CELLULAR FACTORS INVOLVED IN NUCLEAR EXPORT OF VIRAL RNA

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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.