Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens, Greece1
Department of Experimental and Diagnostic Medicine (Section of Microbiology), University of Ferrara, Via Luigi Borsari 46, I-44100 Ferrara, Italy2
Interdepartmental Center for Biotechnology, University of Ferrara, Via Fossato di Mortara 64-B, I-44100 Ferrara, Italy3
Author for correspondence: Penelope Mavromara. Fax +30 1 6423498. e-mail penelopm{at}hol.gr
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Abstract |
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Several studies have indicated that the gE homologue of VZV is phosphorylated at the cytoplasmic tail of the protein by host kinases including casein kinase II (CKII) and casein kinase I (CKI) (Grose et al., 1989 ; Yao et al., 1993
). By analogy with the VZV gE, an acidic region has been identified in the cytoplasmic tail of HSV-1 gE with consensus recognition sites for CKII (Edson, 1993
; Ng et al., 1998
). Moreover, it has been reported that the viral UL13 kinase of HSV-1 is able to catalyse the phosphorylation of gE under both in vivo and in vitro labelling conditions (Ng et al., 1998
). It was also shown that exogenous CKII phosphorylates gE-1 protein in immune complexes from lysates of HSV-1-infected cells (Ng et al., 1998
). However, there is as yet no experimental evidence supporting phosphorylation of the HSV-1 gE protein in vivo in the absence of other viral products, nor have any sites of phosphorylation been identified.
In this study, we characterized further the nature of the phosphorylation event involving HSV-1 gE. Firstly, we demonstrated that gE can be phosphorylated in vivo in stably transfected cells, in the absence of any other viral gene products. Secondly, we provided experimental evidence for the location of the phosphorylation site(s) within the cytoplasmic tail of the polypeptide. Moreover, by using an in vitro kinase assay combined with mutational analysis, we confirmed the involvement of the cellular kinase CKII and established that serine residues 476 and/or 477 in the endodomain of gE-1 are major phosphoacceptor sites.
In order to determine whether gE-1 was phosphorylated in vivo in the absence of other viral proteins, we investigated the extent of phosphorylation of the protein in a stably transformed 293 cell line (embryonal kidney adherent cell line; ATCC CRL 1573) expressing the entire gE-1 protein. A 1·9 kb ApaINruI fragment (nt 141131142974) containing the gE-1-encoding gene (US8) was cloned into the BamHI cloning site of pRP-RSV expression vector under the control of the RSV promoter, yielding plasmid pHPI414 (Fig. 1a). Plasmid pHPI414 was first tested for the expression of gE-1 protein in transiently transfected 293 cells and was used subsequently for the construction of 293gE stable cell lines (Miriagou et al., 1995
). Two clones, designated 4A1 and 6C2, were found to produce sufficient amounts of gE protein (hgE-1) and were selected for further studies. For the phosphorylation experiments, confluent cell monolayers (4x106 cells) of mock-infected 293 cells, 4A1 and 6C2 cell clones and 293 cells infected with HSV-1 (F) were labelled with [32P]orthophosphate (250 µCi/ml) or [35S]methionine (45 µCi/ml) for 2 h. gE proteins were immunoprecipitated with anti-maltose-binding protein (MBP)gE V3 polyclonal serum (Miriagou et al., 1995
), separated on 10% SDSpolyacrylamide gels, transferred to nitrocellulose membranes and autoradiographed. As shown in Fig. 2(a)
, one strong protein band of 66 kDa and a weak, diffuse band of 80 kDa were present in [35S]methionine-labelled 4A1 and 6C2 cell lysates (lanes 4A1 and 6C2). The mobilities of these protein products are in agreement with the sizes of the precursor and the mature forms, respectively, of the viral gE-1 glycoprotein. Similar protein species were observed in immunoprecipitates of HSV-1-infected 293 cells, but were absent from mock-infected 293 cell lysates (lanes F and M, respectively). Two polypeptide bands, of 66 and 80 kDa, were detected in [32P]orthophosphate-labelled cell lysates of HSV-1-infected 293 cells (Fig. 2b
, lane F) as well as of stably gE-transformed cells (Fig. 2b
, lanes 4A1 and 6C2). Neither of these phosphoproteins was observed in mock-infected cells (Fig. 2b
, lane M). These results indicate that both the mature and precursor forms of hgE-1 protein were phosphorylated under in vivo labelling conditions in the absence of any other HSV gene products. Moreover, these data suggest that phosphorylation is an early event in the maturation process of gE-1.
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The HSV-1 gE contains an acidic region between amino acids 468 and 484 carrying putative CKII phosphorylation recognition site(s). Furthermore, recent studies have shown that CKII can catalyse the phosphorylation of the gE-1 protein in vitro, in immunoprecipitates of cells infected with HSV-1 (Ng et al., 1998 ). To examine the possibility that HSV-1 gE contains functional CKII phosphorylation sites in its endodomain, we first established an in vitro CKII kinase assay by using purified gE-1 protein expressed in E. coli as a fusion protein with MBP. Using this system, we addressed the role of the serine residues within the acidic region between amino acids 468 and 484 in CKII-mediated phosphorylation by in vitro mutagenesis.
Two MBPgE fusion proteins were produced: MBPgE-1, containing amino acids 90550 (460 aa) of gE-1, and MBPgE-1t, a tailless gE-1 fusion protein, containing amino acids 90406 (316 aa) of gE-1 (Fig. 1a). Both MBPgE proteins lacked the gE-1 signal sequence, which might be toxic when expressed in E. coli cells (Rose & Shafferman, 1981
). MBPgE-1 protein is encoded by plasmid pHPI413, which was constructed by inserting the 1·5 kb SphINruI fragment (nt 141511142974) into the XmnI cloning site of pMAL-c2. MBPgE-1t protein is encoded by the previously described plasmid pHPI427 (Miriagou et al., 1995
). Expression of the MBPgE-1 and MBPgE-1t fusion proteins was tested by Western blot analysis of protein extracts from E. coli cells transformed with pHPI427 or pHPI413 plasmids by using an anti-MBP polyclonal antiserum (New England Biolabs) and the anti-gE monoclonal antibody II 481-A6 (kindly provided by P. Spear, Northwestern University Medical School, Chicago, IL, USA). A protein band of 78 kDa (MBPgE-1t) or 92 kDa (MBPgE-1) was detected in extracts of cells harbouring pHPI427 and pHPI413 plasmids, respectively. This band was absent in cells harbouring the pMAL-c2 plasmid (data not shown). Both the MBPgE-1t and MBPgE-1 fusion proteins were isolated and used as substrates for the in vitro phosphorylation experiment with CKII. MBP was used as a negative control. Cells from 1·5 ml culture were lysed and the MBPgE proteins were immunoprecipitated by the anti-MBP polyclonal antiserum. The immune complexes were collected on protein ASepharose CL-4B beads and resuspended in 100 µl CKII reaction mixture containing 20 mM TrisHCl, 50 mM KCl, 10 mM MgCl2 and 200 µM cold ATP (New England Biolabs). The reaction was initiated by the addition of 25 U CKII (New England Biolabs) followed immediately by the addition of 20 µCi [
-32P]ATP (NEN, 3000 Ci/mmol) per reaction. The mixture was incubated at 30 °C for 1 h. Proteins were eluted from the protein ASepharose by boiling for 5 min in SDSmercaptoethanol denaturing buffer, separated on 10% SDSPAGE, transferred to nitrocellulose membrane and exposed to autoradiography. As shown in Fig. 3(a)
, the MBPgE-1 protein was phosphorylated in vitro (lane 2), whereas phosphorylation of the truncated MBPgE-1t protein was not detectable (lane 3). Similarly, MBP was not phosphorylated (lane 4). The proteins MBPgE-1, MBPgE-1t and MBP were detected in comparable quantities when they were probed with the anti-MBPgE polyclonal serum V3 (Fig. 3b
, lanes 24). The results shown in Fig. 3(a)
indicate that CKII can phosphorylate E. coli-produced MBPgE-1 fusion protein but not the truncated MBPgE-1t. These data are consistent with the results obtained from the in vivo phosphorylation experiments, as described above, and support the presence of functional CKII sites in the tail of the gE-1 protein.
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Expression of the mutated gE-1 fusion protein (MBPgE-1m) was tested by Western blot as described above and a protein of 92 kDa was detected in extracts of cells harbouring the pHPI438 plasmid (Fig. 3b, lane 1). When the MBPgE-1m protein was used as the substrate in an in vitro phosphorylation experiment with CKII, only trace amounts of the MBPgE-1m protein appeared to be phosphorylated (Fig. 3a
, lane 1). The drastic reduction of phosphorylation associated with the alteration of Ser476Ser477 of the gE-1 tail (>90% compared with wild-type MBPgE-1 protein) suggests that these amino acids represent the main acceptors for the phosphate groups on the gE-1 polypeptide. However, the residual phosphorylation of the mutant protein suggested that the third serine residue is probably also phosphorylated.
This report provides experimental evidence supporting similarities in the nature of phosphorylation between the gE homologues of HSV-1 and VZV. We have shown that HSV-1 gE is phosphorylated in the absence of other viral proteins, which strongly suggests that cellular kinase(s) phosphorylate the gE-1 protein. Furthermore, we showed that the endodomain of gE contains the major phosphorylation sites for cellular kinases, inasmuch as a tailless gE-1 was no longer phosphorylated. In addition, the results showed that the MBPgE-1 fusion proteins produced in E. coli were phosphorylated in vitro by CKII only when the C-terminal portion of the gE-1 protein was intact. Furthermore, when Ser476 and/or Ser477 were mutated, the level of phosphorylation of the gE fusion protein was reduced greatly, supporting the functional importance of these amino acids in the CKII-mediated reaction. The similarities in the nature of phosphorylation of the gE homologues of VZV, HSV-1 and 2 and PRV suggest an important biological function of such post-translational modifications, which remains to be elucidated for all neurotropic herpesviruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 September 1999;
accepted 2 December 1999.