Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules.
The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS). Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells. Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate. Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors. This inhibition was released by treatment with the RNase enzyme. Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import. Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain. Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex. The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.
Fineberg
Konstantin
K
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Fineberg
Tali
T
Graessmann
Adolf
A
Luedtke
Nathan W
NW
Tor
Yitzhak
Y
Lixin
Rui
R
Jans
David A
DA
Loyter
Abraham
A
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D020816:Amino Acid Motifs / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D003600:Cytosol / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000494:pharmacology; D015694:Gene Products, rev / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009687:Nuclear Proteins / Q000494:pharmacology; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D012313:RNA / Q000494:pharmacology*; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D028961:beta Karyopherins / Q000037:antagonists & inhibitors / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; C087018:ArgR protein, Bacteria; D001426:Bacterial Proteins; D015694:Gene Products, rev; D008164:Luminescent Proteins; C099695:MANF protein, human; D009414:Nerve Growth Factors; D009687:Nuclear Proteins; D011506:Proteins; D012097:Repressor Proteins; D028961:beta Karyopherins; C069802:nuclear factor, RRE binding; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D012313:RNA; D004247:DNA