Interaction of retroviral Tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-alpha expression.
BACKGROUND
The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell leukemia viruses and bovine leukemia virus). To better understand the molecular pathways leading to cell transformation, we aimed to identify cellular proteins interacting with Tax.


METHODS
We used a yeast two-hybrid system to identify interacting cellular proteins. Interactions between Tax and candidate interacting cellular proteins were confirmed by glutathione S-transferase (GST) pulldown assays, co-immunoprecipitation, and confocal microscopy. Functional interactions between Tax and one interacting protein, tristetraprolin (TTP), were assessed by analyzing the expression of tumor necrosis factor-alpha (TNF-alpha), which is regulated by TTP, in mammalian cells (HeLa, D17, HEK 293, and RAW 264.7) transiently transfected with combinations of intact and mutant Tax and TTP.


RESULTS
We obtained seven interacting cellular proteins, of which one, TTP, was further characterized. Tax and TTP were found to interact specifically through their respective carboxyl-terminal domains. The proteins colocalized in the cytoplasm in a region surrounding the nucleus of HeLa cells. Furthermore, coexpression of Tax was associated with nuclear accumulation of TTP. TTP is an immediate-early protein that inhibits expression of TNF-alpha at the post-transcriptional level. Expression of Tax reverted this inhibition, both in transient transfection experiments and in stably transfected macrophage cell lines.


CONCLUSIONS
Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis.
Twizere
Jean-Claude
JC
Biologie cellulaire et moléculaire, Faculté Universitaire des Sciences Agronomiques, Gembloux, Belgium.
Kruys
Véronique
V
Lefèbvre
Laurent
L
Vanderplasschen
Alain
A
Collete
Delphine
D
Debacq
Christophe
C
Lai
Wi S
WS
Jauniaux
Jean-Claude
JC
Bernstein
Lori R
LR
Semmes
O John
OJ
Burny
Arsène
A
Blackshear
Perry J
PJ
Kettmann
Richard
R
Willems
Luc
L
D000818:Animals; D002417:Cattle; D018076:DNA, Complementary / Q000032:analysis; D004268:DNA-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D016356:Gene Products, tax / Q000378:metabolism*; D015368:Human T-lymphotropic virus 1; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D001909:Leukemia Virus, Bovine; D018613:Microscopy, Confocal; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism* / Q000821:virology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014162:Transfection; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D016356:Gene Products, tax; D017874:Immediate-Early Proteins; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; C497552:ZFP36 protein, human; D001616:beta-Galactosidase