Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging.
The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.
Wolff
Horst
H
Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstaedterlandstr. 1, 85764 Neuherberg, Germany.
Hadian
Kamyar
K
Ziegler
Manja
M
Weierich
Claudia
C
Kramer-Hammerle
Susanne
S
Kleinschmidt
Andrea
A
Erfle
Volker
V
Brack-Werner
Ruth
R
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001253:Astrocytes / Q000378:metabolism / Q000821:virology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003592:Cytophotometry / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006678:HIV / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D028884:Karyopherins / Q000037:antagonists & inhibitors; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D015678:Viral Structural Proteins / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097573:Red Fluorescent Protein; D000097606:Exportin 1 Protein
D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D015683:Gene Products, gag; D015694:Gene Products, rev; D028884:Karyopherins; D008164:Luminescent Proteins; D011498:Protein Precursors; D018160:Receptors, Cytoplasmic and Nuclear; D015678:Viral Structural Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097573:Red Fluorescent Protein; D000097606:Exportin 1 Protein; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; C054562:yellow fluorescent protein, Bacteria; C038753:leptomycin B