The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins

Vivi Miriagoub,1, Lara Stevanato2, Roberto Manservigi2,3 and Penelope Mavromara1

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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Herpes simplex virus 1 glycoprotein E (gE-1) is highly phosphorylated in culture cells during infection. In this report, it is shown that phosphorylation is mediated by host enzymes in human cells stably transfected with gE, in the absence of other herpesvirus products. In contrast, a tailless gE product (C terminus deletion mutant) is not phosphorylated. By using an in vitro kinase assay combined with linker-insertion mutagenesis, it is shown that casein kinase II catalyses the phosphorylation of the C-terminal domain of the protein. Also, it is demonstrated that the serine residues at positions 476 and/or 477 in the cytoplasmic portion of the protein are the major acceptors for the phosphate groups.


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Phosphorylation of viral glycoproteins is a rare post-translational modification event during virus infections (Grose, 1990 ). Of the 11 known glycoproteins of herpes simplex virus type 1 (HSV-1), at least three, glycoproteins B, E and I, are phosphoglycoproteins (Edson et al., 1987 ; Ng et al., 1998 ). In addition, glycoprotein E (gE-1) undergoes palmitoylation and sulphation (Hope et al., 1982 ; Hope & Marsden, 1983 ; Johnson & Spear, 1983 ). gE serves as an Fc receptor for human IgG and its function is enhanced by association with the gI protein (Baucke & Spear, 1979 ; Para et al., 1982 ; Johnson & Feenstra, 1987 ; Johnson et al., 1988 ). Both glycoproteins are required for the cell-to-cell spread of the virus and are essential for the spread of the infection throughout the host nervous system (Balan et al., 1994 ; Dingwell et al., 1994 ; Dingwell & Johnson, 1998 ). However, neither gE-1 nor gI-1 is essential for propagation of the virus in vitro (Longnecker & Roizman, 1987 ; Longnecker et al., 1987 ). Although the biological significance of the phosphorylation of gE-1 remains elusive, the conservation of phosphorylation in the gE homologues of other neurotropic viruses, such as HSV-2, varicella-zoster virus (VZV) and pseudorabies virus (PRV), combined with the absence of these proteins in the beta- and gammaherpesviruses, suggest an important physiological role in virus pathogenesis.

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 ApaI–NruI fragment (nt 141131–142974) 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% SDS–polyacrylamide 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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Fig. 1. (a) Schematic representation depicting gE coding domains of the hgE-1, hgE-1s, MBP–gE-1, MBP–gE-1t and MBP–gE-1m proteins expressed by pHPI414, pHPI404, pHPI413, pHPI427 and pHPI438 plasmids, respectively (1–5). The star indicates the location of the two serine residues at the C terminus of the gE-1 protein. (b) Primary nucleotide sequences and inferred amino acid sequences of the serine-rich acidic region (aa 468–484) in the cytoplasmic tail of the wild-type gE-1 and the mutated gE-1m proteins. The underlined sequences represent the nucleotides altered by XbaI linker-insertion mutagenesis.

 


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Fig. 2. Expression and phosphorylation of HSV gE-1 protein in stably transformed 293 cells. Recombinant proteins hgE-1 and hgE-1s from cell lysates from A'3-9, 4A1, 6C2 clones and the viral gE-1 protein from 293 cells infected with HSV-1 and the supernatant culture medium from the A'3-9 clone were immunoprecipitated. The cells had been labelled with [35S]methionine (a) or [32P]orthophosphate (b) for 2 h. HSV-1-infected 293 cells were labelled 11–13 h post-infection. The gE-1 proteins from cells and supernatants were immunoprecipitated by the anti-MBP–gE polyclonal antiserum V3, separated on 10% SDS–PAGE, transferred to nitrocellulose membranes and exposed to autoradiography. Lanes: Ms, 293 cell supernatant; M, 293 cell lysate; A'3-9s, A'3-9 cell clone supernatant; A'3-9, A'3-9 cell clone lysate; 4A1, 4A1 cell clone lysate; 6C2, 6C2 cell clone lysate; F, lysate of 293 cells infected with HSV-1. The arrows indicate the positions of the precursor (filled arrow) and mature (open arrow) forms of the recombinant and viral gE-1 proteins. The asterisk indicates the position of the secreted hgE-1s recombinant protein.

 
Having established that HSV-1 gE is phosphorylated in transformed 293 cells, we tested whether the major region for phosphorylation is located in the endodomain of the protein. For this purpose, a 293 cell line (A'3-9) expressing a truncated gE-1 protein (hgE-1s) lacking the last 144 amino acid residues (Miriagou et al., 1995 ) was examined. In [35S]methionine-labelled A'3-9 cells, a protein band of about 45 kDa corresponding to the precursor form of hgE-1s was detected in the cell lysates (Fig. 2a, lane A'3-9), and a band of 54 kDa corresponding to the mature hgE-1s was detected in the culture supernatant. In contrast, immunoprecipitations of 32P-labelled cell extracts indicated that the phosphate failed to be incorporated into either the secreted protein or the cell-associated form of the protein. These results indicate that the major region for gE-1 phosphorylation is located within the C-terminal part of the protein, similar to the VZV gE homologue.

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 MBP–gE fusion proteins were produced: MBP–gE-1, containing amino acids 90–550 (460 aa) of gE-1, and MBP–gE-1t, a tailless gE-1 fusion protein, containing amino acids 90–406 (316 aa) of gE-1 (Fig. 1a). Both MBP–gE proteins lacked the gE-1 signal sequence, which might be toxic when expressed in E. coli cells (Rose & Shafferman, 1981 ). MBP–gE-1 protein is encoded by plasmid pHPI413, which was constructed by inserting the 1·5 kb SphI–NruI fragment (nt 141511–142974) into the XmnI cloning site of pMAL-c2. MBP–gE-1t protein is encoded by the previously described plasmid pHPI427 (Miriagou et al., 1995 ). Expression of the MBP–gE-1 and MBP–gE-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 (MBP–gE-1t) or 92 kDa (MBP–gE-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 MBP–gE-1t and MBP–gE-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 MBP–gE proteins were immunoprecipitated by the anti-MBP polyclonal antiserum. The immune complexes were collected on protein A–Sepharose CL-4B beads and resuspended in 100 µl CKII reaction mixture containing 20 mM Tris–HCl, 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 [{gamma}-32P]ATP (NEN, 3000 Ci/mmol) per reaction. The mixture was incubated at 30 °C for 1 h. Proteins were eluted from the protein A–Sepharose by boiling for 5 min in SDS–mercaptoethanol denaturing buffer, separated on 10% SDS–PAGE, transferred to nitrocellulose membrane and exposed to autoradiography. As shown in Fig. 3(a), the MBP–gE-1 protein was phosphorylated in vitro (lane 2), whereas phosphorylation of the truncated MBP–gE-1t protein was not detectable (lane 3). Similarly, MBP was not phosphorylated (lane 4). The proteins MBP–gE-1, MBP–gE-1t and MBP were detected in comparable quantities when they were probed with the anti-MBP–gE polyclonal serum V3 (Fig. 3b, lanes 2–4). The results shown in Fig. 3(a) indicate that CKII can phosphorylate E. coli-produced MBP–gE-1 fusion protein but not the truncated MBP–gE-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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Fig. 3. In vitro phosphorylation of MBP, MBP–gE-1t, MBP–gE-1 and MBP–gE-1m fusion proteins by CKII with [{gamma}-32P]ATP as the phosphate donor. (a) Autoradiographic images of the MBP–gE fusion proteins phosphorylated in vitro by CKII as described in the text. The fusion proteins were immunoprecipitated with the anti-MBP polyclonal antiserum. The proteins were separated on 10% SDS–PAGE, transferred to nitrocellulose membrane and exposed to autoradiography. The arrowhead indicates the position of the 92 kDa MBP–gE-1 fusion protein. (b) Western blot analysis of the MBP–gE fusion proteins after in vitro phosphorylation by CKII. The immunoprecipitated proteins were probed with the anti-MBP-gE polyclonal antiserum V3. The arrowheads indicate the positions of the 92 kDa MBP–gE-1 and/or MBP–gE-1m (filled arrowhead) and the 78 kDa MBP–gE-1t (open arrowhead) proteins. Positions of molecular mass markers (in kDa) are indicated.

 
In order to identify the sites of CKII-mediated phosphorylation, we used linker-insertion mutagenesis to alter the two serine residues at positions 476 and 477 within the acidic region of the cytoplasmic tail of the gE-1 protein (Fig. 1b). The mutated gE-1 protein was again expressed as fusion protein with MBP. For this purpose, the plasmid pHPI438 was constructed to contain an XbaI linker (5' GCTCTAGAGC 3') in the unique SacI site (nt 142671) of the US8 gene (Fig. 1b). Plasmid pHPI438 expresses the same form of the MBP–gE-1 fusion protein as pHPI413 except for the two serine residues at positions 476 and 477 of gE-1, which were replaced with the amino acid sequence Ala–Leu–Glu–Pro (Fig. 1b).

Expression of the mutated gE-1 fusion protein (MBP–gE-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 MBP–gE-1m protein was used as the substrate in an in vitro phosphorylation experiment with CKII, only trace amounts of the MBP–gE-1m protein appeared to be phosphorylated (Fig. 3a, lane 1). The drastic reduction of phosphorylation associated with the alteration of Ser476–Ser477 of the gE-1 tail (>90% compared with wild-type MBP–gE-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 MBP–gE-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.


   Acknowledgments
 
This work was supported by grants from the National Research Council (CNR) Target Project ‘Biotechologie’, from Telethon (A 100) and from National Research funds from the Greek Ministry of Industry and Technology.


   Footnotes
 
b Present address: Department of Microbiology, Evangelismos Hospital, 45–47 Ipsilantou Street, Athens, Greece.


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Received 14 September 1999; accepted 2 December 1999.