Detection of Epstein–Barr virus BGLF4 protein kinase in virus replication compartments and virus particles

Abstract

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-1d 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.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

“…This diverse pattern, coupled with the presence of vPK transcripts both early and late, implies that vPK could influence multiple steps in viral replication. The pattern of vPK localization is similar to the EBV vPK homolog BGLF4, which is found in the viral replication compartment, with variation in subcellular localization at different times after reactivation of virus (2,52). Likewise, CMV UL97, the vPK homolog, is also colocalized with UL44 (the PPF homolog) in the viral DNA replication compartment; UL97 phosphorylates UL44 (33).…”

“…Interestingly, the antibody readily detected vPK in purified virions. Structural analysis of purified virions of other herpesviruses, including HSV, CMV, EBV, and rhesus rhadinovirus, indicate that the conserved protein kinases of these viruses are also incorporated into mature virus particles (2,12,37,50,52). This finding suggests that this kinase may play a role in phosphorylating viral proteins during virion assembly and/or regulate the functions of viral and cellular proteins upon entry into host cells and during the early stage of de novo infection (47).…”

“…Experimental studies on members of each of the three major groups of herpesviruses have identified a wide array of viral and cellular protein substrates of the conserved viral protein kinases. Structural analysis of purified virions of several herpesviruses, including HSV, CMV, and EBV, indicate that these protein kinases are asso-ciated with virus particles (2,12,37,50,52); thus, the kinases are in a position to influence virion assembly and events that take place after entry of the virion into the cell. A number of studies using kinase-null viral mutants demonstrated the importance of the kinase for regulating viral gene expression, replication, tissue tropism, or infection in animal models (7,11,26,35,41,48).…”

“…A number of studies using kinase-null viral mutants demonstrated the importance of the kinase for regulating viral gene expression, replication, tissue tropism, or infection in animal models (7,11,26,35,41,48). Various studies have implicated the viral protein kinase in influencing viral gene expression (6,58), viral DNA replication (27,52,53), or nucleocapsid egress from the nucleus during virus assembly (26,34,53). The importance of the viral protein kinase (vPK) for HSV and CMV replication is supported by studies showing that changes in this gene can confer resistance to certain antiviral agents (e.g., ganciclovir) (7,26).…”

Characterization of Epstein-Barr virus BGLF4 kinase expression control at the transcriptional and translational levels

“…The substrates of BGLF4 include the viral DNA polymerase accessory factor BMRF1, BZLF1, EBNA-LP, EBNA-2 and cellular translation factor EF-1d, condensin, lamin A/C and the MCM complex (Asai et al, 2009;Chen et al, 2000;Kato et al, 2001Kato et al, , 2003Kudoh et al, 2006;Lee et al, 2007Lee et al, , 2008Yue et al, 2005). BGLF4 localizes at the DNA replication compartment at an early stage and is packaged into virus particles (Wang et al, 2005). BGLF4 is required for optimal DNA replication and nuclear egress as revealed by a small interfering RNA knockdown experiment (Gershburg et al, 2007).…”