AVIAN RETROVIRUS STRUCTURE AND ASSEMBLY

这项建议的目的是了解这些原则 控制逆转录病毒颗粒在血浆中的组装 被感染细胞的细胞膜。病毒颗粒的形成 需要蛋白质-膜相互作用，蛋白质-蛋白质 相互作用和蛋白质-RNA相互作用。此外, 蛋白水解性裂解发生在病毒的成熟过程中 粒子。以禽肉瘤为模型系统 白血病病毒，我们将继续调查其中的每一个 装配的各个方面。 1.导致病毒蛋白水解酶缺陷的点突变 被引入到病毒基因组中，鹌鹑细胞系将被 产生缺陷粒子的衍生品。生物化学 这些未成熟的病毒颗粒的性质可能是 特色化的。此外，对蛋白酶活性的调节将是 从部分纯化的蛋白酶融合蛋白中进行体外研究 从大肠杆菌的表达载体中获得。 2.病毒中Gag蛋白与脂质相互作用的部位 将使用不同的交联剂对膜进行研究。 正常病毒和蛋白酶缺陷型病毒都含有 未裂解的GAG前体蛋白Pr76将被分析。此外， 体外制备的Pr76与鸡膜的相互作用 将被表征以了解这是否与生物学上相关 模特。重建磷脂的研究将继续进行 围绕着脱脂的未成熟病毒核心。 3.确定的5‘端的详细缺失和连接子扫描突变 并对RNA上的3‘区域进行分析，以便学习 一个RNA到底需要什么样的RNA序列 被识别并有效地包装成病毒颗粒。这个 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剂 技巧。这些实验的目的是了解什么是RNA 序列Pr76特定地与哪些结构域相互作用 Pr76参与了这种相互作用。此外，还有一系列 将进行实验以了解什么是Gag蛋白质 与新合成的病毒DNA相互作用。 4.GAG基因形成所需的最小部分 病毒粒子将使用一系列嵌套的 缺失突变体。GAG蛋白在细胞内的作用机制 病毒颗粒与表面的包膜蛋白相互作用 使用几种不同的交联剂进行了研究。最后， 将尝试克隆一种鸡的基因，当 被引入哺乳动物细胞，使这些正常的非 允许细胞组装禽类逆转录病毒颗粒。

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.