HIV-1 rev promotes the nuclear export of unspliced and singly spliced RNAs in a mammalian cell-free export system |
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Authors: | Kim M. Huffman Salvatore J. Arrigo Michael G. Schmidt |
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Affiliation: | (1) Department of Microbiology and Immunology, Medical University of South Carolina, Basic Science Building, Room 201 171 Ashley Avenue, 29425-2230 Charleston, SC, USA |
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Abstract: | Rev has been shown to promote the export of HIV-1 RNAs fromXenopus oocyte nuclei, but a system to examine the direct effect of Rev on HIV-1 RNA export in mammalian somatic cells does not exist. In this report, the development of a cell-free RNA export system using COS cells is described. This system is capable of examining the movement of RNA from nuclei of COS cells transfected with an HIV-1 proviral construct into reconstituted cytosol from nontransfected cells. A reproducible preparation of nuclei free of residual cytoplasmic RNA is demonstrated. Export of RNA from these nuclei into reconstituted cell-free extracts was saturable and dependent on temperature and energy. Further validation of the system was obtained by confirming that the nuclear export of HIV-1-unspliced and partially spliced RNAs was dependent upon the expression of HIV-1 Rev and that the presence of Rev appeared to decrease the export of an HIV-1-spliced RNA. The system was also able to demonstrate that Rev did not appear to significantly enhance the export of an HIV-1 protease-containing RNA that has been shown to be dependent upon Rev for maximal expression. Consequently, the system appears useful for the examination of parameters of nuclear export of HIV-1 and cellular RNAs. |
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Keywords: | HIV-1 Rev Nuclear export Cis acting repressor sequences HIV-1 protease |
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