Department of Tumor Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan1
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan2
Author for correspondence: Yasushi Kawaguchi. Fax +81 3 5803 0241. e-mail kawagchi.creg{at}mri.tmd.ac.jp
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Abstract |
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Introduction |
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Although HSV-1 UL13 and HCMV UL97 have been shown to mediate modification of EF-1 (Kawaguchi et al., 1998
, 1999
), it is not yet known whether conserved protein kinases of herpesviruses (HSV-1 UL13 homologues) are commonly involved in modification of EF-1
because (i) the modification has not been shown by gammaherpesvirus protein kinases and (ii) conserved protein kinases sometimes show other biological activities (Heineman & Cohen, 1995
; Moffat et al., 1998
; Ng et al., 1994
, 1998
; Ogle et al., 1997
; Purves & Roizman, 1992
). We wished, therefore, to examine whether a gammaherpesvirus protein kinase hyperphosphorylates EF-1
. This would further support the hypothesis that EF-1
modification is a conserved function that is expressed by all herpesviruses subfamily members in mammalian cells and that conserved protein kinases encoded by herpesviruses universally mediate this modification. In this report, we present evidence that this is in fact the case. We report here that BGLF4, a viral protein kinase of EpsteinBarr virus (EBV), mediates EF-1
modification at a cellular level.
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Methods |
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Plasmids.
EBV DNA was isolated from B95-8 cells as described previously (Horimoto et al., 1992 ). The entire EBV BGLF4 open reading frame (ORF) was amplified by PCR using viral DNA as a template and the primers GCGAATTCGGAACATGGATGTGAATATG and GCGGATCCTCATCCACGTCGGCCATCTG. The amplified fragments were digested with EcoRI/BamHI and cloned into the EcoRI and BamHI sites of pBluescript II KS+ (Stratagene). The resultant plasmid was designated pBS-BGLF4-stop. pAcGHLT-BGLF4 (Fig. 1C
) was generated by inserting an EcoRINotI fragment of pBS-BGLF4-stop into pAcGHLT-B (Pharmingen). The entire EF-1
ORF without the stop codon was amplified from pBH1003 (Kawaguchi et al., 1997a
) by PCR using the primers GCGAATTCAGAAAAATGGCTACAAACTT and GCGGATCCGATCTTGTTGAAAGCTGCGA. The amplified fragments were digested with EcoRI/BamHI and cloned into the EcoRI and BamHI sites of pBS-Flag-Stop (Kawaguchi et al., 2000
). The resultant plasmid was designated pBS-EF-1
(F)-Stop. The EcoRINotI fragment of EF-1
(F) was inserted into pFASTBAC DUAL (GibcoBRL) to generate pFASTBAC-EF-1
(F). To construct pBS-BGLF4(F), a fragment of EBV viral DNA encoding the entire coding sequence of BGLF4 without the stop codon was amplified by PCR using the primers GCGAATTCGGAACATGGATGTGAATATG and GCGGATCCTCCACGTCGGCCATCTGGAC. This fragment was then cloned into pBS-Flag-Stop in-frame with the Flag epitope. The EcoRINotI fragment of pBS-BGLF4(F) was inserted into the EcoRI and NotI sites of pME18S (kindly provided by K. Maruyama, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan) to yield pME-BGLF4(F) (Fig. 1D
). In pME-BGLF4(F), the expression of the 3' Flag epitope-tagged BGLF4 was driven by the SR
promoter (Takebe et al., 1988
).
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Purification of EBV BGLF4 protein.
Sf9 cells (1·0x106) infected with each baculovirus (Bac-GST-BGLF4 or Bac-GST) in 0·5 ml of ice-cold buffer C (50 mM TrisHCl, pH 7·5, 100 mM NaCl, 5 mM MgCl2, 0·1% Nonidet P-40, 10% glycerol and 1 mM PMSF) were lysed by sonication. After insoluble material was removed by centrifugation, the supernatants were mixed with 50 µl of a 50% slurry of glutathioneSepharose beads (Amersham Pharmacia) for 2 h. The beads were extensively washed with buffer C and eluted with elution buffer (10 mM glutathione and 500 mM TrisHCl, pH 8·0). Next, the eluted supernatants were reacted with Ni2+NTA agarose beads (Qiagen) for 1 h. The beads were then washed three times with buffer C. Purified protein captured on the beads was separated by 10% SDSPAGE and either silver-stained (Fig. 2A) or immunoblotted (Fig. 2B
) with rabbit antiserum containing anti-GST antibody, as described previously (Kawaguchi et al., 1997b
).
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Results |
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The objective of the first series of experiments was to purify the EBV BGLF4 gene product. We expressed and purified BGLF4 as a histidine-tagged GST fusion protein using the baculovirus system, as described in Methods. Purified protein was then electrophoretically separated in a denaturing gel and either silver-stained (Fig. 2A) or immunoblotted with rabbit antiserum containing anti-GST antibody (Fig. 2B
).
The purified supernatants from Sf9 cells infected with either Bac-GST or Bac-GST-BGLF4 each contained one major purified protein with an Mr of 32000 or 78000, as detected by silver staining (Fig. 2A), and these proteins reacted with antiserum containing anti-GST antibody (Fig. 2B
). These results indicated that we had purified the desired GST fusion proteins.
Protein kinase activity of purified BGLF4
Many protein kinases have autophosphorylating activity (Edelman et al., 1987 ). To determine whether purified BGLF4 does in fact possess kinase activity, we examined the ability of purified BGLF4 to autophosphorylate itself in the kinase assay, as described in Methods. The results (Fig. 3
) were as follows: (i) electrophoretically separated purified GST protein, which was incubated in kinase buffer, did not contain any labelled bands (Fig. 3A
, B
). However, in the autoradiographic image of purified BGLF4, a protein band with an apparent Mr of 78000 was labelled (Fig. 3B
). The electrophoretic mobility of labelled BGLF4 was the same as that of purified BGLF4 stained with Ponceau S (Fig. 3A
, B
). (ii) To determine if the labelling of BGLF4 with [
-32P]ATP was due to phosphorylation, labelled BGLF4 was boiled to inactivate kinases and incubated with 50 units of alkaline phosphatase at 37 °C for 30 min. As shown in Fig. 3(D)
, the band of labelled BGLF4 was eliminated by phosphatase treatment, indicating that BGLF4 was labelled with [
-32P]ATP by phosphorylation. Fig. 3(C)
shows the results of Ponceau S staining of the same blot used in the experiment of Fig. 3(D)
. These data showed that BGLF4 levels remained relatively unchanged after boiling and indicated that boiling did not result in the loss of BGLF4. Thus, BGLF4 autophosphorylates itself and possesses protein kinase activity.
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We concluded from these results that BGLF4 protein mediates EF-1 hyperphosphorylation at a cellular level in mammalian cells.
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Discussion |
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Our knowledge regarding the requirements of viruses with respect to host cell biosynthetic processes depends in part on the identification of cellular proteins that interact with viral proteins. Therefore, if many viral proteins commonly interact with the same cellular protein, this protein would be suggested to be important in the life cycle of viruses, as viruses sometimes use similar strategies regardless of their large diversity in size, structure and genome arrangement. For instance, most DNA tumour viruses encode proteins (e.g. adenovirus E1A and E1B, simian virus 40 T antigen and papillomavirus E6 and E7) that interact with and modify key cellular proteins such as retinoblastoma protein and p53, and transform host cells by a common strategy (i.e. by interaction with the cellular regulatory proteins) (Knipe, 1996 ). Our current studies (Kawaguchi et al., 1997a
, 1998
, 1999
) together with reports from other laboratories, which show that HIV Tat also interacts functionally with a cellular protein and affects translation in vivo (Xiao et al., 1998
) and that the RNA polymerase of vesicular stomatis virus associates with the EF-1 complex for its activity (Das et al., 1998
), suggest that EF-1
is one of the cellular proteins that is universally important in various virus replication systems.
This study also provides important information for the field of EBV research. (i) We expressed enzymatically active BGLF4 using the baculovirus expression system and purified it to near homogeneity. The purified protein was labelled with [-32P]ATP in vitro and the labelling was eliminated by phosphatase treatment, indicating that BGLF4 has the ability to autophosphorylate itself. Our experiments using purified BGLF4 also suggested that it functions, without any cofactors, as a protein kinase. These results supplement those of the study by Chen et al. (2000)
in which purification and phosphatase treatment were not performed, and confirm the conclusion that BGLF4 protein is a serine/threonine protein kinase. (ii) It is known that HSV-1 UL13 protein kinase and its HCMV counterpart, UL97, regulate viral gene expression by phosphorylation of various viral and cellular proteins (He et al., 1997
; Kawaguchi et al., 1998
; Ng et al., 1998
; Ogle et al., 1997
; Prichard et al., 1999
; Purves et al., 1992
, 1993
). Furthermore, ORF36 of human herpesvirus-8 (HHV-8), another gammaherpesvirus UL13 homologue, and HCMV UL97 have been reported to phosphorylate ganciclovir and thus induce ganciclovir-mediated cell death (Cannon et al., 1999
; Litter et al., 1992
; Sullivan et al., 1992
). The similarity of BGLF4 to HSV-1 UL13, HCMV UL97 and HHV-8 ORF36 (Cannon et al., 1999
; Chee et al., 1989
; Smith & Smith, 1989
) suggests that BGLF4 plays a role in EBV replication in a manner similar to HSV-1 UL13 and HCMV UL97 and, like HHV-8 ORF36 and HCMV UL97, is a target of anti-viral drugs. Supporting this hypothesis, Chen et al. (2000)
reported that an EBV regulatory protein, EA-D, is a potential substrate of BGLF4. As it is easy to express large amounts of enzymatically active BGLF4 and to obtain highly purified BGLF4 using our system, this method will be useful for the further characterization of BGLF4. For example, to identify additional cellular and viral targets of the protein and to analyse its sensitivity to anti-viral drugs.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 September 2000;
accepted 15 January 2001.