Institute of Haematology and Blood Transfusion, Dept of Experimental Virology, Prague 128 20, Czech Republic1
Institute of Organic Chemistry and Biochemistry, Dept of Protein Biochemistry, Prague 166 10, Czech Republic2
Author for correspondence: Lucie Mareová. Fax +420 2 21977392. e-mail lucie-maresova{at}uiowa.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unlike gH, the gL genes are not well conserved among herpesviruses. Typically, gL has an N-terminal hydrophobic domain characteristic of a signal sequence; however, gL has no other predicted membrane-spanning domains. Not only is VZV gL smaller than HSV-1 gL, but it also lacks a typical N-terminal endoplasmic reticulum (ER) signal sequence (Davison & Scott, 1986 ). On the other hand, VZV gL possesses a 16 residue sequence which is common with members of the ER-targeting family of proteins (Duus et al., 1995
). In the absence of gH, the gL proteins are either secreted, as is the case of HCMV gL (Spaete et al., 1993
) and HSV-1 gL (Dubin & Jiang, 1995
), or they are found as type 2 membrane proteins, like EBV gL (Li et al., 1995
). A model of post-translational VZV gH:gL regulation was proposed whereby the gL chaperone modulated gH expression via retrograde flow from the Golgi apparatus to the ER (Duus & Grose, 1996
).
It has been reported that in certain herpesviruses, additional viral glycoproteins or cellular proteins are required for proper gH:gL interaction (L. Li et al., 1997 ; Wang et al., 1998
). For example, glycoprotein O has been found to be associated with gH:gL in HCMV virions (Huber & Compton, 1997
, 1998
). Moreover, EBV has added a unique glycoprotein, gp42, for proper gH:gL complex formation (gH:gL:gp42). This specific adaptation of EBV is required for infection of B lymphocytes (Li et al., 1995
). The differences in gH:gL can reflect various models of virus penetration into different cell types and a different tropism of the virus. It remains to be determined whether VZV and the other members of the alphaherpesvirus subfamily require another component in addition to the gH:gL complex. In this work, formation of the VZV gH:gL complex was analysed in greater detail through the use of a recombinant vaccinia virus expression system.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The double recombinant VV-gE+gB was generated by recombination of the VV-gE-HA virus with the pgB plasmid. Construction of the VV-gE-HA recombinant, which expresses the gE gene inserted in the HA gene, was described previously by Kutinová et al. (1999) . For the construction of the double recombinant VV-gH+gL, the pgL plasmid containing the gL gene (N
me
kováet al., 1996
) was digested with NruI and HindIII, and its cohesive ends were filled by Klenow enzyme. This fragment, containing the gL gene, the VV 11k promoter and the gpt gene (under control of the VV I3 promoter), was cloned in the NruI site of pVV-HA plasmid (Kutinová et al., 1999
). The resulting plasmid pgL-HA was used for recombination with the single VV-gH recombinant. Vaccinia virus VV-E/L-lacZ, which expresses
-galactosidase, was prepared by recombination with plasmid pMJ601 (Davison & Moss, 1990
).
Antibodies.
Human anti-gH specific V3MAb, which recognizes a conformation-dependent virus-neutralization epitope (Sugano et al., 1991 ), and human anti-gB specific V1MAb (obtained from T. Sugano, Tokyo, Japan) were used at a stock concentration of 1 mg/ml. High VZV titre serum from a herpes zoster patient was precleared by absorption with VV antigens (N
me
ková et al., 1996
), and shown to be free of anti-VV antibodies by ELISA with VV antigens. Rabbit antiserum 60A was raised against a synthetic peptide fragment of the VZV gL. The immunogenic peptide GEEHKSGDIRD predicted by the ANTIGEN program (PCgene, Intelli Genetics) corresponds to amino acid residues 111121 of the gL sequence. The synthetic peptide coupled to keyhole limpet haemocyanin (Sigma) was used for immunization according to a standard protocol (Harlow & Lane, 1988
).
Radioimmunoprecipitation procedures.
Overnight-cultured CV-1 cells (6x105) were starved for 3 h in methionine/cysteine-deficient Dulbeccos modified Eagle medium (Sigma) (labelling medium) supplemented with 10% foetal bovine serum and L-glutamine (0·58 µg/ml). Infection with VV recombinant viruses was performed at an m.o.i. of 5 p.f.u. per cell. A mixture of [35S]methionine/[35S]cysteine of 83 µCi (Pro-Mix, Amersham) in 1 ml of labelling medium was added at the same time. The cell cultures were incubated for 18 h, then harvested, washed with PBS and lysed for 30 min on ice with 0·2 ml of lysis buffer (50 mM TrisHCl pH 8·0, 150 mM NaCl, 1% NP-40) containing the protease inhibitor cocktail (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 44 µg/ml PMSF, 50 µg/ml TLCK and 90 µg/ml TPCK). The cell lysates were clarified by centrifugation at 19000 g for 10 min at 4 °C.
Prior to harvesting the culture medium was removed from radiolabelled CV-1 cells and immediately treated with the protease inhibitor cocktail, and centrifuged gently at 300 g for 10 min at 4 °C to remove dead cells and other cellular debris. The medium was then centrifuged at 100000 g for 1 h. The supernatant was removed and concentrated tenfold in Centricon-10 concentrator tubes (Amicon) at 4 °C and incubated with the same volume of 2x lysis buffer for 30 min on ice.
Both the lysates and the media were precleared by incubation with 10 µl of human VZV-negative serum for 1 h on ice. The immune complexes were removed by 10 µl Protein ASepharose beads (Sigma). For immunoprecipitation of VZV glycoproteins, 25 µl of either MAbs or serum from the zoster patient was added and the samples were incubated for 1 h on ice. Precipitates were collected on Protein ASepharose beads. After overnight incubation, the beads were washed in lysis buffer, resuspended in Laemmli sample buffer and heated at 95 °C for 10 min.
Immunoprecipitated proteins were analysed by gradient (7·520% polyacrylamide) SDSPAGE under reducing conditions (Laemmli sample buffer with 5% -mercaptoethanol) (Laemmli, 1970
). Protein mobility was calibrated by unlabelled and 14C-radiolabelled molecular mass standards (Biorad, Amersham). Gels were incubated with Amplify (Amersham), dried and exposed to autoradiographic film Hyperfilm-
max (Amersham).
Treatment with glycosidases.
The immune complexes collected on Protein ASepharose beads were washed with lysis buffer and PBS. For endoglycosidase H (Endo H) digestion, proteins were dissociated from the beads by heating at 95 °C for 6 min in 35 mM Na/phosphate, pH 5·4, 0·7% SDS, 3·5 mM dithiothreitol. The eluted material was then diluted with 4 vols of 35 mM Na/phosphate, pH 5·4 and incubated for 24 h at 37 °C with or without 20 mU Endo H (Boehringer) per ml. For peptide:N-glycosidase F (PNGase F) digestion, proteins were dissociated from the beads by heating at 95 °C for 6 min in 0·1 M Na/phosphate, pH 8·6, 0·2% SDS, 10 mM EDTA, 0·1 mM PMSF, 5% -mercaptoethanol. The eluted material was then diluted with 2 vols of 0·5% octyl
-D-glucopyranoside, and incubated for 24 h at 37 °C with or without 3 U PNGase F (Boehringer) per ml. Deglycosylated proteins were precipitated with 80% acetone.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Release of the VZV gH:gL complex into culture media
To test whether VZV glycoproteins were secreted from the CV-1 cells, we analysed the culture media of cells infected with the VV recombinants and their various coinfection mixtures (Fig. 5). In cells that coexpressed gH+gL, gH was secreted in the complex with gL (lane 4). The molecular mass of the secreted gH (precipitated by serum from a zoster patient or V3MAb) was approximately 114 kDa, which was lower by 45 kDa than the mature intracellular gH (118 kDa) (Fig. 4B
, lane 2 and Fig. 4A
, lanes 3 and 6). An intermediate 97 kDa form of gH produced by the simple VV-gH recombinant in cell lysates (Fig. 2
, lane 4) was not identified in the culture medium (Fig. 5
, lane 3). Results in Figs 4
and 5
show that gL was detected in the culture medium only if coprecipitated by V3MAb or serum from a zoster patient. This secreted gL form (Fig. 4A
, lanes 3 and 6) had the same molecular mass of 18 kDa as that of gL coprecipitated (by V3MAb or zoster patient serum) in the complex with gH from the cell lysates (Fig. 4A
, lanes 3 and 6 and Fig. 4C
, lane 2). The 60A antiserum was not able to precipitate any gL molecule from the culture medium of the cells infected with the VV-gL recombinant alone (Fig. 4A
, lanes 2 and 5). Thus, gL was detected in media only when associated with the secreted gH form.
|
Finding the VZV glycoproteins in culture medium cannot be attributed solely to the CV-1 cells damaged upon VV infection. We did not detect any -galactosidase activity as a leakage marker in the culture medium of the control experiment with infection of the VV-lacZ recombinant virus alone (Fig. 6
).
|
An analysis of gL expressed after VV-gH+gL infection revealed that the 18 kDa gL form precipitated from the cell lysates was digested by Endo H (Fig. 4C, lane 2 and Fig. 4A
, lane 3) with a decrease in molecular mass of about 4 kDa. An analogous shift in mobility was observed after PNGase F digestion (Fig. 4A
, lane 6). This finding suggested that one high-mannose/hybrid-type N-linked oligosaccharide was removed. Furthermore, the glycosidase results were the same for both intracellular and secreted 18 kDa gL (Fig. 4C
, lane 2 and Fig. 4A
, lanes 3 and 6), indicating that processing of gL was not changed by trafficking of the gH:gL complex to the culture media.
The deglycosylation pattern of both forms of gL detected by the 60A antiserum was tested after single VV-gL expression in the cell lysates (Fig. 4A, lanes 2 and 5). The same downward shift to approximately 14 kDa occurred for both the 18 kDa and 19 kDa gL form after digestion by either glycosidase. Thus, both forms of gL differ in processing of one high mannose or hybrid-type N-linked oligosaccharide. Since the analogous result about deglycosylation of 18 kDa gL was obtained from single VV-gL and double VV-gH+gL expression (Fig. 4A
, lanes 23 and 56, respectively), we infer that the maturation of gL was not influenced by coexpression and complex formation with gH.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A heterogeneous molecular pattern of VZV gL was identified for CV-1 cells infected with a single VV-gL recombinant. Two gL forms, of 18 kDa and 19 kDa, were detected by the 60A antiserum recognizing amino acid residues 111121 of the gL sequence. Analysis of the oligosaccharide moiety revealed that both forms of gL differed only in the processing of one N-linked oligosaccharide of high mannose/hybrid-type sensitive to Endo H. It has been shown previously that gL does not change its molecular mass if expressed in the presence of monensin, which indicates that gL maturation does not require processing in the trans-Golgi system (Duus & Grose, 1996 ). Our data on Endo H sensitivity localized the final oligosaccharide processing step of gL within the early part of the medial-Golgi system, according to the spectrum of glycosylation reactions within the Golgi system (Kornfeld & Kornfeld, 1985
).
The heterogeneity of gL forms has not been studied in detail to date. Only one band of gL was previously observed in the VVT7 cotransfection systems with VZV glycoproteins, but two closely migrating gL species have appeared in cultured cells infected with natural VZV (Forghani et al., 1994 ). We postulated that the processing of gL forms reflected a difference in their trafficking status. The localization could explain a selective distribution of the gL forms in complex with gH. Since both forms differed only in a subtle nuance in their glycosylation, the two gL forms may have the same potential for interacting with gH. If it is assumed that mature gL is found in complex with mature gH, then the 18 kDa gL form should be considered as the mature form; the 19 kDa gL form would represent a premature gL containing non-trimmed oligosaccharides. Our results concerning the two gL forms are supported by diverse trafficking routes of VZV gL in the model proposed by Duus & Grose (1996)
, which includes the GolgiER recycling of gL as well as the transport of the gH:gL complex through the Golgi apparatus to the outer cell membrane.
A hypothesis on the structure of the gH:gL complex can be drawn from the fact that 60A antiserum recognized only the non-complexed 19 kDa gL molecule. The gL amino acid sequence 111121 is exposed on the surface of the gL molecule, but is hidden in the gH:gL complex. This region is in the relative vicinity of amino acid sequence 73100, which is highly conserved for herpesviral gL homologues (Yoshida et al., 1994 ) and is a candidate for the ER-targeting motif on gL responsible for its GolgiER recycling (Duus & Grose, 1996
). We hypothesize that the ER-targeting motif on gL can be masked during complex formation with gH. Afterwards, the complex escapes the recycling, and allows gH to travel to the surface of the cell.
Our data showed that individually expressed gH was not secreted. Unlike gL of HSV-1, which is secreted from cells when expressed in the absence of gH (Dubin & Jiang, 1995 ), we did not detect VZV gL in the media, unless it was coexpressed with gH. The differences between HSV and VZV gL include the following points. First, HSV-1 gL was found to be membrane-associated, most likely as a result of complex formation with gH (Dubin & Jiang, 1995
), whereas the VZV gL was not present on the surface of gH/gL cotransfected cells (Duus & Grose, 1996
). Second, coexpression of HSV-1 gH and gL is required for normal post-translational processing and intracellular transport of both gH and gL glycoproteins. Our experiments support earlier results where VZV gL processing was not dependent on gH. Third, gL VZV is half the size of HSV-1 gL.
Finally, we propose a model to explain VZV gH secretion. This model is based on the earlier work by Dubin & Jiang (1995) in which they truncated HSV-1 gH of its transmembrane domain and cytoplasmic tail. When this truncated gH construct was cotransfected with gL, both HSV-1 gL and gH were found in the medium. Similarly, we suggest that VZV gH is proteolytically cleaved at a similar location, although the site was not located.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boyle, D. B. & Coupar, B. E. (1988). A dominant selectable marker for the construction of recombinant poxviruses.Gene 65, 123-128.[Medline]
Davison, A. J. & Moss, B. (1990). New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins.Nucleic Acids Research 18, 4285-4286.[Medline]
Davison, A. J. & Scott, J. E. (1986). The complete DNA sequence of varicella-zoster virus.Journal of General Virology 67, 1759-1816.[Abstract]
Dubin, G. & Jiang, H. (1995). Expression of herpes simplex virus type 1 glycoprotein L (gL) in transfected mammalian cells: evidence that gL is not independently anchored to cell membranes.Journal of Virology 69, 4564-4568.[Abstract]
Duus, K. M. & Grose, C. (1996). Multiple regulatory effects of varicella-zoster virus (VZV) gL on trafficking patterns and fusogenic properties of VZV gH.Journal of Virology 70, 8961-8971.[Abstract]
Duus, K. M., Hatfield, C. & Grose, C. (1995). Cell surface expression and fusion by the varicella-zoster virus gH:gL glycoprotein complex: analysis by laser scanning confocal microscopy.Virology 210, 429-440.[Medline]
Ecker, J. R. & Hyman, R. W. (1982). Varicella zoster virus DNA exists as two isomers.Proceedings of the National Academy of Sciences, USA 79, 156-160.[Abstract]
Forghani, B., Ni, L. & Grose, C. (1994). Neutralization epitope of the varicella-zoster virus gH:gL glycoprotein complex.Virology 199, 458-462.[Medline]
Forrester, A., Farrell, H., Wilkinson, G., Kaye, J., Davis, P. N. & Minson, T. (1992). Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.Journal of Virology 66, 341-348.[Abstract]
Fuller, A. O. & Lee, W. C. (1992). Herpes simplex virus type 1 entry through a cascade of viruscell interactions requires different roles of gD and gH in penetration.Journal of Virology 66, 5002-5012.[Abstract]
Harlow, E. & Lane, D. (1988). Antibody: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Huber, M. T. & Compton, T. (1997). Characterization of a novel third member of the human cytomegalovirus glycoprotein Hglycoprotein L complex.Journal of Virology 71, 5391-5398.[Abstract]
Huber, M. T. & Compton, T. (1998). The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein Hglycoprotein L-containing envelope complex.Journal of Virology 72, 8191-8197.
Hutchinson, L., Browne, H., Wargent, V., Davis, P. N., Primorac, S., Goldsmith, K., Minson, A. C. & Johnson, D. C. (1992). A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.Journal of Virology 66, 2240-2250.[Abstract]
Kaye, J. F., Gompels, U. A. & Minson, A. C. (1992). Glycoprotein H of human cytomegalovirus (HCMV) forms a stable complex with the HCMV UL115 gene product.Journal of General Virology 73, 2693-2698.[Abstract]
Klupp, B. G., Baumeister, J., Karger, A., Visser, N. & Mettenleiter, T. C. (1994). Identification and characterization of a novel structural glycoprotein in pseudorabies virus, gL.Journal of Virology 68, 3868-3878.[Abstract]
Kornfeld, R. & Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides.Annual Review of Biochemistry 54, 631-664.[Medline]
Kutinová, L., Ludvíková, V., Simonová, V., Otavová, M., Krystofová, J., Hainz, P., Press, M., Kunke, D. & Vonka, V. (1995). Search for optimal parent for recombinant vaccinia virus vaccines. Study of three vaccinia virus vaccinal strains and several virus lines derived from them.Vaccine 13, 487-493.[Medline]
Kutinová, L., Ludvíková, V., Mareová, L., N
me
ková,
., Brou
ek, J., Hainz, P. & Vonka, V. (1999). Effect of virulence on immunogenicity of single and double vaccinia virus recombinants expressing differently immunogenic antigens: antibody-response inhibition induced by immunization with a mixture of recombinants differing in virulence.Journal of General Virology 80, 2901-2908.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680-685.[Medline]
Li, L., Nelson, J. A. & Britt, W. J. (1997). Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex.Journal of Virology 71, 3090-3097.[Abstract]
Li, Q., Turk, S. M. & Hutt, F. L. (1995). The EpsteinBarr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells.Journal of Virology 69, 3987-3994.[Abstract]
Li, Q., Buranathai, C., Grose, C. & Hutt, F. L. (1997). Chaperone functions common to nonhomologous EpsteinBarr virus gL and varicellazoster virus gL proteins.Journal of Virology 71, 1667-1670.[Abstract]
Liu, D. X., Gompels, U. A., Nicholas, J. & Lelliott, C. (1993). Identification and expression of the human herpesvirus 6 glycoprotein H and interaction with an accessory 40K glycoprotein.Journal of General Virology 74, 1847-1857.[Abstract]
Massaer, M., Haumont, M., Place, M., Bollen, A. & Jacobs, P. (1993). Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus.Journal of General Virology 74, 491-494.[Abstract]
Montalvo, E. A. & Grose, C. (1986). Neutralization epitope of varicella zoster virus on native viral glycoprotein gp118 (VZV glycoprotein gpIII).Virology 149, 230-241.[Medline]
Nme
ková,
., Ludvíková, V. , Mare
ová, L., Krystofová, J., Hainz, P. & Kutinová, L. (1996). Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene.Journal of General Virology 77, 211-215.[Abstract]
Olson, J. K. & Grose, C. (1997). Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.Journal of Virology 71, 4042-4054.[Abstract]
Olson, J. K. & Grose, C. (1998). Complex formation facilitates endocytosis of the varicella-zoster virus gE:gI Fc receptor.Journal of Virology 72, 1542-1551.
Peeters, B., de Wind, N., Broer, R., Gielkens, A. & Moormann, R. (1992). Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus.Journal of Virology 66, 3888-3892.[Abstract]
Perkus, M. E., Panicali, D., Mercer, S. & Paoletti, E. (1986). Insertion and deletion mutants of vaccinia virus.Virology 152, 285-297.[Medline]
Rodriguez, J. E., Moninger, T. & Grose, C. (1993). Entry and egress of varicella virus blocked by same anti-gH monoclonal antibody.Virology 196, 840-844.[Medline]
Sambrook, J., Maniatis, T. & Fritsch, E. F. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shida, H. & Matsumoto, S. (1983). Analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope.Cell 33, 423-434.[Medline]
Spaete, R. R., Perot, K., Scott, P. I., Nelson, J. A., Stinski, M. F. & Pachl, C. (1993). Coexpression of truncated human cytomegalovirus gH with the UL115 gene product or the truncated human fibroblast growth factor receptor results in transport of gH to the cell surface.Virology 193, 853-861.[Medline]
Stokes, A., Alber, D. G., Greensill, J., Amellal, B., Carvalho, R., Taylor, L. A., Doel, T. R., Killington, R. A., Halliburton, I. W. & Meredith, D. M. (1996). The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.Virus Research 40, 91-107.[Medline]
Sugano, T., Tomiyama, T., Matsumoto, Y.-i., Sasaki, S., Kimura, T., Forghani, B. & Masuho, Y. (1991). A human monoclonal antibody against varicella-zoster virus glycoprotein III.Journal of General Virology 72, 2065-2073.[Abstract]
Wang, X., Kenyon, W. J., Li, Q., Mullberg, J. & Hutt, F. L. (1998). EpsteinBarr virus uses different complexes of glycoproteins gH and gL to infect B lymphocytes and epithelial cells.Journal of Virology 72, 5552-5558.
Ye, M., Duus, K. M., Peng, J., Price, D. H. & Grose, C. (1999). Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase.Journal of Virology 73, 1320-1330.
Yoshida, S., Lee, L. F., Yanagida, N. & Nazerian, K. (1994). Identification and characterization of a Mareks disease virus gene homologous to glycoprotein L of herpes simplex virus.Virology 204, 414-419.[Medline]
Zhu, Z., Hao, Y., Gershon, M. D., Ambron, R. T. & Gershon, A. A. (1996). Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.Journal of Virology 70, 6563-6575.[Abstract]
Received 29 October 1999;
accepted 18 February 2000.