BGLF4 is the only serine/threonine protein kinase identified in Epstein–Barr virus (EBV); it is known to phosphorylate viral DNA polymerase processivity factor, EA-D (BMRF1), EBNA-LP, EBNA-2, cellular EF-1δ and nucleoside analogue ganciclovir. However, the expression and biological functions of BGLF4 have not yet been clearly demonstrated in EBV-infected cells. To reveal authentic functions of BGLF4 protein within viral-replicating cells, a panel of specific monoclonal antibodies was generated and characterized. The major immunogenic regions of BGLF4 were mapped to aa 27–70 and 327–429. Using these antibodies, the expression kinetics and localization of BGLF4 were analysed in reactivated EBV-positive lymphoid and epithelial cells. BGLF4 was expressed as a phosphoprotein at the early lytic stage and was detected predominantly in the nucleus of EBV-positive cells, but small amounts of BGLF4 were observed in cytosolic and heavy membrane fractions at the late phase of virus replication. Additionally, it was demonstrated that BGLF4 co-localizes with viral DNA polymerase processivity factor, EA-D (BMRF1), in the virus replication compartment and that it is a virion component. Finally, possible functional domains at the N terminus of BGLF4 were analysed and it was found that aa 1–26 of BGLF4 are dispensable for EA-D phosphorylation, whereas deletion of aa 27–70 reduced kinase activity.
ChangY.,
ChengS. D.,
TsaiC. H.2002; Chromosomal integration of Epstein–Barr virus genomes in nasopharyngeal carcinoma cells. Head Neck 24:143–150[CrossRef]
ChenM. R.,
TsaiC. H.,
WuF. F.,
KanS. H.,
YangC. S.,
ChenJ. Y.1999; The major immunogenic epitopes of Epstein–Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines. J Gen Virol 80:447–455
ChenM. R.,
ChangS. J.,
HuangH.,
ChenJ. Y.2000a; A protein kinase activity associated with Epstein–Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. J Virol 74:3093–3104[CrossRef]
ChenM. R.,
HuangH.,
FenC. Y.,
ChenJ. Y.2000b; A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo. J Virol Methods 85:35–41[CrossRef]
GershburgE.,
PaganoJ. S.2002; Phosphorylation of the Epstein–Barr virus (EBV) DNA polymerase processivity factor EA-D by the EBV-encoded protein kinase and effects of the l-riboside benzimidazole 1263W94. J Virol 76:998–1003[CrossRef]
GershburgE.,
MarschallM.,
HongK.,
PaganoJ. S.2004; Expression and localization of the Epstein–Barr virus-encoded protein kinase. J Virol 78:12140–12146[CrossRef]
HanksS. K.,
HunterT.1995; Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596
JohannsenE.,
LuftigM.,
ChaseM. R.,
WeickselS.,
Cahir-McFarlandE.,
IllanesD.,
SarracinoD.,
KieffE.2004; Proteins of purified Epstein–Barr virus. Proc Natl Acad Sci U S A 101:16286–16291[CrossRef]
KatoK.,
YokoyamaA.,
TohyaY.,
AkashiH.,
NishiyamaY.,
KawaguchiY.2003; Identification of protein kinases responsible for phosphorylation of Epstein–Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function. J Gen Virol 84:3381–3392[CrossRef]
KawaguchiY.,
KatoK.,
TanakaM.,
KanamoriM.,
NishiyamaY.,
YamanashiY.2003; Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta. J Virol 77:2359–2368[CrossRef]
KieffE.,
RickinsonA. B.2001; Epstein–Barr virus and its replication. In Fields Virology , 4th edn. pp 2511–2573 Edited by
KnipeD. M.,
HowleyP. M.
Philadelphia: Lippincott Williams & Wilkins;
KiehlA.,
DorskyD. I.1991; Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells. Virology 184:330–340[CrossRef]
KolaskarA. S.,
TongaonkarP. C.1990; A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174[CrossRef]
KrajewskiS.,
TanakaS.,
TakayamaS.,
SchiblerM. J.,
FentonW.,
ReedJ. C.1993; Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 53:4701–4714
KroskyP. M.,
BaekM. C.,
CoenD. M.2003; The human cytomegalovirus UL97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress. J Virol 77:905–914[CrossRef]
LiaoG.,
WuF. Y.,
HaywardS. D.2001; Interaction with the Epstein–Barr virus helicase targets Zta to DNA replication compartments. J Virol 75:8792–8802[CrossRef]
MichelD.,
MertensT.2004; The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host. Biochim Biophys Acta1697169–180[CrossRef]
MorrisonE. E.,
WangY. F.,
MeredithD. M.1998; Phosphorylation of structural components promotes dissociation of the herpes simplex virus type 1 tegument. J Virol 72:7108–7114
PurvesF. C.,
RoizmanB.1992; The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation of the regulatory protein α 22. Proc Natl Acad Sci U S A 89:7310–7314[CrossRef]
PurvesF. C.,
OgleW. O.,
RoizmanB.1993; Processing of the herpes simplex virus regulatory protein α 22 mediated by the UL13 protein kinase determines the accumulation of a subset of α and γ mRNAs and proteins in infected cells. Proc Natl Acad Sci U S A 90:6701–6705[CrossRef]
TakadaK.,
HorinouchiK.,
OnoY.,
AyaT.,
OsatoT.,
TakahashiM.,
HayasakaS.1991; An Epstein–Barr virus-producer line Akata: establishment of the cell line and analysis of viral DNA. Virus Genes 5:147–156[CrossRef]
TsaiC. H.,
GlaserR.1991; A comparison of Epstein–Barr virus specific proteins expressed by three Epstein–Barr virus isolates using specific monoclonal antibodies. Intervirology 32:376–382
TsaiC. H.,
WilliamsM. V.,
GlaserR.1991; Characterization of two monoclonal antibodies to Epstein–Barr virus diffuse early antigen which react to two different epitopes and have different biological function. J Virol Methods 33:47–52
TsaiC. H.,
LiuM. T.,
ChenM. R.,
LuJ.,
YangH. L.,
ChenJ. Y.,
YangC. S.1997; Characterization of monoclonal antibodies to the Zta and DNase proteins of Epstein–Barr virus. J Biomed Sci 4:69–77[CrossRef]
TsurumiT.1993; Purification and characterization of the DNA-binding activity of the Epstein–Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system. J Virol 67:1681–1687
van ZeijlM.,
FairhurstJ.,
BaumE. Z.,
SunL.,
JonesT. R.1997; The human cytomegalovirus UL97 protein is phosphorylated and a component of virions. Virology 231:72–80[CrossRef]
WaltherR. F.,
AtlasE.,
CarriganA.7 other authors2005; A serine/threonine-rich motif is one of three nuclear localization signals that determine unidirectional transport of the mineralocorticoid receptor to the nucleus. J Biol Chem 280:17549–17561[CrossRef]
WestphalE. M.,
MauserA.,
SwensonJ.,
DavisM. G.,
TalaricoC. L.,
KenneyS. C.1999; Induction of lytic Epstein–Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo. Cancer Res 59:1485–1491
WolfD. G.,
HonigmanA.,
LazarovitsJ.,
TavorE.,
PanetA.1998; Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase. Arch Virol 143:1223–1232[CrossRef]
WolfD. G.,
CourcelleC. T.,
PrichardM. N.,
MocarskiE. S.2001; Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. Proc Natl Acad Sci U S A 98:1895–1900[CrossRef]
YueW.,
GershburgE.,
PaganoJ. S.2005; Hyperphosphorylation of EBNA2 by Epstein–Barr virus protein kinase suppresses transactivation of the LMP1 promoter. J Virol 79:5880–5885[CrossRef]