1 Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan
2 Department of Veterinary Microbiology, Graduate School of Agricultural and Life Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
3 Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
4 PRESTO, Japan Science and Technology Corporation, Tachikawa, Tokyo 190-0012, Japan
Correspondence
Yasushi Kawaguchi
at Nagoya University Graduate School of Medicine
ykawagu{at}med.nagoya-u.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EBV nuclear antigen leader protein (EBNA-LP), the subject of this study, is a latency-associated phosphoprotein that is expressed first, together with EBNA-2, following the infection of B cells with EBV in vitro (Alfieri et al., 1991; Petti et al., 1990
). It consists of a multi-repeat domain (W1W2) and a unique C-terminal domain (Y1Y2) and has five regions (CR1CR5) that are evolutionarily conserved among related primate gammaherpesviruses (Fig. 1
A, B) (Peng et al., 2000a
). As described above, EBNA-LP is considered critical for EBV-induced B-cell immortalization based on the observation that EBNA-LP mutant viruses show severely impaired transforming activity (Allan et al., 1992
; Hammerschmidt & Sugden, 1989
; Mannick et al., 1991
). However, the mechanisms by which EBNA-LP facilitates EBV-induced B cell immortalization remain to be elucidated. Several groups have shown a number of potential interactions of EBNA-LP with cellular proteins including pRb, p53, the 70 kDa family of heat shock proteins (Hsp70), HS1-associated protein X1, bcl-2,
- and
-tubulins, Hsp27, HA95, protein kinase A and oestrogen-related receptor 1 (Han et al., 2001
, 2002
; Igarashi et al., 2003
; Kawaguchi et al., 2000
; Kitay & Rowe, 1996b
; Mannick et al., 1995
; Matsuda et al., 2003
; Szekely et al., 1993
). In LCLs, EBNA-LP is localized to discrete nuclear foci (called ND10) that also contain Hsp70, an antigenically distinct form of pRb and CBP/p300 (Bandobashi et al., 2001
; Jiang et al., 1991
; Szekely et al., 1995
, 1996
). The plethora of interactions between EBNA-LP and various cellular proteins imply that EBNA-LP is a multifunctional protein that controls various components of the cellular machinery and that the functions of EBNA-LP in the virus life-cycle result from the sum of these interactions. Whatever the significance of these interactions, it is now generally accepted that a major activity of EBNA-LP is to stimulate EBNA-2-mediated transcriptional activation of viral and cellular genes such as LMP-1 and cyclin D2 (Harada & Kieff, 1997
; Nitsche et al., 1997
; Sinclair et al., 1994
). The mechanism by which EBNA-LP expresses its coactivator function has recently been investigated and several lines of evidence described below provide insight into the biochemical role for EBNA-LP in this function. Thus, we previously demonstrated that the conserved region CR2 (Fig. 1B
) is a multifunctional domain mediating self-association, nuclear localization and nuclear matrix association of the protein (Tanaka et al., 2002
; Yokoyama et al., 2001a
). We also mapped the major sites of phosphorylation of EBNA-LP by cellular kinase(s) to serine 35 (Ser-35) in the W2 repeat region (Fig. 1B
) (Yokoyama et al., 2001b
). The introduction of amino acid substitutions into CR2 or Ser-35 severely impaired the ability of EBNA-LP to induce the expression of LMP-1 in concert with EBNA-2 in B cells (Yokoyama et al., 2001a
, b
). The requirement for CR2 and Ser-35 for the functions of EBNA-LP was also reported by Peng et al. (2000b)
and McCann et al. (2001)
. Recently, Han et al. (2001
, 2002)
reported that HA95 and PKA form a complex with EBNA-LP and modulate the coactivator function of EBNA-LP. Taken together, these observations indicate that the modification, cellular localization and protein complex formation of EBNA-LP are critical for the regulation of its coactivator function.
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids.
pGEX-W2, pGEX-W2S35A and pGEX-W1W2 were generated by cloning the EcoRINotI fragments of pM-W2(H), pM-W2-S35A(H) and pM-EBNA-LPR1d1(H) (Yokoyama et al., 2001b), respectively, into the EcoRI and NotI sites of pGEX4T-1 (Amersham-Pharmacia) in frame with glutathione S-transferase (GST). The construction of the expression plasmid pME-EBNA-LPR3(F) or pME-EBNA-LPR3SA(F) containing Flag epitope-tagged wild-type EBNA-LP cDNA with three copies of the W repeat domain or a mutant version of Flag epitope-tagged EBNA-LP cDNA in which Ser-35 in each W repeat domain is substituted with alanine (Fig. 1C
) has been described elsewhere (Yokoyama et al., 2001b
). Here we refer to the gene products of pME-EBNA-LPR3(F) and pME-EBNA-LPR3SA(F) as EBNA-LPR3(F) and EBNA-LPR3SA(F), respectively. The expression plasmid pCMVcdc2 for human cdc2 was kindly provided by S. van den Heuvel (van den Heuvel & Harlow, 1993
). Construction of pAcGHLT-BGLF4 (see Fig. 3A
) and pME-BGLF4(F) has been described previously (Kato et al., 2001
). To generate pAcGHLT-BGLF4K102I (Fig. 3A
), the lysine at position 102 of BGLF4 was replaced with isoleucine using a QuikChange site-directed mutagenesis kit (Stratagene) with the oligonucleotide 5'-CAGATAATGCCACGGTCATACTCTATGACTCTGTG-3' and its complementary oligonucleotide, according to the manufacturer's directions. To construct pAS-BHRF1, a fragment containing the entire coding sequence of EBV BHRF1 was amplified using pRcCMV-BHRF1 as a template (kindly provided by G. Chinnadurai). The amplified fragment was digested with EcoRI and SalI and cloned into the EcoRI and SalI sites of pAS2 (Clontech) in frame with the DNA-binding domain of GAL4. pMAL-BHRF1 was generated by cloning the EcoRISalI fragment of pAS2-BHRF1 into the EcoRI and SalI sites of pMAL-c (New England BioLabs) in frame with maltose-binding protein (MBP). To construct pMAL-EBNA-LPR1, a fragment encoding the entire coding sequence of EBNA-LP with a single W1W2 repeat was amplified by PCR using pACT-EBNA-LPR1 (Tanaka et al., 2002
) as a template. The amplified fragment was digested with EcoRI and XbaI and cloned into the EcoRI and XbaI sites of pMAL-c in frame with MBP.
|
Purification of recombinant BGLF4 proteins from insect cells.
The GST, GSTBGLF4 or GSTBGLF4K102I fusion proteins were purified from Sf9 cells infected with the recombinant baculoviruses BacGST, BacGSTBGLF4 or BacGSTBGLF4K102I, respectively, as described previously (Kato et al., 2001; Kawaguchi et al., 2003
).
Production and purification of MBP or GST fusion proteins expressed in E. coli.
GST fusion proteins were expressed in E. coli BL21 transformed with either pGEX-W2, pGEX-W2S35A or pGEX-W1W2 and purified on glutathioneSepharose beads (Amersham-Pharmacia) as described previously (Kawaguchi et al., 1997a). MBP fusion proteins were expressed in E. coli XL-1 Blue transformed with either pMAL-BHRF1 or pMAL-EBNA-LPR1 and purified on amylose resin (New England BioLabs) according to the procedure followed for the purification of GST fusion proteins except that PBS containing 1 % Tween-20 was used instead of PBS containing 1 % Triton X-100.
In vitro kinase assays.
Purified MBP fusion proteins or GST fusion proteins captured on amylose or glutathioneSepharose beads were subjected to in vitro kinase assays. These assays were done to determine whether certain MBP or GST fusion proteins could serve as substrates for cdc2 (New England Biolabs) or GSTBGLF4, as described previously (Kawaguchi et al., 2003).
Phosphatase treatment.
After the in vitro kinase assays, the MBP fusion proteins or GST fusion proteins captured on the amylose or glutathioneSepharose beads were subjected to phosphatase treatment as described elsewhere (Kawaguchi et al., 2003).
Transfection, metabolic labelling and immunoprecipitation.
COS-7 cells were transfected with appropriate combinations of expression plasmids using the DEAE-dextran method as described preciously (Kawaguchi et al., 2000). The transfected cells were labelled with [32P]orthophosphate (Amersham Pharmacia) and then subjected to immunoprecipitation as described elsewhere (Kawaguchi et al., 1998
; Yokoyama et al., 2001b
).
Immunoblotting.
The electrophoretically separated proteins transferred to nitrocellulose sheets were reacted with appropriate antibodies as described previously (Kawaguchi et al., 1997b, 2000
, 2001
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To test this hypothesis, we expressed chimeric proteins consisting of GST fused either to the W2 domain of EBNA-LP (GSTW2) containing Ser-35, to a mutated W2 domain in which Ser-35 was substituted with alanine (GSTW2S35A), or to the W1W2 domain (GSTW1W2) in E. coli transformed with pGEX-W2, pGEX-W2S35A or pGEX-W1W2, respectively (Fig. 2A). GST alone and these GST fusion proteins captured on glutathioneSepharose beads were used as substrates for in vitro kinase assays in the absence or presence of purified cdc2.
|
To examine whether the labelling of GSTW2 with [-32P]ATP in the presence of cdc2 was due to phosphorylation, the labelled GSTW2 was boiled to inactivate kinases and then incubated with alkaline phosphatase. As shown in Fig. 2(E)
, the labelling of GSTW2 by incubation with cdc2 was eliminated by phosphatase treatment, indicating that GSTW2 was labelled with [
-32P]ATP by phosphorylation. The elimination of the labelling was not due to the degradation of GSTW2 by boiling or the phosphatase reaction, as shown by the observation that GSTW2 levels did not decrease after boiling or phosphatase treatment (Fig. 2D
). Taken together, this series of experiments indicates that cdc2 protein kinase specifically phosphorylates Ser-35 of EBNA-LP in vitro.
Cdc2 protein kinase mediates the phosphorylation of Ser-35 in EBNA-LP at the cellular level
To test whether cdc2 protein kinase phosphorylates Ser-35 of EBNA-LP at the cellular level, we examined the effect of cdc2 in co-transfection assays with wild-type EBNA-LP or its mutant in which Ser-35 in each W repeat domain was substituted with alanine (Fig. 1C). COS-7 cells transfected with appropriate combinations of expression plasmids using the DEAE-dextran method were labelled with [32P]orthophosphate. Immunoprecipitates obtained by incubation of a mouse monoclonal antibody to Flag epitope (M2; Sigma) with the labelled lysates were solubilized, electrophoretically separated in a denaturing gel and subjected to immunoblotting with anti-Flag antibody (M2) and autoradiography. The bands of immunoblots and autoradiographs were quantified with an image analyser, LAS-1000, using the software Image Gauge (Fuji Film). The relative amounts of phosphorylated EBNA-LP in autoradiographs were normalized against those of EBNA-LP in immunoblots (Fig. 2F
).
Since EBNA-LP is phosphorylated by endogenous cellular protein kinase(s) (Yokoyama et al., 2001b), EBNA-LPR3(F) was labelled with 32Pi even when it was expressed alone (Fig. 2F
, line 2). The level of EBNA-LP phosphorylation was reduced when Ser-35 was substituted with alanine (Fig. 2F
, lines 2 and 4), but EBNA-LPR3SA(F) was consistently labelled with 32Pi (data not shown) as we previously reported (Yokoyama et al., 2001b
).
Overexpression of cdc2 caused a significant increase in the phosphorylation of EBNA-LPR3(F) (Fig. 2F, lines 1 and 2), while this increase in phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine [EBNA-LPR3SA(F)] (Fig. 2F
, lines 3 and 4). These results indicate that cdc2 mediates the phosphorylation of Ser-35 in EBNA-LP at the cellular level.
EBV-encoded protein kinase BGLF4 mediates the phosphorylation of EBNA-LP at Ser-35
Recently, we demonstrated that cdc2 and conserved protein kinases encoded by herpesviruses, including herpes simplex virus type 1 (HSV-1) UL13 and EBV BGLF4, phosphorylate the same amino acid residues of target proteins (Kawaguchi et al., 2003). These observations together with the data obtained here and described above led us to hypothesize that an EBV-encoded conserved protein kinase, BGLF4, targets Ser-35 of EBNA-LP for phosphorylation. A series of experiments was designed to test this hypothesis.
In the first series of experiments, we generated and purified a kinase-negative mutant of BGLF4. We previously developed systems to express large amounts of recombinant BGLF4 in insect cells using a recombinant baculovirus and to obtain highly purified BGLF4 with enzymatic activity (Kato et al., 2001). In the present study, we attempted to test whether BGLF4 phosphorylates EBNA-LP in an in vitro assay using purified BGLF4. However, one might argue that the protein kinase activity detected in such experiments is due to contaminating kinase(s) that are physically associated with GSTBGLF4 or fortuitously pulled down by the affinity resins. To eliminate this possibility as far as possible, we constructed a mutant that had no intrinsic protein kinase activity but has probably retained its overall structure. To this end, we generated a recombinant baculovirus (BacBGLF4K102I) expressing BGLF4 fused to GST in which lysine-102 (Lys-102) of BGLF4 was replaced with isoleucine by site-directed mutagenesis. We chose Lys-102 in BGLF4 for the site-directed mutagenesis because of the following observations: (i) Lys-102 in BGLF4 corresponds to an invariant lysine in subdomain II of known protein kinases (Fig. 1D
) that is required for kinase activity (Hanks et al., 1988
); (ii) mutations of the corresponding lysines in BGLF4 homologues of pseudorabiesvirus (PRV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and HSV-1 were shown to result in the loss or reduction of kinase activity (de Wind et al., 1992
; He et al., 1997
; Kawaguchi et al., 2003
; Kenyon et al., 2001
); and (iii) it has been reported that overexpression of BGLF4 in mammalian cells mediates the hyperphosphorylation of viral protein EA-D, while that of a mutant of BGLF4 in which Lys-102 was replaced with glutamine did not (Gershburg & Pagano, 2002
).
GST, GSTBGLF4 or GSTBGLF4K102I was purified from Sf9 cells infected with either BacGST, BacGSTBGLF4 or BacGSTBGLF4K102I as described in Methods, electrophoretically separated in denaturing gels and either silver stained (Fig. 3B) or immunoblotted with rabbit antiserum containing anti-GST antibody (Kawaguchi et al., 2001
) (Fig. 3C
). Purified GST, GSTBGLF4 or GSTBGLF4K102I contained one major purified protein with an Mr of 32 000 (GST) or 78 000 (GSTBGLF4 or GSTBGLF4K102I), respectively (Fig. 3B
), which reacted with antiserum containing anti-GST antibody (Fig. 3C
). Purified GSTBGLF4 and GSTBGLF4K102I were also subjected to in vitro kinase assays to examine their enzymatic activity. When the purified proteins were incubated with [
-32P]ATP, the wild-type fusion protein was labelled by autophosphorylation (Fig. 3E
, lane 1), while the mutant was not (Fig. 3E
, lane 2). Furthermore, the labelling of the wild-type was eliminated by treatment with phosphatase (Fig. 3E
, lane 3). The expression of each GST fusion protein and the identity of the radiolabelled band were verified by CBB staining as shown in Fig. 3(D)
. These results indicate that: (i) the desired GST fusion protein was indeed purified; (ii) the kinase-negative mutant with a single amino acid substitution was obtained; and (iii) Lys-102 in BGLF4 is required for the kinase activity.
Next, we performed in vitro kinase assays using the purified recombinant BGLF4 proteins to test whether EBNA-LP is a BGLF4 substrate. A full-length EBNA-LP with a single W1W2 repeat or BHRF1 fused to MBP was purified from E. coli and purified MBP fusion protein (MBPEBNA-LPR1 or MBPBHRF1) captured on amylose resin served as substrate for in vitro kinase assays in the presence of the purified wild-type GSTBGLF4 and the kinase-negative mutant GSTBGLF4K102I. As shown in Fig. 4(B), MBPEBNA-LPR1 was labelled with [
-32P]ATP by a reaction with purified GSTBGLF4 (Fig. 4B
, lane 2), while MBPBHRF1 was not (Fig. 4B
, lane 1). When the kinase-negative mutant GSTBGLF4K102I was used instead of GSTBGLF4 in in vitro kinase assays, neither MBPBHRF1 nor MBPEBNA-LP was labelled (Fig. 4B
, lanes 3 and 4). The labelling of MBPEBNA-LPR1 by incubation with GSTBGLF4 was eliminated by phosphatase treatment (Fig. 4D
). The expression of each MBP fusion protein and the identity of the radiolabelled band of MBPEBNA-LP were verified by CBB staining as shown in Fig. 4(A)
and (C). This series of experiments indicates that BGLF4 specifically phosphorylates EBNA-LP in vitro.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cdc2 targets Ser-35 of EBNA-LP for phosphorylation
EBNA-LP is a phosphoprotein whose phosphorylation state is dependent on the cell cycle in latently infected cells (Kitay & Rowe, 1996a; Petti et al., 1990
). We previously identified Ser-35 as a major phosphorylation site of EBNA-LP by peptide mapping of purified EBNA-LP using mass spectrometry and mutational analyses of the protein (Yokoyama et al., 2001b
). Substitution of Ser-35 with an alanine codon resulted in a substantial reduction in the ability of EBNA-2 to transactivate the expression of LMP-1 in EBV-infected cells, while substitution with glutamic acid, which is known to mimic constitutive phosphorylation, restored the wild-type phenotype, indicating that Ser-35 is a functional phosphorylation site (Yokoyama et al., 2001b
). Although it has been reported that multiple sites of EBNA-LP can also be phosphorylated by cdc2 or casein kinase II in vitro (Kitay & Rowe, 1996a
), Ser-35 is the only functional site that has been shown to be phosphorylated in vivo and to be critical for the function of EBNA-LP (Yokoyama et al., 2001b
). In the present study, we have demonstrated that cdc2 phosphorylates the functional site Ser-35 in EBNA-LP, suggesting that cdc2 regulates the coactivator function of EBNA-LP by phosphorylating Ser-35. Our results are consistent with the published observations that phosphorylation of EBNA-LP in infected cells is maximal at G2/M when the activity of cdc2 is specifically up-regulated and that EBNA-LP is phosphorylated at serine residues only in infected cells (Kitay & Rowe, 1996a
; Smits & Medema, 2001
).
EBV-encoded protein kinase BGLF4 mediates the phosphorylation of EBNA-LP at Ser-35
Herpesviruses contain viral genes that encode protein kinases (Chee et al., 1989; Chung et al., 1989
; McGeoch & Davison, 1986
; Smith & Smith, 1989
). Among them, the amino acid sequences that encode a subset of the viral protein kinases exemplified by HSV-1 UL13 are conserved in all members of the family Herpesviridae (Chee et al., 1989
; Smith & Smith, 1989
). Here we refer to these viral protein kinases as CHPKs (conserved herpesvirus protein kinases). Recently we reported that the cellular protein kinase cdc2 and CHPKs phosphorylate the same amino acid residues of target proteins, suggesting that CHPKs mimic cdc2 enzymatically in infected cells. These conclusions are supported by two series of observations. First, cdc2 and CHPKs including HSV-1 UL13 and EBV BGLF4 phosphorylate the same site, Ser-133, of cellular elongation factor 1
, which has been reported to be commonly phosphorylated by CHPKs in herpesvirus-infected cells (Kato et al., 2001
; Kawaguchi et al., 1998
, 1999
, 2003
). Secondly, HSV-1 UL13 phosphorylates Ser-209 of the casein kinase II
subunit, which was reported to be targeted by cdc2 for phosphorylation (Kawaguchi et al., 2003
; Litchfield et al., 1991
). These observations prompted us to examine the possibility that EBV BGLF4 phosphorylates EBNA-LP at Ser-35. In the present study, we obtained evidence that this is in fact the case, further supporting our hypothesis that CHPKs mimic cdc2 in infected cells. One may wonder whether BGLF4 in fact interacts with and phosphorylates EBNA-LP in EBV-infected cells since EBNA-LP has been considered a latency-associated protein (Kieff & Rickinson, 2001
), while BGLF4 has been reported to be expressed in lytically infected cells (Gershburg & Pagano, 2002
). One possible explanation, based on the functional similarities of herpesvirus conserved gene products, is that BGLF4 can be a component of capsid tegument structures like the other CHPKs (Cunningham et al., 1992
; Overton et al., 1992
; Stevenson et al., 1994
; van Zeijl et al., 1997
), and is brought into the infected cells by virions and phosphorylates newly synthesized EBNA-LP, which is expressed first following infection of B cells by EBV (Alfieri et al., 1991
). Conceivably, it is beneficial to the virus for the virion-associated protein kinase to be brought into infected cells and to phosphorylate Ser-35 of EBNA-LP to express its coactivator function independent of the condition of target host cells, since in vivo EBV infects resting B cells, where the activity of cdc2, which would mediate the functional phosphorylation of EBNA-LP, is down-regulated (Smits & Medema, 2001
).
BGLF4K102I is a kinase-negative mutant
There is one puzzling aspect of the studies reported earlier with regard to the invariant catalytic lysine of BGLF4. Chen et al. (2000) previously found that in BGLF4 an amino acid substitution of Lys-102, which is predicted to be the catalytic site of the protein kinase based on analogy with known protein kinases (Hanks et al., 1988
), has no effect on the ability of the protein to autophosphorylate. In contrast, Gershburg & Pagano (2002)
demonstrated that wild-type BGLF4, which was overexpressed in mammalian cells, mediated the hyperphosphorylation of a viral protein, EA-D, while site-directed mutagenesis of Lys-102 impaired the ability of the protein to mediate the hyperphosphorylation. Furthermore, several laboratories have demonstrated that the corresponding lysines of the other BGLF4 homologues of herpesviruses, including PRV, VZV, HSV-1 and HCMV, cannot be mutated without loss or reduction of their protein kinase activity (de Wind et al., 1992
; He et al., 1997
; Kawaguchi et al., 2003
; Kenyon et al., 2001
). In the present study, we obtained evidence that amino acid substitution of Lys-102 of BGLF4 abolished the ability of the protein to autophosphorylate itself or trans-phosphorylate EBNA-LP. These results indicate that Lys-102 is essential for the activity of BGLF4 and suggest that it corresponds to the invariant catalytic lysine conserved in various protein kinases.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allan, G. J., Inman, G. J., Parker, B. D., Rowe, D. T. & Farrell, P. J. (1992). Cell growth effects of EpsteinBarr virus leader protein. J Gen Virol 73, 15471551.[Abstract]
Bandobashi, K., Maeda, A., Teramoto, N., Nagy, N., Szekely, L., Taguchi, H., Miyoshi, I., Klein, G. & Klein, E. (2001). Intranuclear localization of the transcription coadaptor CBP/p300 and the transcription factor RBP-Jk in relation to EBNA-2 and -5 in B lymphocytes. Virology 288, 275282.[CrossRef][Medline]
Ben-Sasson, S. A. & Klein, G. (1981). Activation of the EpsteinBarr virus genome by 5-aza-cytidine in latently infected human lymphoid lines. Int J Cancer 28, 131135.[Medline]
Chee, M. S., Lawrence, G. L. & Barrell, B. G. (1989). Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase. J Gen Virol 70, 11511160.[Abstract]
Chen, M. R., Chang, S. J., Huang, H. & Chen, J. Y. (2000). A protein kinase activity associated with EpsteinBarr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. J Virol 74, 30933104.
Chung, T. D., Wymer, J. P., Smith, C. C., Kulka, M. & Aurelian, L. (1989). Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). J Virol 63, 33893398.[Medline]
Cohen, J. I., Wang, F., Mannick, J. & Kieff, E. (1989). EpsteinBarr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc Natl Acad Sci U S A 86, 95589562.[Abstract]
Cunningham, C., Davison, A. J., Dolan, A., Frame, M. C., McGeoch, D. J., Meredith, D. M., Moss, H. W. & Orr, A. C. (1992). The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein kinase. J Gen Virol 73, 303311.[Abstract]
de Wind, N., Domen, J. & Berns, A. (1992). Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells. J Virol 66, 52005209.[Abstract]
Gershburg, E. & Pagano, J. S. (2002). Phosphorylation of the EpsteinBarr 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, 9981003.
Hammerschmidt, W. & Sugden, B. (1989). Genetic analysis of immortalizing functions of EpsteinBarr virus in human B lymphocytes. Nature 340, 393397.[CrossRef][Medline]
Han, I., Harada, S., Weaver, D., Xue, Y., Lane, W., Orstavik, S., Skalhegg, B. & Kieff, E. (2001). EBNA-LP associates with cellular proteins including DNA-PK and HA95. J Virol 75, 24752481.
Han, I., Xue, Y., Harada, S., Orstavik, S., Skalhegg, B. & Kieff, E. (2002). Protein kinase A associates with HA95 and affects transcriptional coactivation by EpsteinBarr virus nuclear proteins. Mol Cell Biol 22, 21362146.
Hanks, S. K., Quinn, A. M. & Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 4252.[Medline]
Harada, S. & Kieff, E. (1997). EpsteinBarr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J Virol 71, 66116618.[Abstract]
He, Z., He, Y. S., Kim, Y., Chu, L., Ohmstede, C., Biron, K. K. & Coen, D. M. (1997). The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines. J Virol 71, 405411.[Abstract]
Hudewentz, J., Bornkamm, G. W. & zur Hausen, H. (1980). Effect of the diterpene ester TPA on EpsteinBarr virus antigen- and DNA synthesis in producer and nonproducer cell lines. Virology 100, 175178.[Medline]
Igarashi, M., Kawaguchi, Y., Hirai, K. & Mizuno, F. (2003). Physical interaction of EpsteinBarr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization. J Gen Virol 84, 319327.
Jiang, W. Q., Szekely, L., Wendel-Hansen, V., Ringertz, N., Klein, G. & Rosen, A. (1991). Co-localization of the retinoblastoma protein and the EpsteinBarr virus-encoded nuclear antigen EBNA-5. Exp Cell Res 197, 314318.[Medline]
Kato, K., Kawaguchi, Y., Tanaka, M. & 10 other authors (2001). EpsteinBarr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1 (EF-1
): EF-1
is universally modified by conserved protein kinases of herpesviruses in mammalian cells. J Gen Virol 82, 14571463.
Kawaguchi, Y., Bruni, R. & Roizman, B. (1997a). Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery. J Virol 71, 10191024.[Abstract]
Kawaguchi, Y., Van Sant, C. & Roizman, B. (1997b). Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. J Virol 71, 73287336.[Abstract]
Kawaguchi, Y., Van Sant, C. & Roizman, B. (1998). Eukaryotic elongation factor 1 is hyperphosphorylated by the protein kinase encoded by the U(L)13 gene of herpes simplex virus 1. J Virol 72, 17311736.
Kawaguchi, Y., Matsumura, T., Roizman, B. & Hirai, K. (1999). Cellular elongation factor 1 is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses. J Virol 73, 44564460.
Kawaguchi, Y., Nakajima, K., Igarashi, M. & 8 other authors (2000). Interaction of EpsteinBarr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP. J Virol 74, 1010410111.
Kawaguchi, Y., Tanaka, M., Yokoymama, A., Matsuda, G., Kato, K., Kagawa, H., Hirai, K. & Roizman, B. (2001). Herpes simplex virus 1 alpha regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1. Proc Natl Acad Sci U S A 98, 18771882.
Kawaguchi, Y., Kato, K., Tanaka, M., Kanamori, M., Nishiyama, Y. & Yamanashi, Y. (2003). Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1. J Virol 77, 23592368.
Kaye, K. M., Izumi, K. M. & Kieff, E. (1993). EpsteinBarr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc Natl Acad Sci U S A 90, 91509154.[Abstract]
Kenyon, T. K., Lynch, J., Hay, J., Ruyechan, W. & Grose, C. (2001). Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63. J Virol 75, 88548858.
Kieff, E. & Rickinson, A. B. (2001). EpsteinBarr Virus and its replication. In Fields Virology, 4th edn, pp. 25112573. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott-Williams & Wilkins.
Kitay, M. K. & Rowe, D. T. (1996a). Cell cycle stage-specific phosphorylation of the EpsteinBarr virus immortalization protein EBNA-LP. J Virol 70, 78857893.[Abstract]
Kitay, M. K. & Rowe, D. T. (1996b). Proteinprotein interactions between EpsteinBarr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins. Virology 220, 9199.[CrossRef][Medline]
Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J. & Krebs, E. G. (1991). Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2. J Biol Chem 266, 2038020389.
Luka, J., Kallin, B. & Klein, G. (1979). Induction of the EpsteinBarr virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94, 228231.[Medline]
McCann, E. M., Kelly, G. L., Rickinson, A. B. & Bell, A. I. (2001). Genetic analysis of the EpsteinBarr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function. J Gen Virol 82, 30673079.
McGeoch, D. J. & Davison, A. J. (1986). Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses. Nucleic Acids Res 14, 17651777.[Abstract]
Mannick, J. B., Cohen, J. I., Birkenbach, M., Marchini, A. & Kieff, E. (1991). The EpsteinBarr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. J Virol 65, 68266837.[Medline]
Mannick, J. B., Tong, X., Hemnes, A. & Kieff, E. (1995). The EpsteinBarr virus nuclear antigen leader protein associates with hsp72/hsc73. J Virol 69, 81698172.[Abstract]
Marchini, A., Cohen, J. I., Wang, F. & Kieff, E. (1992). A selectable marker allows investigation of a nontransforming EpsteinBarr virus mutant. J Virol 66, 32143219.[Abstract]
Marin, O., Meggio, F., Draetta, G. & Pinna, L. A. (1992). The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2. FEBS Lett 301, 111114.[CrossRef][Medline]
Matsuda, G., Nakajima, K., Kawaguchi, Y., Yamanashi, Y. & Hirai, K. (2003). EpsteinBarr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1. Microbiol Immunol 47, 9199.[Medline]
Nitsche, F., Bell, A. & Rickinson, A. (1997). EpsteinBarr virus leader protein enhances EBNA-2-mediated transactivation of latent membrane protein 1 expression: a role for the W1W2 repeat domain. J Virol 71, 66196628.[Abstract]
Overton, H. A., McMillan, D. J., Klavinskis, L. S., Hope, L., Ritchie, A. J. & Wong-kai-in, P. (1992). Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion. Virology 190, 184192.[Medline]
Peng, R., Gordadze, A. V., Fuentes Panana, E. M., Wang, F., Zong, J., Hayward, G. S., Tan, J. & Ling, P. D. (2000a). Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses. J Virol 74, 379389.
Peng, R., Tan, J. & Ling, P. D. (2000b). Conserved regions in the EpsteinBarr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2. J Virol 74, 99539963.
Petti, L., Sample, C. & Kieff, E. (1990). Subnuclear localization and phosphorylation of EpsteinBarr virus latent infection nuclear proteins. Virology 176, 563574.[Medline]
Ragona, G., Ernberg, I. & Klein, G. (1980). Induction and biological characterization of the EpsteinBarr virus (EBV) carried by the Jijoye lymphoma line. Virology 101, 553557.[Medline]
Rickinson, A. B. & Kieff, E. (2001). EpsteinBarr virus. In Fields Virology, 4th edn, pp. 25752627. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott-Williams & Wilkins.
Saemundsen, A. K., Kallin, B. & Klein, G. (1980). Effect of n-butyrate on cellular and viral DNA synthesis in cells latently infected with EpsteinBarr virus. Virology 107, 557561.[Medline]
Sinclair, A. J., Palmero, I., Peters, G. & Farrell, P. J. (1994). EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by EpsteinBarr virus. EMBO J 13, 33213328.[Abstract]
Smith, R. F. & Smith, T. F. (1989). Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and EpsteinBarr virus. J Virol 63, 450455.[Medline]
Smits, V. A. & Medema, R. H. (2001). Checking out the G2/M transition. Biochim Biophys Acta 1519, 112.[Medline]
Stevenson, D., Colman, K. L. & Davison, A. J. (1994). Characterization of the putative protein kinases specified by varicella-zoster virus genes 47 and 66. J Gen Virol 75, 317326.[Abstract]
Szekely, L., Selivanova, G., Magnusson, K. P., Klein, G. & Wiman, K. G. (1993). EBNA-5, an EpsteinBarr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc Natl Acad Sci U S A 90, 54555459.[Abstract]
Szekely, L., Jiang, W. Q., Pokrovskaja, K., Wiman, K. G., Klein, G. & Ringertz, N. (1995). Reversible nucleolar translocation of EpsteinBarr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion. J Gen Virol 76, 24232432.[Abstract]
Szekely, L., Pokrovskaja, K., Jiang, W. Q., de The, H., Ringertz, N. & Klein, G. (1996). The EpsteinBarr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies. J Virol 70, 25622568.[Abstract]
Tanaka, M., Yokoyama, A., Igarashi, M., Matsuda, G., Kato, K., Kanamori, M., Hirai, K., Kawaguchi, Y. & Yamanashi, Y. (2002). Conserved region CR2 of EpsteinBarr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association. J Virol 76, 10251032.
Tomkinson, B., Robertson, E. & Kieff, E. (1993). EpsteinBarr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. J Virol 67, 20142025.[Abstract]
van den Heuvel, S. & Harlow, E. (1993). Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262, 20502054.[Medline]
van Zeijl, M., Fairhurst, J., Baum, E. Z., Sun, L. & Jones, T. R. (1997). The human cytomegalovirus UL97 protein is phosphorylated and a component of virions. Virology 231, 7280.[CrossRef][Medline]
Yokoyama, A., Kawaguchi, Y., Kitabayashi, I., Ohki, M. & Hirai, K. (2001a). The conserved domain CR2 of EpsteinBarr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization. Virology 279, 401413.[CrossRef][Medline]
Yokoyama, A., Tanaka, M., Matsuda, G. & 8 other authors (2001b). Identification of major phosphorylation sites of EpsteinBarr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA-2 is regulated by phosphorylation. J Virol 75, 51195128.
zur Hausen, H., O'Neill, F. J., Freese, U. K. & Hecker, E. (1978). Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature 272, 373375.[Medline]
zur Hausen, H., Bornkamm, G. W., Schmidt, R. & Hecker, E. (1979). Tumor initiators and promoters in the induction of EpsteinBarr virus. Proc Natl Acad Sci U S A 76, 782785.[Abstract]
Received 25 June 2003;
accepted 3 September 2003.