Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.
Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.
Daher
A
A
Molecular Oncology Group/McGill AIDS Centre, Lady Davis Institute for Medical Research, 3755 Côte Ste Catherine, Montréal H3T 1E2, Québec, Canada.
Longuet
M
M
Dorin
D
D
Bois
F
F
Segeral
E
E
Bannwarth
S
S
Battisti
P L
PL
Purcell
D F
DF
Benarous
R
R
Vaquero
C
C
Meurs
E F
EF
Gatignol
A
A
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D019281:Dimerization; D017353:Gene Deletion; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D019892:eIF-2 Kinase / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
D000596:Amino Acids; D008156:Luciferases; D012313:RNA; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase; D000099253:Trans-Activation Responsive RNA-Binding Protein