Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7.
The Epstein-Barr nuclear antigen-1 (EBNA1) protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes; has been implicated in host cell immortalization; and avoids proteasomal processing and cell-surface presentation. To gain insight into how EBNA1 fulfills these functions, we have profiled cellular protein interactions with EBNA1 using EBNA1 affinity chromatography and tandem affinity purification (TAP) of EBNA1 complexes from human cells (TAP-tagging). We discovered several new specific cellular protein interactions with EBNA1, including interactions with HAUSP/USP7, NAP1, template-activating factor-I beta/SET, CK2, and PRMT5, all of which play important cell regulatory roles. The ubiquitin-specific protease USP7 is a known target of herpes simplex virus, and the USP7-binding region of EBNA1 was mapped to amino acids 395-450. A mutation in EBNA1 that selectively disrupted binding to USP7 was found to cause a 4-fold increase in EBNA1 replication activity but had no effect on EBNA1 turnover and cell-surface presentation. The results suggest that USP7 can regulate the replication function of EBNA1 and that EBNA1 may influence cellular events by sequestering key regulatory proteins.
Holowaty
Melissa N
MN
Department of Medical Genetics and Microbiology University of Toronto, Ontario, Canada.
Zeghouf
Mahel
M
Wu
Hong
H
Tellam
Judy
J
Athanasopoulos
Vicki
V
Greenblatt
Jack
J
Frappier
Lori
L
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011496:Protein Methyltransferases / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases; D012965:Sodium Chloride / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015533:Transcriptional Activation; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7
D019309:Epstein-Barr Virus Nuclear Antigens; D012965:Sodium Chloride; D011496:Protein Methyltransferases; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases; D010450:Endopeptidases; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; C417029:EBV-encoded nuclear antigen 1