Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm.
Expression of gag/pol and env genes of human immunodeficiency virus requires the viral Rev protein. Mutant Rev proteins, displaying a transdominant phenotype (TDRev), were shown to inhibit Rev function. To investigate the underlying mechanism of this inhibition, the green fluorescent protein (GFP) of Aequorea victoria was fused to Rev and TDRev, which allowed the study of their trafficking and interactions in living human cells. Both Rev-GFP and TDRev-GFP were shown to retain appropriate nucleolar localization and function. Upon actinomycin D treatment, Rev-GFP was transported to the cytoplasm within 1.5 hr, while TDRev, although partially dissociated from the nucleolus, was retained in the nucleus. Coexpression of Rev-GFP and TDRev in the same cell demonstrated that TDRev inhibited the transport of Rev-GFP from the nucleus to the cytoplasm. This inhibition was specific for Rev, since the export of the functionally analogous Rex protein of the human T-cell leukemia virus type I was not inhibited by TDRev. These results indicate that Rev and TDRev form heteromultimers in the nucleolus and that this interaction prevents Rev's export from the nucleus to the cytoplasm. In addition to providing a model for the function of TDRev, these results also demonstrate the successful application of protein fusions to GFP to study localization and trafficking of proteins in living mammalian cells.
D001483:Base Sequence; D001692:Biological Transport; D002461:Cell Line, Transformed; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D003609:Dactinomycin / Q000494:pharmacology; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D016370:Gene Products, rex / Q000378:metabolism; D049452:Green Fluorescent Proteins; D016655:HIV Core Protein p24 / Q000096:biosynthesis; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015694:Gene Products, rev; D016370:Gene Products, rex; D016655:HIV Core Protein p24; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D003609:Dactinomycin