CELLULAR FACTORS INVOLVED IN NUCLEAR EXPORT OF VIRAL RNA

描述：（改编自申请人的摘要）HIV Rev蛋白 不同地控制病毒蛋白质的转录后表达。 Rev 及其同源RNA，RRE，可能与细胞成分， 参与细胞的后转录基因调控。 顺式作用， 来自D型逆转录病毒的组成型转运元件（CTE）能够 替代REV功能。 目前尚不清楚这些途径是否以及何时 参与REv-和CTE-介导的RNA转运的聚合。 阐明 这些过程将阐明细胞运输的基本方面 机制，并可能是重要的抗病毒策略的发展。 他们先前已经鉴定并部分纯化了两种40 Kd的蛋白质 和65 Kd，其特异性结合功能CTE RNA。 最近，他们 已经鉴定出一种额外的高分子量蛋白质， 到功能性CTE RNA。 他们进一步纯化了这种蛋白质， 部分序列与RNA解旋酶A（Hel A）完全匹配。 然后他们获得了完整的DNA克隆，发现尽管Hel A是 通常主要位于细胞核中，它穿梭于 细胞质中存在功能性CTE RNA或表达两种REv 的RRE。 RNA解旋酶A可以作为穿梭蛋白是一个新的发现。 提案的主要目标是分离CTE并对其进行功能表征 结合蛋白，并确定它们在CTE和REv介导的 途径。 其具体目标是：（一）在功能上确定 RNA解旋酶A在病毒mRNA转运中的作用-他们将绘制结合 Hel A和CTE RNA的结构域，并确定影响CTEBP的突变 结合也会影响其解旋酶活性或穿梭能力 对CTE的回应 (ii)为了确定Hel A是否在细胞中广泛发挥作用， 病毒mRNA的转录后调节-我们将确定HEL A是否 与Rev和RRE复合物发生物理相互作用，如果它对 其他RNA核输出元件（例如肝炎病毒的PRE）或其他 逆转录病毒RNA。 (iii)为了克隆和表征40 Kd和65 Kd的基因， Kd CTE结合蛋白-两种蛋白质都已部分纯化。 他们 将确定这些蛋白质的部分氨基酸序列， cDNA克隆探针。 衍生的cDNA与CTE或Rev的相关性， 将确定RRE功能。 (iv)确定的相关性 REV或CTE介导的反式激活中的特异性细胞蛋白， 核酶失活-我们将设计条件表达载体， 靶向CTEBP基因以及已发表的Rev和RRE结合的核酶 细胞基因，并确定是否这些基因的核糖酶失活 将影响Rev和CTE介导的基因表达。

DESCRIPTION: (Adapted from Applicant's Abstract) The HIV Rev protein differtially controls viral protein expression post-transcriptionally. Rev and its cognate RNA, RRE, likely interface with cellular components that are involved in cellular post-transciptional gene regulation. A cis-acting, constitutive transport element (CTE) from type-D retroviruses is able to substitute for REV function. It is not clear if and when the pathways involved in REv- and CTE-mediated RNA transport converge. Elucidation of these processes will shed light on fundamental aspects of cellular transport mechanisms and may be important for development of antiviral strategies. They have previously identified and partially purified two proteins of 40 Kd and 65 Kd that specifically bind to functional CTE RNA. More recently, they have identified an additional, high molecular weight protein that also binds to functional CTE RNA. They have further purified this protein and obtained a partial sequence, which completely matched that of RNA Helicase A (Hel A). They then obtained the complete DNA clone, and found that although Hel A is normally localized predominantly in the nucleus, it shuttles to the cytoplasm in the presence of functional CTE RNA or expression of both REv and RRE. That RNA Helicase A can be a shuttle protein is a novel discovery. The major goal of proposal is to isolate and functionally characterize CTE binding proteins and to define their role in CTE-anad REv- mediated pathways. Their specific aims are: (i) To functionally characterize the role of RNA Helicase A in viral mRNA transport - They will map the binding domains of Hel A and CTE RNA, and determine if mutations that affect CTEBP binding will also affect its helicase activity or its ability to shuttle in response to CTE. (ii) To determine if Hel A functions broadly in the post-transcriptional regulation of viral mRNA - We will determine if HEL A physically interacts with the Rev and RRE complex, and if it responds to other RNA nuclear export elements (e.g. the PRE of hepatitis virus) or other retroviral RNA. (iii) To clone and characterize genes for the 40 Kd and 65 Kd CTE binding proteins - Both proteins have been partially purified. They will determine partial amino acid sequences of these proteins to derive probe for cDNA cloning. The relevance of the derived cDNA to CTE or Rev and RRE function will be determined. (iv) To determine the relevance of specific cellular proteins in REV- or CTE-mediated transactivation, using ribozyme inactivation- We will design conditional expresssion vectors for ribozymes targeting the CTEBP genes as well as published Rev and RRE binding cellular genes, and determine if inactivation of these genes by ribozmes will impact on both Rev- and CTE-mediated gene expression.