ASSEMBLY OF MURINE LEUKEMIA VIRUSES

鼠白血病病毒的组装

• 批准号: 3175241
• 负责人: Ronald B Luftig
• 金额: $ 12.9万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1990-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3175241
• 关键词: Retroviridae disease; affinity chromatography; autoradiography; cytoskeleton; density gradient ultracentrifugation; electron microscopy; embryo /fetus tissue /cell culture; erythroleukemia; gel electrophoresis; immunofluorescence technique; ion exchange chromatography; membrane activity; monoclonal antibody; murine leukemia virus; nucleocapsid; oncogenic virus; paper chromatography; phosphorylation; protease inhibitor; proteolysis; radiotracer; viral leukemogenesis; virus RNA; virus genetics; virus replication; viruslike particle

The morphogenesis of murine retroviruses involves an initial interaction between two 35s viral RNA genomes and about 2000 Pr65gag polyprotein precursor molecules to form a nucleoprotein complex and the subsequent association of the Pr65gag/RNA complex at the cell surface with lipids and additional envelope proteins to form a nascent, immature particle. Specific proteolytic cleavages of Pr65gag and envelope proteins during and/or after budding by specific proteases then leads to maturation of the virion. We intend to analyze three aspects of this assembly problem: i) The mechanism by which the about 2000 Pr65gag precursor molecules are brought to and then inserted into the plasma membrane. The recent discovery that the NH2 terminus of Pr65gag is myristylated has led us to look at when this modification occurs, e.g., on polysomes? on cytoskeletons? on membranes? Also, what happens to virus production when cerulenin, an inhibitor of fatty acid synthesis, is used? Further, since p15, the NH2 terminal protein on Pr65gag has a stretch of 20 hydrophobic amino acids at its NH2 terminus, we want to know what, if any, is more important for insertion of Pr65gag into the membrane, viz., the myristic acid modification or the presence of about 20 hydrophobic amino acids? Appropriate mutations and deletions in cloned p15 MuLV DNA (corresponding to the NH2 terminus) will be constructed and the DNA will then be used to answer these questions via transfection or microinjection experiments with appropriate host cells. ii) The manner in which the Pr65gag molecules are associated with the cytoskeleton, e.g., is it necessary to be both myristylated and under-phosphorylated? Also, how does this association lead to an alteration of the actin component of the cytoskeleton? iii) The molecular basis by which the "immature", Pr65gag enriched C-type particles are converted by proteolytic cleavage to "mature", C-type virus particles. This involves a further study of the murine eetrovirus specific protease activity. Toward this end there now appears good evidence that the protease is virus-coded. It is located on the genome after gag and before pol. We are currently attempting to clone the putative protease gene and isolate the gene product in sufficient quantities, so that the above reaction and concomitant maturation can be characterized in molecular detail.

鼠逆转录病毒的形态发生涉及初始相互作用 在两个35S病毒RNA基因组和大约2000 pr65gag多蛋白之间 前体分子形成核蛋白复合物，随后 PR65GAG/RNA复合物在细胞表面与脂质和 附加的包膜蛋白形成一个新生的，不成熟的颗粒。 PR65GAG和包膜蛋白的特定蛋白水解切割 和/或在特定蛋白酶发芽之后会导致成熟 病毒。 我们打算分析此组装问题的三个方面： i）大约2000 pr65gag前体分子的机制是 带到然后插入质膜。 最近 发现PR65GAG的NH2末端是肉豆蔻酰化已导致我们进入 查看这种修饰何时发生，例如在多聚体上？ 在 细胞骨架？ 在膜上？ 另外，当病毒生产发生了什么时候 使用脂肪酸合成的抑制剂Cerulenin？ 此外，从那以后 p15，PR65GAG上的NH2末端蛋白具有20个疏水性 在其NH2末端的氨基酸，我们想知道什么（如果有）更多 对于将PR65GAG插入到膜中，即Myristic很重要 酸性改性还是存在约20种疏水氨基酸？ 克隆的P15 MULV DNA中的适当突变和缺失（相应 将构建到NH2末端），然后将DNA用于 通过转染或微注射实验回答这些问题 适当的宿主细胞。 ii）PR65GAG分子与 细胞骨架，例如，有必要同时被肉豆蔻酰化和 磷酸化不足？ 另外，这种关联如何导致 细胞骨架的肌动蛋白成分的改变？ iii）“未成熟”，PR65GAG富含C型的分子基础 颗粒通过蛋白水解裂解转化为“成熟”，C型病毒 颗粒。 这涉及对鼠Eetrovirus特异性病毒的进一步研究 蛋白酶活性。 为此，现在似乎有充分的证据表明 蛋白酶是病毒编码的。 它位于堵嘴后的基因组上 pol之前。 我们目前正在尝试克隆推定的蛋白酶 基因并分离足够量的基因产物，以便 以上反应和伴随的成熟可以在分子中表征 细节。