HIV-1 rev promotes the nuclear export of unspliced and singly spliced RNAs in a mammalian cell-free export system

Abstract

Rev has been shown to promote the export of HIV-1 RNAs from Xenopus 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.

Nuclear Export of Simian Immunodeficiency Virus Vpx Protein

“…Sensitivity of Vpx nuclear export to LMB has led us to consider the possibility of its interaction with the CRM-1 pathway, which is a fundamental nuclear transport system used for the export of RNAbinding proteins and specific subclasses of RNA (4,22). In addition, HIV-1 Rev, Gag, and Vpr proteins are exported into the cytoplasm by CRM-1-dependent pathways (10,47).…”