CELLULAR FACTORS INVOLVED IN NUCLEAR EXPORT OF VIRAL RNA

描述：(改编自申请者摘要)HIV Rev蛋白 在转录后不同地控制病毒蛋白的表达。雷夫 和它的同源RNA，RRE，可能与细胞成分相结合 参与细胞转运后的基因调控。一位顺位演员， D型逆转录病毒的构成运输元件(CTE)能够 替代REV函数。目前尚不清楚这些路径是否以及何时 参与了REV和CTE介导的RNA运输的融合。澄清： 这些过程将阐明细胞运输的基本方面。 抗病毒机制，并可能对抗病毒策略的发展很重要。 他们之前已经鉴定并部分纯化了两种40kD的蛋白质 65kD，与功能CTE RNA特异性结合。最近，他们 已经确定了一种额外的高分子量蛋白质，它也可以结合 转化为具有功能的CTE RNA。他们进一步纯化了这种蛋白质，并获得了 与RNA解旋酶A(Hel A)完全匹配的部分序列。 然后他们获得了完整的DNA克隆，并发现尽管Hel A是 通常主要定位在核中，它穿梭到 细胞质中存在功能性CTE RNA或同时表达两种REV 和RRE。RNA解旋酶A可以是一种穿梭蛋白是一项新的发现。 该提案的主要目标是分离CTE并对其功能进行表征 结合蛋白及其在CTE-Anad Rev介导中的作用 小路。它们的具体目标是：(1)从功能上描述 RNA解旋酶A在病毒mRNA运输中的作用-他们将映射结合 Hel A和CTE RNA的结构域，并确定是否影响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)确定HELA是否广泛地在 病毒mRNA的转录后调控-我们将确定HEL A是否 物理上与REV和RRE复合体相互作用，如果它对 其他核糖核酸核输出成分(如肝炎病毒前病毒)或其他 逆转录病毒RNA。(Iii)克隆和鉴定40 kD和65 kD的基因 KD CTE结合蛋白-这两种蛋白都已部分纯化。他们 将确定这些蛋白质的部分氨基酸序列以推导出 用于cdna克隆的探针。获得的cDNA与CTE或REV和 将确定RRE功能。(四)确定 REV或CTE介导的反式激活中的特定细胞蛋白，使用 核酶失活-我们将设计条件表达载体 针对CTEBP基因的核酶以及已发表的REV和RRE结合 细胞基因，并确定这些基因是否被核酶失活 将对REV和CTE介导的基因表达产生影响。

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.