THE ROLE OF INTRACELLULAR TRAFFIC IN HIV INFECTION

细胞内运输在 HIV 感染中的作用

• 批准号: 3839619
• 负责人: J A HANOVER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3839619
• 关键词: Xenopus oocyte; antisense nucleic acid; chimeric proteins; gene expression; glucocorticoids; glycoproteins; human immunodeficiency virus; intracellular transport; membrane structure; molecular cloning; nuclear membrane; receptor expression; regulatory gene; reporter genes; transfection /expression vector; virus RNA; virus protein; virus replication

Transport across the nuclear membrane is necessary at several steps in the life cycle of HIV. Once in the cytoplasm, the viral RNA is converted to double stranded DNA which must enter the nucleus. Viral regulatory proteins enter the nucleus while viral transcripts are exported into the cytoplasm. The viral REV protein has been identified as a key regulator of transport of HIV envelope mRNA. Several approaches have been taken to understand how REV may function. Cell lines are being prepared which express a REV/glucocorticoid receptor chimera which enters the nucleus in an inducible fashion; these cell lines also contain a construct containing a REV response element controlling expression of a chloramphenicolacetyltransferase (CAT) gene. This will allow CAT expression to be conveniently followed allowing a direct measure of export of CAT mRNA into the cytoplasm. A means has also been devised for generating nuclei in vitro around exogenously added DNA. The method uses extracts from Xenopus laevis eggs. The nuclei assembled in such extracts mimic interphase nuclei in many ways and carry out active nuclear transport. These preparations allow examination of the mechanism of REV action and the movement of other molecules involved in the HIV life cycle. In other studies, the structure of the nuclear pore has been examined. The nuclear pore requires glycoprotein components for proper morphology and function. Previous studies indicate the overexpression of nuclear pore glycoproteins may be toxic to cells. To circumvent these problems, the cDNA encoding the major nuclear glycoprotein p62 has been placed under the control of a glucocorticoid responsive expression vector. By expressing the sense and antisense constructs, it should be possible to study the effects of suppression or overexpression of this nuclear pore component. A number of the components of the pore complex have now been molecularly cloned. Understanding how retroviral products cross the nuclear envelope is critical to attempts to regulate or inhibit the critical steps in the HIV life cycle.

跨核膜转运在细胞核转运过程中的几个步骤中是必需的 艾滋病毒的生命周期。 一旦进入细胞质，病毒RNA就会转化为 双链DNA必须进入细胞核。 病毒监管 蛋白质进入细胞核，而病毒转录物则输出到细胞核 细胞质。 病毒 REV 蛋白已被确定为关键调节因子 HIV 包膜 mRNA 的运输。 已采取多种方法 了解 REV 如何发挥作用。 正在制备细胞系 表达 REV/糖皮质激素受体嵌合体，进入细胞核 诱导时尚；这些细胞系还含有一个含有 REV 响应元件控制 a 的表达 氯霉素乙酰转移酶（CAT）基因。 这将允许 CAT 方便地遵循表达式，允许直接测量出口 CAT mRNA 进入细胞质。 还设计了一种方法 在体外围绕外源添加的 DNA 生成细胞核。 该方法使用 从非洲爪蟾卵中提取。 在此类提取物中组装的细胞核 以多种方式模拟间期核并进行活性核 运输。 这些制剂可以检查 REV 的机制 HIV生命周期中涉及的其他分子的作用和运动。 在其他研究中，已经检查了核孔的结构。 这 核孔需要糖蛋白成分才能保持正确的形态 功能。 先前的研究表明核孔的过度表达 糖蛋白可能对细胞有毒。 为了规避这些问题， 编码主要核糖蛋白p62的cDNA已被置于 糖皮质激素反应性表达载体的控制。 通过表达 有义和反义结构，应该可以研究 这种核孔成分的抑制或过度表达的影响。 孔隙复合体的许多成分现已被分子化 克隆的。 了解逆转录病毒产物如何穿过核膜 对于尝试调节或抑制关键步骤至关重要 艾滋病毒生命周期。