The applicant has observed that a single nuclear factor (NF-RRE) of approximately 56 kD binds to the proximal region on the RRE. Furthermore, the presence of both Rev and NF-RRE result in formation of a single complex comprising both proteins and RRE. The major goal of the applicant is to study the structure and function of NF-RRE and its role in Rev-mediated transactivation as well as post-transcriptional gene regulation. In addition, the investigator will attempt to identify and characterize similar nuclear factors that are involved in analogous pathways of other human retroviruses. The first specific aim is to attempt to identify and characterize nuclear factors that bind HIV/RRE and human T cell lymphotropic virus (HTLV)/RxRE sequences. The investigator will try to determine if the same or different nuclear factors recognize these various RNA target sequences. The applicant proposes to fine map the binding sites on RRE and RxRE for Rev and nuclear factor(s) using mutagenesis as well as fingerprinting and footprinting techniques. The second goal of Dr. Wong-Staal is to attempt a partial purification of NF-RRE protein and to prepare monospecific and monoclonal antibodies to it. These reagents will be used in initial functional characterization of the protein and possibly to generate probes for cloning of the NF-RRE gene. Next, the applicant proposes to clone and express the NF-RRE gene. This will be attempted using several alternative strategies to achieve cloning of the gene, depending on whether accessory protein(s) are required for NF-RRE-RRE binding. Once obtained, the gene will be expressed in prokaryotic vectors to obtain large amounts of purified protein and in eukaryotic vectors for functional analyses. The last goal of the investigator is the functional characterization of NF-RRE. Nucleotide sequence determination, immunodepletion and reconstitution studies using in vitro splicing and spliceosome assembly assays and expression of anti-sense or transdominant mutant constructs in mammalian cells will be carried out. These will be done to try to determine the structure and function of NF-RRE and its role in mRNA splicing/transport and Rev-mediated transactivation.
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