POST-TRANSCRIPTIONAL REGULATION OF HIV GENE EXPRESSION

HIV 基因表达的转录后调控

• 批准号: 3307776
• 负责人: Karen L. Beemon
• 金额: $ 20.72万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307776
• 关键词: Rous sarcoma virus; fusion gene; gel mobility shift assay; gene expression; genetic regulatory element; human immunodeficiency virus 1; molecular cloning; posttranscriptional RNA processing; site directed mutagenesis; tissue /cell culture; transfection; ultraviolet radiation; virus RNA

A major focus of this proposal is to elucidate the mechanism by which the large number of alternatively spliced HIV mRNAs are generated. HIV has three size classes of mRNAs, all generated from a single primary RNA transcript: 9kb unspliced mRNA, 4-5kb singly-spliced mRNAs, and 2kb multiply-spliced mRNAs. The hypothesis to be tested is that each of the individual splicing events must be either inefficient or regulated in some way. The efficiency of individual HIV 5' and 3' splice sites will be tested in vivo in combination with known, efficient splice sites. These experiments will be carried out with and without Rev (and the RRE). If some of the HIV splice sites are observed to be used efficiently in the chimeric constructs, we will search for cis-acting sequences that could moderate their splicing efficiency (either in the vicinity of the splice site or at some distance from it). HIV cis-acting elements involved in control of RNA processing will be compared to elements previously studied in RSV, including cis-acting negative regulators of splicing (NRS) within the gag gene and near the src 3' splice site, as well as an inefficient env 3' splice site. A cis-acting sequence has been identified in the env region of HIV that inhibits splicing of a heterologous intron in an orientation-dependent manner. Function of this HIV element will be further compared to that of the RSV gag NRS in in vitro splicing assays. If pre- mRNA splicing is blocked by this element, spliceosome formation will be monitored in native gels and by immune precipitation using antibodies to splicing factors. Factors binding to the sequence will also be identified using UV-crosslinking or gel shift analysis. Mutagenesis studies will be carried out to determine the critical sequences in the element, and its secondary and tertiary structure will be determined. It has been proposed that cis repressive sequences (CRS) play a role in nuclear retention of HIV unspliced and singly-spliced RNA in the absence of Rev, although the mechanism is unknown. The potential role of CRS elements in splicing will be studied both in vivo, using a heterologous intron construct, and in an in vitro splicing system. The HIV env sequence that inhibits splicing will also be tested for CRS activity. Unspliced, RSV NRS-containing heterologous RNA is confined to the nucleus in association with components of the splicing apparatus; we will determine if Rev in trans and the RRE in cis will elicit cytoplasmic appearance of this RNA. Lastly, the effect of premature translation termination within the gag region of HIV on unspliced RNA stability will be probed. Insertion of termination codons within the analogous region of RSV results in a marked decrease in unspliced RNA accumulation, due to decreased RNA stability. Any role of the Rev protein in RNA stability of these mutants will also be evaluated.

该提案的主要重点是阐明 产生大量的剪接HIV mRNA。 艾滋病毒有 三个尺寸类别的mRNA，全部由单个主要RNA产生 成绩单：9KB未上贴mRNA，4-5KB单张mRNA和2KB 倍增的mRNA。 要检验的假设是每个 单个剪接事件必须在某些人中效率低下或调节 方式。 单个HIV 5'和3'剪接站点的效率将是 在体内与已知的，有效的剪接位点结合使用。 这些 实验将在有或没有转速（以及RRE）的情况下进行。 如果 观察到一些HIV剪接位点有效地用于 嵌合构建体，我们将搜索可以的顺式作用序列 调节它们的剪接效率（要么在剪接附近 站点或与之一定的距离）。 HIV顺式作用元素涉及 将RNA处理的控制与先前研究的元素进行比较 在RSV中，包括拼接的负面调节因子（NRS） GAG基因和SRC 3'剪接位点附近，以及效率低下的Env 3'拼接网站。 在Env中已经确定了顺式作用序列 HIV区域抑制了在 方向依赖性方式。 该HIV元素的功能将进一步 与体外剪接测定中的RSV GAG NR相比。 如果预先 mRNA剪接被该元素阻塞，剪接体形成将是 用天然凝胶监测和通过使用抗体的免疫沉淀 剪接因子。 与序列结合的因素也将被鉴定 使用UV-跨链接或凝胶移位分析。 诱变研究将是 进行以确定元素中的临界序列及其 将确定二级和三级结构。 有人提出，顺式抑制序列（CRS）在 在没有的情况下 Rev，尽管该机制未知。 CRS元素的潜在作用 在剪接中，将使用异源内含子研究既有体内 在体外剪接系统中构造。 HIV env序列 抑制剪接也将测试CRS活性。 未填充，RSV 含NRS的异源RNA仅限于核的核 带有剪接设备的组成部分；我们将确定REV是否 trans和顺式中的RRE将引起该RNA的细胞质外观。 最后，插科打术中过早翻译终止的效果 将探测HIV的艾滋病毒区域。 插入 RSV类似区域内的终止密码子导致标记 由于RNA稳定性的降低，未植物的RNA积累降低。 Rev蛋白在这些突变体的RNA稳定性中的任何作用也将是 评估。