A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro

Abstract

The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.Protein kinases are known to be involved in the regulation of a wide variety of eukaryotic cellular functions including cell metabolism, cell cycle control, hormone response, and control of transcription and translation. Studying viral protein kinases might therefore lead to an understanding of the mechanisms of virus replication and virus-cell interactions. Most of the protein kinases of the retroviruses are Tyr protein kinases, such as v-src and v-erb, which may contribute to the growth transformation phenotype of the virally infected host cells (for a review, see reference 32). The first protein kinase gene demonstrated in a eukaryotic DNA virus was that contained in the unique short (US) regions of the related human and porcine alphaherpesviruses, herpes simplex virus type 1 (HSV-1), and pseudorabies virus (20). Other protein kinases have been reported in DNA viruses, including protein kinase B1 of the poxviruses (45, 46) and ORF9 of baculovirus (42).Phosphorylation of cellular and viral proteins, which has been observed during lytic infection of cells by herpesviruses, seems to be a common phenomenon which involves a number of different protein kinase activities (21). Two groups of viral protein kinase activities, US3 and UL13, have been identified in alphaherpesviruses. The US3 gene of HSV-1 (37) and the VZV66 gene of varicella-zoster virus (VZV) (19) were predicted to encode protein kinases on the basis of their strong similarity to the family of eukaryotic serine/threon...

The regulatory role of protein phosphorylation in human gammaherpesvirus associated cancers

“…Interestingly, most of viral kinases not only phosphorylate other viral and cellular proteins, but also autophosphorylated themselves, which facilitates viral replication or production within the host cells . For instance, the EBV-encoded BGLF4 is a protein kinase, and is able to phosphorylate a number of viral proteins including BZLF1 (Asai et al, 2006), EA-D (Chen et al, 2000), and EBNA2 during the latency . Surprisingly, during the viral lytic replication, BGLF4 also phosphorylates EBNA-2 in a manner similar to the cellular kinase cdk1, and the hyperphosphorylation of EBNA-2 will in turn inhibit its normal ability to transactivate the EBV LMP1 promoter, and eventually lead to induction of EBV lytic replication (Yue et al, 2005).…”

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

“…EBV BGLF4 kinase belongs to the conserved herpesviral UL protein kinases, which have been identified in alpha-, betaand gammaherpesviruses (Chee et al, 1989;Gershburg & Pagano, 2008). 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).…”

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

“…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).…”

“…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.…”

“…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 itself ; EBV nuclear antigen (EBNA)-2, a viral transcriptional regulator of viral and cellular genes (Yue et al, 2005); EBNA-LP, a viral transcriptional co-activator of EBNA-2 ; BZLF1, a viral transcriptional regulator involved in the switch from latent to lytic infection (Asai et al, 2006); and cellular translation elongation factor 1d . BGLF4 could regulate functions governed by these substrates by phosphorylating the substrates.…”