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 functionally 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 sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had 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 hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.
Bogerd
H P
HP
Howard Hughes Medical Institute and Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Fridell
R A
RA
Benson
R E
RE
Hua
J
J
Cullen
B R
BR
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D016370:Gene Products, rex / Q000378:metabolism*; D015368:Human T-lymphotropic virus 1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
C119724:AGFG1 protein, human; D002352:Carrier Proteins; D016370:Gene Products, rex; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins