Identification and characterization of Risp
The aim of this study was to characterize the role of Rev-interacting cellular proteins in\ncontrolling the function of Rev in the host cell. The HIV-1 protein Rev plays an essential role in\nthe temporal regulation of the virus gene expression by stimulating the expression of viral\nstructural proteins. Rev enhances the nucleocytoplasmic transport and the translation of unspliced\nand single spliced viral mRNAs by binding with high affinity to a specific target element on the\nHIV-RNA. It was assumed that interaction with cellular factors is essential for Rev function. At\nthe onset of this study, only a few potential cofactors were known with no clearly defined\nfunctional relevance. So we decided to search for new Rev-interacting factors using the yeast\ntwo-hybrid system.\nIn this work a new Rev-interacting protein has been identified, by screening a Jurkat T cell cDNA\nlibrary. The protein was termed Risp (Rev-interacting shuttle protein), because it shuttles\nbetween the nuclear and the cytoplasmic compartments.\nThe Risp gene is widely expressed in human cells and conserved among various species, most\nprobably as part of a larger gene. High amino acid homology (99%) with the C-terminal part of a\nlarge brain cDNA clone for KIAA0592 protein has been found, whereas no obvious homology to\nproteins with known function was observed. However, a weak and partial similarity appeared\nwith several RNA-/DNA-binding and shuttle proteins. This might indicate that the Risp protein -\nor the larger protein containing it - could be a member of a new family of nucleocytoplasmic\nshuttle proteins with RNA-/DNA-binding function.\nNext, the intracellular localization and shuttling of Risp was investigated. In HeLa cells Risp-\nGFP localized in both nuclear and cytoplasmic compartments, but clearly accumulated in the\ncytoplasm, indicating the presence of a strong nuclear export signal (NES). The identification of\na NES sequence was confirmed by deletion analysis of Risp and by nuclear microinjection of\nBSA-fusion proteins conjugated to peptides from the C-terminal part of Risp.\nTreatment with leptomycin B, a drug which has been shown to specifically block Crm1\n(exportin) mediated export, resulted in nuclear accumulation of Risp-GFP, showing that the\nnuclear export of Risp, like that of Rev, is Crm1-dependent.\nUsing bioinformatic tools able to detect weak homologies with high specificity, sequence\ncomparisons between Risp and all currently known Rev interacting factors were performed. This\nanalysis for the first time revealed a common motif shared between Rev and Rev-interacting\ncellular factors, termed RIP. The region of Risp harboring the RIP motif was neither essential nor\nsufficient for the Rev-binding in the yeast two hybrid system, suggesting no direct correlation\nbetween RIP and the Rev-binding ability.\nPreliminary experiments suggested, that Risp, as a Rev-interacting protein, is able to inhibit Revtrans-\nactivation, while Risp does not interfere with Tat in a Tat-trans-activation assay. The overexpression\nof Risp-GFP reduced the production of the Rev-dependent structural viral protein\np24gag up to 70%.\nIn addition a previously unrecognized sequence motif in the activation domain of Rev with\nintrinsic nuclear import activity was found and tested in transfection and microinjection assays.\nThis motif (\u201cPPXXR\u201d) is conserved in various RNA-binding proteins and was proposed to\nmediate nuclear translocation of the cellular functional homologue of HIV-1 Rev Sam68.