interaction of Pr76 with viral RNA will be studied in vitro, and in vivo using protease-defective particles and cross-linking techniques. The intent of these experiments is to learn what RNA sequences Pr76 interacts specifically with, and what domains on Pr76 are involved in this interaction. Also, a series of experiments will be carried out to learn what gag proteins interact with newly synthesized viral DNA. 4. The minimum portion of the gag gene needed for formation of a virus particle will be determined using a series of nested deletion mutants. The mechanism by which gag protein inside the virus particle interacts with env protein on the surface will be investigated using several different crosslinking agents. Finally, an attempt will be made to clone a chicken gene that when introduced into mammalian cells, allows these normally non- permissive cells to assemble avian retrovirus particles.

该提案的目标是了解原则控制血浆中逆转录病毒颗粒的组装被感染细胞的膜。病毒颗粒的形成需要蛋白质-膜相互作用、蛋白质-蛋白质相互作用，以及蛋白质-RNA 相互作用。此外，病毒成熟过程中发生蛋白水解裂解颗粒。使用禽肉瘤作为模型系统白血病病毒，我们将继续研究其中每一个装配方面。 1. 导致病毒蛋白酶缺陷的点突变被引入病毒基因组，鹌鹑细胞系将被产生有缺陷的颗粒。生化这些可能未成熟的病毒颗粒的特性将是特点。此外，蛋白酶活性的调节将体外研究，来自部分纯化的蛋白酶融合蛋白从大肠杆菌中的表达载体获得。 2. 病毒中gag蛋白与脂质相互作用的位点将使用不同的交联剂来研究膜。正常病毒和蛋白酶缺陷病毒均含有将分析未切割的 gag 前体蛋白 Pr76。更远，体外制备的 Pr76 与鸡膜的相互作用将被表征以了解这是否具有生物学相关性模型。将继续研究重构磷脂围绕脱脂的未成熟病毒核心。 3. 定义的5'的详细删除和连接子扫描诱变和 RNA 上的 3' 区域将进行，以便学习准确地说，RNA 需要哪些 RNA 序列识别并有效包装成病毒颗粒。这 Pr76 与病毒 RNA 的相互作用将在体外进行研究，并在体内使用蛋白酶缺陷颗粒和交联技术。这些实验的目的是了解RNA是什么序列 Pr76 专门与哪些域相互作用？ Pr76 参与了这种相互作用。另外，还有一系列将进行实验来了解什么是gag蛋白与新合成的病毒 DNA 相互作用。 4. 形成所需的gag基因的最小部分将使用一系列嵌套来确定病毒颗粒缺失突变体。内含gag蛋白的机制病毒颗粒与表面的env蛋白相互作用使用几种不同的交联剂进行了研究。最后，将尝试克隆鸡基因，当引入哺乳动物细胞，使这些通常非允许细胞组装禽逆转录病毒颗粒。