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-dom inant (TD) mutant Rex proteins have been reported, however, the mechanism o f 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 gree n fluorescent protein (GFP) we observed that Rexp21-GFP displayed a cytopla smic localization but was actively shuttling between the nucleus and the cy toplasm in live human cells. The presence of Rexp21-GFP inhibited the nucle ar export of Res and HIV-1 Rev as assayed by cotransfection and microinject ion experiments, However, Rex-GFP or Rexp21-GFP did not form heteromultimer s with nuclear Res mutants in Fire. In contrast, shuttling was essential fo r tr ans-dominance. Thus, we propose that TD Res mutants do not function by retaining WT Res in the nucleus by protein-protein interactions, as demons trated for Rev, but to titrate factors essential for Rex/Rev export, Our fi ndings demonstrate differences between the regulatory proteins Res and Rev and implicate a novel strategy to generate highly TD Res mutants also appli cable to other proteins.