HIV-1 Rev promotes the nuclear export of unspliced and singly spliced RNAs in a mammalian cell-free export system.
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.
Huffman
K M
KM
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425-2230, USA.
Arrigo
S J
SJ
Schmidt
M G
MG
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D017931:DNA Primers / Q000235:genetics; D004734:Energy Metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D012313:RNA