Identification of a domain in human immunodeficiency virus type 1 Rev thatis 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 mR NA expression is tightly linked to its ability to shuttle between the nucle us 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/R-BD) required for binding to the Rev-responsive eleme nt (RRE) located on viral unspliced and singly spliced mRNAs. Structure pre dictions and biophysical measurements indicate that Rev consists of an unst ructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail con taining the NES. We present evidence that the loop portion of the helix-loo p-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained usi ng a protein kinase CK2 phosphorylation assay indicated that the loop regio n is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation o f Rev in SC35-positive nuclear bodies that resembled nuclear bodies that fo rm in response to inhibition of transcription. Accumulation of the Delta Lo op mutant in nuclear bodies depended on the presence of an intact NES, sugg esting that both the loop and the NES play a role in controlling intranucle ar compartmentalization of Rev and its association with splicing factors.