Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway

Abstract

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) pulldown in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I: C)-stimulated IFN-␤ promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-␤ 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-␤ 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 prolinedependent 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.The innate immune response is the first-line defense against viral infection. The production of interferons (IFNs) and other cytokines to prevent virus replication and spread is at the center of the antiviral response and requires the activation of multiple transcription activators. The family of IFN regulatory factors (IRFs) is defined by a highly conserved amino-terminal DNA binding domain (DBD) containing five tryptophan repeats and a unique C-terminal domain, the IRF association domain (IAD) (29). IRF3 and IRF7 are two major direct transducers of virus-mediated signaling that induce type I IFNs. IRF3 is a constitutively expressed phosphoprotein of 427 amino acids, which can shuttle into and out of the nucleus in its inactive form. Upon virus infection, cellular TBK-1-and IKKε-mediated phosphorylation of serines 385 and 386 and the serine/threonine cluster between amino acids 396 and 405 of IRF3 lead to its conformational change and activation (19,29,65).The activated IRF3 then undergoes homodimerization or heterodimerization with IRF7, nuclear localization, and association with the coactivator CBP/P300 (29). The phosphor...

EBV Lytic-Phase Protein BGLF5 Contributes to TLR9 Downregulation during Productive Infection

“…Various immunoevasive capabilities have also been assigned to the immediate-early transactivator BZLF1, including association with the NF-kB subunit p65, resulting in its nuclear location while impairing its transcriptional ability (50), inhibition of IRF7, thereby hampering type I IFN production during viral reactivation (51), and downregulation of the TNFR gene promoter activity compromising the effects of TNF-a on an infected cell (52). More recently, the EBV tegument protein LF2 was shown to block dimerization of IRF7, whereas the virionassociated kinase BGLF4 was found to curtail IRF3 transactivation ability (53,54). These latter examples indicate that EBV particles can begin countering the host innate immune response upon cellular entry without the need for prior gene expression.…”

Viral Serine/Threonine Protein Kinases

“…The CHPK of HSV-1, HCMV, EBV, KSHV, and MHV-68 were all found to subvert the type I IFN response by suppressing the activity of interferon regulatory factor 3 (IRF-3) (82,225). The viral kinase interacts with activated IRF-3, thereby interfering with IRF-3 recruitment to the IFN promoter (82,225).…”

“…The CHPK of HSV-1, HCMV, EBV, KSHV, and MHV-68 were all found to subvert the type I IFN response by suppressing the activity of interferon regulatory factor 3 (IRF-3) (82,225). The viral kinase interacts with activated IRF-3, thereby interfering with IRF-3 recruitment to the IFN promoter (82,225). Although the interaction of EBV CHPK with IRF-3 leads to phosphorylation of IRF-3 (225), the kinase activity of the CHPK does not appear to be absolutely required for its effect on the IFN response (82).…”

The Epstein-Barr Virus Protein Kinase BGLF4 and the Exonuclease BGLF5 Have Opposite Effects on the Regulation of Viral Protein Production

“…In addition, BGLF4 was found to interact with EBNA1 (53). BGLF4's targets also include cellular proteins such as the translation elongation factor 1␦ (EF-1␦) (28) and members of the interferon regulatory factor 3 signaling pathway (46).…”