Identification and characterization of Risp

Ceccherini-Silberstein, Francesca (2001): Identification and characterization of Risp: a new nucleocytoplasmic shuttle protein interacting with the HIV-1 Rev protein. Dissertation, LMU München: Fakultät für Biologie

Abstract

The aim of this study was to characterize the role of Rev-interacting cellular proteins in controlling the function of Rev in the host cell. The HIV-1 protein Rev plays an essential role in the temporal regulation of the virus gene expression by stimulating the expression of viral structural proteins. Rev enhances the nucleocytoplasmic transport and the translation of unspliced and single spliced viral mRNAs by binding with high affinity to a specific target element on the HIV-RNA. It was assumed that interaction with cellular factors is essential for Rev function. At the onset of this study, only a few potential cofactors were known with no clearly defined functional relevance. So we decided to search for new Rev-interacting factors using the yeast two-hybrid system. In this work a new Rev-interacting protein has been identified, by screening a Jurkat T cell cDNA library. The protein was termed Risp (Rev-interacting shuttle protein), because it shuttles between the nuclear and the cytoplasmic compartments. The Risp gene is widely expressed in human cells and conserved among various species, most probably as part of a larger gene. High amino acid homology (99%) with the C-terminal part of a large brain cDNA clone for KIAA0592 protein has been found, whereas no obvious homology to proteins with known function was observed. However, a weak and partial similarity appeared with several RNA-/DNA-binding and shuttle proteins. This might indicate that the Risp protein - or the larger protein containing it - could be a member of a new family of nucleocytoplasmic shuttle proteins with RNA-/DNA-binding function. Next, the intracellular localization and shuttling of Risp was investigated. In HeLa cells Risp- GFP localized in both nuclear and cytoplasmic compartments, but clearly accumulated in the cytoplasm, indicating the presence of a strong nuclear export signal (NES). The identification of a NES sequence was confirmed by deletion analysis of Risp and by nuclear microinjection of BSA-fusion proteins conjugated to peptides from the C-terminal part of Risp. Treatment with leptomycin B, a drug which has been shown to specifically block Crm1 (exportin) mediated export, resulted in nuclear accumulation of Risp-GFP, showing that the nuclear export of Risp, like that of Rev, is Crm1-dependent. Using bioinformatic tools able to detect weak homologies with high specificity, sequence comparisons between Risp and all currently known Rev interacting factors were performed. This analysis for the first time revealed a common motif shared between Rev and Rev-interacting cellular factors, termed RIP. The region of Risp harboring the RIP motif was neither essential nor sufficient for the Rev-binding in the yeast two hybrid system, suggesting no direct correlation between RIP and the Rev-binding ability. Preliminary experiments suggested, that Risp, as a Rev-interacting protein, is able to inhibit Revtrans- activation, while Risp does not interfere with Tat in a Tat-trans-activation assay. The overexpression of Risp-GFP reduced the production of the Rev-dependent structural viral protein p24gag up to 70%. In addition a previously unrecognized sequence motif in the activation domain of Rev with intrinsic nuclear import activity was found and tested in transfection and microinjection assays. This motif (“PPXXR”) is conserved in various RNA-binding proteins and was proposed to mediate nuclear translocation of the cellular functional homologue of HIV-1 Rev Sam68.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 20. Juli 2001