Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 rex mutants.
The HTLV-1 Rex protein is an essential shuttle protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans-dominant (TD) mutant Rex proteins have been reported, however, the mechanism of trans-dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green fluorescent protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells. The presence of Rexp21-GFP inhibited the nuclear export of Rex and HIV-1 Rev as assayed by cotransfection and microinjection experiments. However, Rex-GFP or Rexp21-GFP did not form heteromultimers with nuclear Rex mutants in vivo. In contrast, shuttling was essential for trans-dominance. Thus, we propose that TD Rex mutants do not function by retaining WT Rex in the nucleus by protein-protein interactions, as demonstrated for Rev, but to titrate factors essential for Rex/Rev export. Our findings demonstrate differences between the regulatory proteins Rex and Rev and implicate a novel strategy to generate highly TD Rex mutants also applicable to other proteins.
Heger
P
P
Institute for Medical and Clinical Virology, University of Erlangen-Nürnberg, Erlangen, Germany.
Rosorius
O
O
Hauber
J
J
Stauber
R H
RH
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism*; D016370:Gene Products, rex / Q000502:physiology*; D005799:Genes, Dominant; D017930:Genes, Reporter; D016355:Genes, pX; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D010641:Phenotype; D055503:Protein Multimerization; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015533:Transcriptional Activation; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D016370:Gene Products, rex; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins