Identification of a domain in human immunodeficiency virus type 1 rev that is required for functional activity and modulates association with subnuclear compartments containing splicing factor SC35.
The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the DeltaLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.
D'Agostino
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DM
Dipartimento di Scienze Oncologiche e Chirurgiche, Sezione di Oncologia, University of Padova, Italy. dmdago@ux1.unipd.it
Ferro
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Zotti
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Meggio
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F
Pinna
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LA
Chieco-Bianchi
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Ciminale
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D000163:Acquired Immunodeficiency Syndrome / Q000821:virology*; D000595:Amino Acid Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics* / Q000378:metabolism; D015757:Genes, rev; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014779:Virus Replication / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D054321:rev Gene Products, Human Immunodeficiency Virus; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors