The essential viral protein Rev was the first characterized factor to direct the export of mRNA from the nucleus to the cytoplasm. It has been demonstrated that Rev is a valid target for antiviral strategies. The multimerization of Rev protein is crucial for the life cycle of HIV-1. The development and study of Rev-independent HIV and SIV molecular clones has shown that even partial inhibition of Rev function can severely diminish the pathogenic potential of these viruses. Therefore, a drug that inhibits Rev function would be valuable for AIDS therapy as a new class of compounds. In addition, the study of Rev-independent SIV virus clones in macaques is a valuable tool to understand mechanisms of protection from disease development, since the Rev-independent clones remain non-pathogenic after more than 7 years of infection. We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have analyzed in detail the structure and function of the RTE element, and we have identified the cellular factor responsible for binding to RTE and linking it to the NXF1 export pathway. This protein, the RNA binding motif protein 15 (RBM15), had no previous assigned function. RBM15 recognizes RTE directly and specifically in vitro, and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. We also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. We propose that RTE evolved as a high-affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts. Consistent with these data, the group of Barbara Felber has now shown that the RTE site within full-length, active retrotransposons is essential for retrotransposition. Our results contribute significantly to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules. The understanding of the regulatory mechanisms of HIV gene expression has many important practical applications. We have applied this knowledge to the development of improved DNA vaccination approaches. We also showed that the combination of CTE and RTE elements in the same mRNA can be used to increase gene expression by more than one order of magnitude. We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can produce bioactive cytokine after DNA delivery in mice and macaques. We have constructed new retroviral vectors that efficiently terminate transcription/translation when inserted in gene regions, and have constructed ordered libraries of mouse embryonic stem cells that can be used to produce mice having any desired knock out. This technology streamlines the production and study of KO mice and can assist in the rapid understanding of gene function. The method was validated by developing mouse KOs for 57 GPCR genes and several nuclear receptor genes, most of which did not exist in the literature.
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