Post-translational modification of Rta of Epstein-Barr virus by SUMO-1.
Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.
Chang
Li-Kwan
LK
Faculty of Biological Medicine and Environmental Biology and Graduate Institute of Biochemistry, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung, Taiwan.
Lee
Yu-Hsiu
YH
Cheng
Tai-Shan
TS
Hong
Yi-Ren
YR
Lu
Pei-Jung
PJ
Wang
Janng J
JJ
Wang
Wen-Hung
WH
Kuo
Chung-Wen
CW
Li
Steven S-L
SS
Liu
Shih-Tung
ST
D001665:Binding Sites; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D015151:Immunoblotting; D019169:Jurkat Cells; D008239:Lysine / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014764:Viral Proteins; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C068382:BRLF1 protein, Human herpesvirus 4; D017874:Immediate-Early Proteins; D025842:SUMO-1 Protein; D015534:Trans-Activators; D014764:Viral Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine