TAR RNA SPLICING IN SIV

恒河猴免疫缺陷病毒(SIVmac)是一种 HIV-1的遗传和生物学亲缘关系。这种关系使SIV 猴子感染是最重要的动物模型之一 研究人类艾滋病。本文中概述的实验的总体目标 建议理解TAR RNA剪接在 SIVmac的生命周期。这些实验是长期努力的一部分 定义和评估病毒的不同方面的贡献 基因调控对艾滋病病毒的病理生物学有一定作用。 三条证据表明，焦油剪接是一个关键 SIV生命周期中的监管事件。第一，病毒的比例 含有剪接焦油的mRNAs在整个过程中减少 感染。第二，含有可防止TAR的突变的病毒 剪接显示病毒产生的动力学显著延迟。 第三，含有剪接的TAR前导序列的mRNAs 比含有未剪接TAR的类似mRNAs有效翻译 领袖。具体目标1是确定TAR剪接在 SIVmac生命周期。含有突变的病毒，可抑制或 增强TAR剪接将在细胞中同时引入 野生型病毒病毒复制和基因的水平和模式 表达将通过检查病毒粒子的产生和 病毒RNA和蛋白的表达。此外， TAR剪接时细胞的生理状态将在 细胞因子和佛波酯处理的细胞。具体目标2是确定 拼接和拼接在翻译能力上的差异 未剪接的含焦油的mRNAs由顺式和/或反式作用介导 机制。将进行进一步的研究，以确定焦油 剪接也会影响其他水平的病毒基因表达，包括 TAT介导的转录、mRNA稳定性和核输出。特定的 目标3是确定剪接的TAR RNA的水平是否受到调控 通过病毒或细胞基因产物。含有焦油的报告基因 内含子和受调控基因所需的所有顺式作用序列 累积的剪接的mRNAs将被共同导入细胞 以及表达各种SIVmac蛋白的质粒和 我们将比较剪接和未剪接的mRNAs。

Simian immunodeficiency virus from rhesus macaques (SIVmac) is a close genetic and biological relative of HIV-1. This relationship makes SIV infection of monkeys one of the most significant animal models for the study of human AIDS. The broad goal of the experiments outlined in this proposal is to understand the role that TAR RNA splicing plays in the life cycle of SIVmac. These experiments are part of a long term effort to define and evaluate the contribution that different aspects of viral gene regulation make to the pathobiology of the AIDS viruses. Three lines of evidence indicate that TAR splicing is a pivotal regulatory event in the SIV life cycle. First, the proportion of viral MRNAS containing a spliced TAR decreases throughout the course of infection. Second, viruses containing mutations which prevent TAR splicing display significantly delayed kinetics of virus production. Third, MRNAS containing a spliced TAR leader sequence are more efficiently translated than similar MRNAS containing an unspliced TAR leader. Specific Aim 1 is to determine the role of TAR splicing in the SIVmac life cycle. Viruses containing mutations that either inhibit or enhance TAR splicing will be introduced into cells in parallel with a wild-type virus. The levels and patterns of virus replication and gene expression will be monitored by examining virion production and the expression of viral RNA and protein. In addition, the influence of the physiological state of the cell on TAR splicing will be examined in cytokine and phorbol ester treated cells. Specific Aim 2 is to determine whether the differences in translational competence between spliced and unspliced TAR-containing MRNAS is mediated by a cis and/or trans acting mechanism. Further studies will be conducted to determine whether TAR splicing also effects other levels of virus gene expression, including tat-mediated transcription, MRNA stability and nuclear export. Specific Aim 3 is to determine whether the level of spliced TAR RNA is regulated by viral or cellular gene products. A reporter gene containing the TAR intron and all of the cis-acting sequences necessary for the regulated accumulation of the spliced MRNAS will be co-transfected into cells along with plasmids expressing various SIVmac proteins and the levels of spliced and unspliced MRNAS will be compared.