Epstein-Barr virus BGLF4 kinase suppresses the interferon regulatory factor 3 signaling pathway.
The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pull-down in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I:C)-stimulated IFN-beta promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-beta mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-beta promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three proline-dependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.
Wang
Jiin-Tarng
JT
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Doong
Shin-Lian
SL
Teng
Shu-Chun
SC
Lee
Chung-Pei
CP
Tsai
Ching-Hwa
CH
Chen
Mei-Ru
MR
D047369:Chromatin Immunoprecipitation; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D047468:Immunoprecipitation; D050838:Interferon Regulatory Factor-3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000502:physiology*
C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D014764:Viral Proteins; D016899:Interferon-beta; C406063:BGLF4 protein, Epstein-Barr virus; D017346:Protein Serine-Threonine Kinases