Epstein–Barr virus protein kinase BGLF4 interacts with viral transactivator BZLF1 and regulates its transactivation activity

Abstract

BGLF4 is a serine/threonine protein kinase encoded by Epstein-Barr virus. One of the physiological substrates of BGLF4 is viral transactivator BZLF1. In the present study, it was demonstrated that alanine substitution of the serine residue at position 209 (S209A) in BZLF1 eliminated phosphorylation of the protein by BGLF4 in vitro. The S209A mutation in BZLF1, as well as a K102I mutation in BGLF4, which inactivated catalytic activity of the viral kinase, also inhibited formation of a stable BGLF4-BZLF1 complex and downregulation of BZLF1 autotransactivation activity mediated by BGLF4. These results indicate that formation of a stable complex of BGLF4-BZLF1 enables downregulation of BZLF1 autoregulation activity and it appears that BGLF4 phosphorylation of BZLF1 may be involved in these processes. INTRODUCTIONEpstein-Barr virus (EBV) BGLF4 is a serine/threonine protein kinase and is packaged in the virion tegument, the structural component between the virion nucleocapsid and envelope (Asai et al., 2006; Johannsen et al., 2004; Wang et al., 2005). BGLF4 is conserved in all members of the family Herpesviridae and is the only protein kinase identified in the EBV genome (Chee et al., 1989;Chen et al., 2000;Kato et al., 2001Smith & Smith, 1989). BGLF4 is expressed in the early phase of the lytic infection cycle (Gershburg & Pagano, 2002) and is detected mainly in the nuclei of EBV-infected cells (Gershburg et al., 2004; Wang et al., 2005). Experiments of BGLF4 knockdown by RNA interference in lytically infected cells have demonstrated that BGLF4 has a role(s) in viral replication, especially in the regulation of expression of a subset of late proteins and nuclear egress of nucleocapsids (Gershburg et al., 2007). BGLF4 has also been suggested to function in viral DNA replication. Thus, BGLF4 co-localizes with viral DNA replication compartments in the nuclei of EBV-infected cells (Asai et al., 2006; Wang et al., 2005). BGLF4 also phosphorylates a DNA replication licensing factor, minichromosome maintenance protein 4 (MCM4), at sites whose phosphorylation has been reported to inactivate the DNA helicase activity of the MCM4-MCM6-MCM7 complex (Kudoh et al., 2006). This MCM4-MCM6-MCM7 inhibitory effect on DNA helicase activity may contribute to blocking host chromosome DNA replication in lytically infected cells. In addition, overexpression of BGLF4 in the absence of other EBV proteins induces premature host chromosome condensation, which might provide more extrachromosomal space to facilitate lytic viral DNA replication (Lee et al., 2007).Identification of BGLF4 substrates has suggested additional roles for this protein kinase in EBV infection. So far, in addition to MCM4, as described above, the following have been reported to be substrates for BGLF4: EBV BMRF1, a DNA polymerase processing factor (Chen et al., 2000;Gershburg & Pagano, 2002) . BGLF4 could regulate functions governed by these substrates by phosphorylating the substrates. Among the substrates listed above, BGLF4-mediated phosphorylation of EBNA-LP and EB...

Transcriptional Repression by Sumoylation of Epstein-Barr Virus BZLF1 Protein Correlates with Association of Histone Deacetylase

“…We assume that BGLF4 PK may suppress BZLF1 sumoylation through phosphorylation of another factor. One possibility is that PK might structurally inhibit SUMO conjugation of BZLF1 through their interaction because K102I mutation of BGLF4 PK disables its association with BZLF1 (57).…”

“…Because the BGLF4 PK of EBV, the structural and functional homolog of KSHV vPK, interacts with and phosphorylates BZLF1 (56,57), its effects on BZLF1 sumoylation were examined (data not shown). EBV BZLF1 sumoylation was also decreased by the virus BGLF4 PK, but unlike K-bZIP, phosphorylation of EBV BZLF1 at Ser-209 by BGLF4 (57) did not influence the SUMO modification (data not shown).…”

Repression of CIITA by the Epstein–Barr virus transcription factor Zta is independent of its dimerization and DNA binding

“…Covalent addition of SUMO at K12 (Hagemeier et al, 2010;Murata et al, 2010) was assessed using the non-sumoylatable mutant version, Zta K12R. Both of these post-translational modifications have been described as transcriptionally repressive (Asai et al, 2009;Hagemeier et al, 2010;Murata et al, 2010). As none of these Zta mutants compromised the The CIITA promoter-luciferase plasmids (either 2286 to +54 or 2214 to +54) and either control, His-Zta or His-Zta mutant expression vectors were introduced into Raji BL cells by electroporation.…”

“…The involvement of phosphorylation at S209 by the viral protein kinase BGLF4 (Asai et al, 2009) was investigated using the phospho-mimetic mutant version of Zta, S209D, and the phosphorylation dead mutant version, Zta S209A. Covalent addition of SUMO at K12 (Hagemeier et al, 2010;Murata et al, 2010) was assessed using the non-sumoylatable mutant version, Zta K12R.…”

“…On: Thu, 10 May 2018 13:10:44 modifications of Zta have been shown to reduce the ability of Zta to regulate gene expression, specifically phosphorylation at residue S209 (Asai et al, 2009) and sumoylation through residue K12 (Hagemeier et al, 2010;Murata et al, 2010).…”