AVIAN RETROVIRUS STRUCTURE AND ASSEMBLY

本提案的目的是了解 控制逆转录病毒颗粒在血浆中的组装 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 蛋白质与RNA的相互作用 此外，本发明还提供了一种方法， 蛋白水解裂解发生在病毒的成熟过程中 粒子 使用作为模型系统的禽肉瘤和 白血病病毒，我们将继续调查每一个这些 组装方面。 1. 使病毒蛋白酶缺陷的点突变将 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 这些可能不成熟的病毒颗粒的性质将是 表征了 此外，蛋白酶活性的调节将是 体外研究，从部分纯化的蛋白酶融合蛋白 从E.杆菌 2. 病毒中gag蛋白与脂质相互作用的位点 膜将被研究，使用不同的交联剂。 正常病毒和蛋白酶缺陷型病毒都含有 分析未切割的gag前体蛋白Pr 76。 此外，本发明还 体外制备Pr 76与鸡膜相互作用 将被表征以了解这是否是生物学相关的 模型 将继续研究重构磷脂 去脂的未成熟病毒核心周围 3. 定义的5'的详细缺失和接头扫描突变 和3'区域的RNA将进行，为了学习 一个RNA需要什么样的RNA序列 被识别并有效地包装成病毒颗粒。 的 Pr 76与病毒RNA的相互作用将在体外进行研究， 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 序列Pr 76特异性相互作用，以及 Pr 76参与了这种相互作用。 此外，一系列 将进行实验以了解哪些gag蛋白 与新合成的病毒DNA相互作用。 4. gag基因的最小部分需要形成 将使用一系列嵌套的 缺失突变体 细胞内的gag蛋白 病毒颗粒与表面上的env蛋白相互作用， 使用几种不同的交联剂进行研究。 最后， 将尝试克隆一种鸡的基因， 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。

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.