Department of Biochemistry, Health Sciences Centre, McMaster University, 1200 Main St W., Hamilton, Ontario, Canada L8N 3Z51
Author for correspondence: Hara Ghosh.Fax +1 905 522 9033. e-mail ghosh{at}fhs.mcmaster.ca
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Abstract |
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Introduction |
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Analyses of the amino acid sequences of gB homologues of herpesviruses show that the carboxy-terminal hydrophobic region is conserved and a number of residues are non-variant within the Herpesviridae. The gB glycoproteins of the Alphaherpesvirinae, however, show a very high degree of homology in this region and the membrane-anchoring segment 3 shows the highest number of conserved amino acids (Pereira, 1994 ). Complementation experiments showed that the gB glycoprotein of HSV-1 could be substituted with the corresponding homologue from either BHV-1 (Misra & Blewett, 1991
) or pseudorabies virus (PrV) (Mettenleiter & Spear, 1994
). Also, gB-null PrV could be complemented by gB protein of BHV-1 (Kopp & Mettenleiter, 1992
; Rauh et al., 1991
). It was thus suggested that homologues of gB may possess common structural and functional domains. The highly conserved residues in the carboxy-terminal hydrophobic region of gB may, therefore, be essential for the biological activity of herpesviruses.
In this report, we have attempted to determine the roles of amino acids that are highly conserved within the carboxy-terminal hydrophobic region of the gB homologues of herpesviruses in virus infectivity by mutagenesis of these residues. Conserved alanines in the membrane-anchoring segment 3 of Alphaherpesvirinae were substituted by a number of neutral polar residues. Two glycines in segment 1 as well as glycine, phenylalanine and proline residues in segment 2, all of which are non-variant in the gB homologues of Herpesviridae, were also mutated. The mutant proteins were localized in the nuclear envelope. Complementation with a gB null-virus showed that mutants G746N, G766N, F770S and P774L showed negligible infectivity whereas G743R showed reduced infectivity. Virus particles generated from the complementation experiment containing these mutant glycoproteins also showed a markedly decreased rate of entry. The results suggest that the non-variant residues present in the carboxy-terminal hydrophobic domain of herpesvirus gB protein may be important for infectivity.
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Methods |
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Transfection, labelling and characterization of mutant protein.
Subconfluent monolayers of COS-1 cells were transfected by the Ca3(PO4)2 method, as described previously (Gilbert et al., 1994 ). The transfected cells were radiolabelled at 24 h post-transfection and the mutant proteins were immunoprecipitated with a polyclonal anti-gB antibody. Proteins were analysed by SDSPAGE.
Intracellular transport and localization.
Transport of the mutant proteins from ER to the Golgi apparatus was determined by the acquisition of endoglycosidase H (Endo H) resistance (Gilbert et al., 1994 ). Transfected COS-1 cells were fixed with 2% paraformaldehyde for cell surface immunofluorescence. For internal immunofluorescence, cells were fixed with 2% paraformaldehyde and then treated with 1% Triton X-100. The cells were treated with a rabbit polyclonal anti-gB antibody and stained with FITC-conjugated goat anti-rabbit IgG (Gilbert et al., 1994
).
Oligomerization assay.
The oligomeric state of wild-type and mutant gB-1 proteins was determined by SDSPAGE analysis of the heat-dissociable, detergent-stable oligomers (Claesson-Welsh & Spear, 1986 ). Oligomeric forms of gB-1 were also detected by immunoprecipitation with a dimer-specific monoclonal antibody DL16 (Laquerre et al., 1998
).
Complementation assay.
Complementation of the gB-null HSV-1, K082, was carried out in Vero cells as described previously (Cai et al., 1988b ; Rasile et al., 1993
). Briefly, 0·53·0 µg pKBXX plasmid encoding the gB mutants was mixed with DEAE-dextran in a serum-free growth medium. This mixture was added to 8x105 Vero cells and incubated at 37 °C for 24 h. After removal of DNA and washing, the cells were incubated at 37 °C in regular growth medium. At 1618 h post-transfection, the cells were infected with 2x106 p.f.u. of K082 virus and incubated for 2 h at 37 °C. Virions remaining outside the cell were removed by treatment with a glycine buffer (0·1 M glycine, pH 3·0). The cells were incubated at 37 °C for 24 h and then harvested. Virus stocks were prepared and titres of the progeny virus particles were determined on the gB-1-expressing VB38 cell line and Vero cells. The VB38 cells (kindly donated by D. Johnson, Oregon Health Sciences University, Portland, OR, USA) were constructed by integrating the HSV-1 gB gene under the control of its own promoter into Vero cells using histidinol as a selection marker.
Radiolabelling of progeny virus particles.
Vero cells were transfected with pKBXX plasmids followed by infection with K082 virus as described for the complementation assay. Cells were labelled with [35S]methionine for 24 h and the virus was harvested. The labelled virus stock was clarified by centrifugation at 3000 r.p.m. for 5 min. Virus particles were pelleted through a cushion of 30% sucrose solution by centrifugation in a Beckman SW41 Ti rotor at 40000 r.p.m. for 2 h at 4 °C. The pelleted virus was suspended in lysis buffer; half of the recovered radioactivity was immunoprecipitated with dimer-specific anti-gB antibody DL16 and analysed by SDSPAGE.
Virus penetration assay.
The rate of virus penetration was determined by measuring the rate at which viruses adsorbed to cells become resistant to inactivation by exposure to a low pH buffer (Highlander et al., 1989 ). Confluent VB38 cells were incubated at 4 °C with 250 p.f.u. progeny virus generated from the complementation experiment. The cells were washed three times to remove unadsorbed virus and incubated at 37 °C in the presence of complete medium when the adsorbed viruses entered the cell. At various times after being shifted, viruses that had not entered the cells were inactivated by exposure to 2 ml glycine buffer (0·1 M, pH 3·0) for 1 min while the control cells were treated with Tris-buffered saline (TBS) (pH 7·4). The cells were washed and overlaid with medium containing 0·5% methyl cellulose and incubated at 37 °C. After 3648 h, cells were fixed and stained and the plaques were counted. The percentage of penetrated virus was calculated from the p.f.u. obtained with the sample treated with low pH buffer versus the total plaque number (100% entry) as obtained from the non-buffer-treated sample.
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Results |
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Expression, intracellular transport and oligomerization of mutant gB proteins
COS-1 cells transfected with pXM vector encoding the mutant or the wild-type gB protein were metabolically labelled with [35S]methionine, and proteins immunoprecipitated with anti-gB antiserum were analysed by PAGE. Fig. 2 shows that all of the mutants of gB proteins were expressed and co-migrated with wild-type gB which is 110120 kDa.
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Intracellular localization of mutant glycoprotein by immunofluorescence staining
The gB glycoprotein has been shown to be localized in the ER, Golgi complex, cell surface and the nuclear envelope by indirect immunofluorescence or immunoelectron microscopy (Gilbert & Ghosh, 1993 ; Gilbert et al., 1994
; Torrisi et al., 1992
). The intracellular localization of the mutant proteins was determined by indirect immunofluorescence staining of COS-1 cells transfected with wild-type or mutant gB gene. In agreement with an earlier report, wild-type gB was localized in the nuclear envelope and in the perinuclear structures representing Golgi complex and ER (Gilbert et al., 1994
). All 13 mutant gB proteins showed similar distinct nuclear rim staining (Fig. 3
). The mutants also showed perinuclear labelling in addition to the nuclear rim staining indicating ER and Golgi localization. Examination for cell surface immunofluorescence showed staining of the plasma membrane of cells expressing all mutants except P774L. The mutants G743R and F770S showed a much weaker staining at the cell surface. The rate of intracellular transport was also determined by acquisition of Endo H resistance. Results of Endo H digestion showed that mutant P774L was completely sensitive to Endo H digestion even after a chase of 2 h while mutants G743R and F770S showed partial resistance. All of the other mutants showed Endo H resistance pattern similar to wild-type gB protein (data not shown).
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Discussion |
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Significant reduction in the rate of entry of viruses containing mutations in segment 1 and 2 suggests the involvement of non-variant residues in virus penetration. In the case of gB glycoprotein of HCMV (Bold et al., 1996 ; Tugizov et al., 1995
) or BHV-1 (Li et al., 1997
), putative fusogenic domains spanning segments 1 and 2 or segment 2 have been proposed. However, in the case of HSV-1 gB protein, no fusogenic domain has yet been established. Since the non-variant residues in gB protein that affect the entry of HSV-1 are also conserved in the fusogenic domains of HCMV or BHV-1 gB protein it may be hypothesized that hydrophobic segment 2 either alone or in combination with segment 1 may serve as an internal fusogenic domain of HSV-1 gB. Segments 1 and 2 are not embedded in the viral envelope but peripherally associated with the membrane and thus available for insertion into a target membrane. The presence of internal fusion peptide in viral fusion protein such as vesicular stomatitis virus G protein (Zhang & Ghosh, 1994
) or Semliki Forest virus E1 glycoprotein (Levy-Mintz & Kielian, 1992
) has already been established. Since glycine residues present in the fusion peptides of a number of viral fusion proteins (Hernandez et al., 1996
), namely influenza virus HA (Skehel et al., 1995
), Semliki Forest virus E1 (Levy-Mintz & Kielian, 1992
), vesicular stomatitis virus G (Zhang & Ghosh, 1994
), human immunodeficiency virus gp 41 (Freed & Martin, 1995
) and paramyxovirus F1 (Sergel-Germans et al., 1994
), were shown to be required for fusogenic activity, the non-variant glycines in segments 1 and 2 of the hydrophobic domain of gB protein may be important for membrane fusion. Proline and phenylalanine are also present in fusion peptides and have been implicated in fusogenic activity (Hernandez et al., 1996
). Studies involving site-specific monoclonal antibodies of HSV-1 (Highlander et al., 1988
; Navarro et al., 1992
) and HCMV (Zheng et al., 1996
) gB protein suggest that the mid-region of the ectodomains, for example, residues 241441 of HSV-1 gB, may be required for virus penetration and cell fusion. It was further shown that the cytoplasmic region of HSV-1 and HCMV gB protein also contained important determinants for virus entry and membrane fusion (syncytia induction) (Gage et al., 1993
; Highlander et al., 1988
; Zheng et al., 1996
). It was suggested that transmembrane region of gB protein may participate in transduction of signals from fusogenic domains in the cytoplasmic tail to the fusogenic regions in the ectodomain. Thus, the substitution of the non-variant residues within the hydrophobic region can affect the transduction of signals involved in fusogenic activity.
The entry of herpesviruses requires the involvement of four transmembrane glycoproteins gB, gD, gH and gL (Cai et al., 1988a ; Davis-Poynter et al., 1994
; Ligas & Johnson, 1988
; Roop et al., 1993
). It is suggested that in the viral envelope, they may form hetero-oligomers (Handler et al., 1996a
, b
; Zhu & Courtney, 1988
) which are involved in virus entry. The multi-subunit complex containing gB protein involved in virus entry and cell fusion could further interact with cellular co-receptors (Montgomery et al., 1996
; Terry-Allison et al., 1998
) or with specific cellular proteins which would induce conformational change of the fusogenic region. Changes in conformation from a non-fusogenic to fusogenic state by exposure to low pH (Gaudin et al., 1995
; Hernandez et al., 1996
) or binding to co-receptors (Berger, 1997
) have been documented for other viral fusion proteins. Thus, one can hypothesize that, in the case of herpesviruses, the multi-subunit complex involved in entry and fusion may be formed by interactions via the transmembrane segment as well as the ecto- and cytoplasmic domains of gB protein. Recent studies have indeed shown that interactions between the transmembrane segments within lipid bilayers are essential for functional assembly of integral membrane proteins (Casson & Bonfacino, 1992
; Shai, 1995
). Disruption of the specific amino acid sequence or local peptide structure of gB protein within the viral membrane may thus affect the biological activity. Recently a system using gB, gD, gH and gL proteins of HSV-1 has been used to demonstrate in vitro fusion of cells (Turner et al., 1998
). Using this system, it may be possible to test if mutations of non-variant residues present in the hydrophobic region of gB-1 protein also affect fusion. Alternately, one can introduce these specific mutations in the fusogenic domains of gB protein of BHV-1 (Li et al., 1997
) or HCMV (Bold et al., 1996
; Tugizov et al., 1995
) and determine the fusogenic activity of the mutant proteins.
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Acknowledgments |
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Footnotes |
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c Present address: University of Ottawa, Ottawa, ON, Canada.
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References |
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Received 7 April 1999;
accepted 2 September 1999.