PROTEIN-SEQUENCE REQUIREMENTS FOR FUNCTION OF THE HUMAN T-CELL LEUKEMIA-VIRUS TYPE-1 REX NUCLEAR EXPORT SIGNAL DELINEATED BY A NOVEL IN-VIVO RANDOMIZATION-SELECTION ASSAY

The Rex protein of human T-cell leukemia virus type 1, like the functi onally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequ ence is shown to function also as a protein nuclear export signal (NES ). Rex sequence libraries containing randomized forms of the activatio n domain/ NES were screened for retention of the ability to bind Rab/h RIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activa tion domains. In contrast, randomized sequences that failed to bind Ra b/hRIP lacked Rex activity. The selected sequences included one with h omology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor or. A highl y variant, yet fully active, activation domain sequence selected on th e basis of Rab/hRIP binding retained full NES function even though thi s sequence preserved only a single leucine residue. In contrast, nonfu nctional activation domain mutants that were unable to bind Rab/hRIP h ad also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/ Rex class of retroviral regulatory proteins and strongly support the h ypothesis that the Rab/hRIP cofactor plays a critical role in mediatin g the biological activity of these NESs. In addition, these data sugge st a consensus sequence for NESs of the Rev/Rex class.