ANALYSIS OF INTRACELLULAR TRAFFICKING AND INTERACTIONS OF CYTOPLASMICHIV-1 REV MUTANTS IN LIVING CELLS

The HIV-I Rev protein is an essential nuclear regulatory viral protein . Rev mutants that are able to block wild-type (WT) Rev activity in tr ans have been reported and used in antiviral approaches. Not only nucl ear but also cytoplasmic Rev mutants were described and suspected to b e transdominant by retaining WT Rev in the cytoplasm. To investigate t heir potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing m utations in the N-terminal multifunctional domain. Using a novel dual- color autofluorescent protein-tagging system, we found that coexpressi on of the nucleolar blue-tagged WT Rev protein together with green-lab eled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but on the contrary, in colocalization of the mutant s to the nucleolus. A combination of mutations abolished the interacti on with WT Rev, defining two domains important for Rev protein interac tion. The identified domains were also essential for specific Rev resp onsive element (RRE) RNA binding and nuclear retention. Inactivation o f the nuclear export signal Shifted the steady-state distribution of t he mutants from the cytoplasm to the nucleus, indicating their capabil ity for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE -specific RNA binding are prerequisites for effective transdominance.