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- Role of One N-linked Oligosaccharide Chain on Canine Herpesvirus gD in Its Biological
Activity
Ken MAEDA, Naoaki YOKOYAMA1), Kentaro FUJITA1), Xuenan XUAN2), and Takeshi MIKAMI 1)*
Department of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753, 1) Department
of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113, and 2) Research Center
for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080,
Japan
(Received 4 June 1997/Accepted 7 August 1997)
ABSTRACT. The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those
of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu))
was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one
predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2
strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a
panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an
MW of approximately 47–51 and 39–44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2)
(approximately 51–55 and 41–46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in
Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates
of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs
possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids
possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-
inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live
vaccine. — KEY WORDS : canine herpesvirus, glycoprotein D, hemagglutinin, N-glycosylation site, YP11mu strain.
J. Vet. Med. Sci. 59(12): 1123–1128, 1997
We reported previously that canine herpesvirus (CHV) in the virus penetration process remains to be further
gD agglutinated canine red blood cells (RBC) and that this analyzed.
hemagglutination (HA) activity was inhibited by monoclonal Xuan et al. [25] reported that one plaque-selected CHV,
antibodies (MAbs) against CHV gD [16, 24, 25]. Similarly, YP11mu strain, possessed a smaller molecular weight (MW)
we reported that gD of feline herpesvirus type 1 (FHV-1) of gD than those of other strains including YP2 strain.
agglutinated feline RBC and cells expressing the gD However its HA activity and reactivity with antibodies
adsorbed the RBC [13, 14]. Further, insect cells expressing against CHV were similar to those of other strains.
FHV-1 gD on their cell surface were adhered to several cell Therefore, it seems that mutation in the gD of YP11mu
lines originating from F elidae b ut not those from other strain does not affect biological activities of the gD. By
animals [15]. Therefore, we speculated that the FHV-1 gD genetical analysis of this mutation, it is expected to obtain
might restrict receptor(s) of cells from Felidae. One MAb further information on functional region of gD.
25C9 against FHV-1 gD recognized CHV gD by indirect In this communication, we identified the mutated region
immunofluorescence assay (IFA) and immunoblot analysis, on the gD of YP11mu strain and expressed the gD in COS-
and inhibited HA activity of CHV [12]. CHV gD 7 and insect ( Spodoptera frugiperda; Sf9) cells. The CHV
agglutinates only canine RBC [16, 19, 24] while FHV-1 gD expressed in COS-7 cells specifically adsorbed canine
agglutinates only feline RBC [4, 13, 14, 18]. The reason of RBC and extracts of CHV gD expressed in Sf9 cells
these different HA activities has never been studied. agglutinated canine RBC. Further, antibodies raised in mice
In herpes simplex virus (HSV), gD seems to have specific immunized with recombinant CHV gDs neutralized CHV
receptors on the surface of cells [8, 9]. In particular, infection in vitro .
Brunetti e t al. r eported that gD binds to mannose-6-
phosphate receptors [2] and that this interaction is important MATERIALS AND METHODS
for virus entry into cells and cell-to-cell transmission [1].
The gDs of alphaherpesviruses are also important for virus Viruses and cells: Three isolates from our laboratory,
penetration to cells [3, 7, 10, 20]. However, the role of gD YP2, YP11, and the plaque-selected YP11 (YP11mu) [25],
two isolates from other laboratories, GCH-1 and Pirene [29],
and two reference strains, F-205V and Glasgow CHV2 of
* C ORRESPONDENCE TO: Dr. MIKAMI, T., Department of Veterinary
CHV were used in this study. All CHV strains were grown
Microbiology, Faculty of Agriculture, The University of
in Madin-Darby canine kidney (MDCK) cells for extraction
Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113, Japan.
- K. MAEDA, ET AL.
1124
of viral DNA as described previously [16]. COS-7 cells gD and an MAb 25C9 against FHV-1 gD were previously
were cultured in DMEM supplemented with 10% FCS and produced and characterized [5, 12, 25, 26].
antibiotics. Recombinant Autographa californica nuclear IFA : For detection of CHV gD in IFA, transfected cells
polyhedrosis viruses (rAcNPVs) were grown in Sf9 cells in were smeared on glass slides, air-dried and then fixed with
acetone. The fixed cells were incubated for 30 min at 37 °C
TC100 medium (GIBCO, Grand Island, N. Y.) supplemented
with 10% FCS, 0.3% tryptose phosphate broth (Difco, with MAbs against CHV gD or FHV-1 gD. After
Detroit, Mich.), and antibiotics. Two rAcNPVs, AccgD incubation, the slides were washed 3 times with PBS, and
(YP2) which expressed CHV gD of YP2 strain in insect then anti-mouse immunoglobulins (G+M+A) rabbit antibody
cells and AcYM [16], were used. conjugated with fluorescein isothiocyanate (FITC) (Cappel,
Construction of plasmids : Using two primers, 5’- PA, U.S.A.) was applied. After incubation for 30 min at
37° C, the slides were washed again, mounted in buffered
GGGAATTCATGATTAAACTTCTATTTAT-3’ (CGD-UP)
and 5’-TTCTCGAGCTAAACATTTGTTGTTAATT-3' glycerol, and examined by fluorescence microscopy.
(CGD-DOWN), the gene encoding CHV gD YP11mu was For membrane immunofluorescence, transfected cells
ampilified, digested with restriction enzymes EcoRI and were suspended in ice-cold PBS containing 3% FCS and
XhoI, and then cloned into E coRI and X ho I sites of 0.1% sodium azide, and then reacted with MAbs for 30 min
at 4 ° C. After washing three times by ice-cold PBS
pBluescript KS-, and designated as pBS-cgD (YP11mu)
[16]. For expression in COS-7 and Sf9 cells, plasmids pME- containing 3% FCS and 0.1% sodium azide, FITC-
cgD (YP11mu) and pAccgD (YP11mu), respectively, were conjugated anti-mouse immunoglobulins were added and
the cells were reincubated at 4 ° C. After further washings
also constructed from pBS-cgD (YP11mu) into pME18S
[23] and pAcYM1 [17], respectively, as described previously for three times, the cells were resuspended in glycerol and
[15]. As a control, two expression plasmids, pME-cgD mounted for immunofluorescence microscopy.
(YP2) which expressed CHV gD (YP2) in COS-7 cells [16] Immunoblot analysis : SDS-polyacrylamide gel
and pME-fgD which expressed FHV-1 gD in COS-7 cells electrophoresis (PAGE) was carried out according to the
[13], were used. discontinuous Laemmli buffer system [12]. All samples
DNA sequencing: To identify the mutated nucleotide were dissolved in the buffer (62.5 mM Tris-HCl, pH 6.8,
sequences of CHV gD (YP11mu), DNA sequencing of pBS- 20% glycerol, and 0.001% bromophenol blue), and then
cgD (YP11mu) was done with a model 370A Applied disrupted by heating for 2 min at 100 °C. Polypeptides were
Biosystems autosequencer, as described previously [16]. separated on an SDS-polyacrylamide gel and
Polymerase chain reaction (PCR) amplification : To electrophoretically transferred to polyvinylidene difluoride
clarify the deletion of YP11mu, two primers, 5’- membrane (Immunobilon, Millipore, MA, U.S.A.). The
blotting papers were incubated for 30 min at 37° C with a
TTACCATCGAGGCCACATAT-3’ (CGD790F) and
5’-GGTGTTGGGGTAGTAGTATC-3’ (CGD902R), were mixture of four MAbs, 11F7, 09D1, 10C10, and 05B7,
prepared. The viral DNAs of all CHV strains used were against CHV gD, or an MAb 10C10. Afterwards, they
amplified by 30 cycles of denaturation (94° C, 1 min), were washed three times, and incubated with anti-mouse
annealing (60°C, 1 min), and polymerization (72° C, 2 min). immunoglobulins (G+M+A) peroxidase conjugate (Cappel,
PA, U.S.A.) for 30 min at 37°C. The reaction was visualized
The amplified fragments were subjected to electrophoresis
on 12% polyacrylamide gel. by addition of a diaminobenzidine-hydrogen peroxidase
Expression in COS-7 cells : COS-7 cells were transfected substrate.
with the constructed plasmids according to the methods HAD and HA tests: HAD and HA activities of expressed
described previously [21] with minor modifications. Briefly, CHV gDs were tested as described previously [16].
when COS-7 cells were grown in a 100 mm dish, 7.5 µg of Immunization of mice: Sf9 cells were infected with AccgD
plasmid DNA prepared in 5 ml of DMEM/DEAE-dextran (YP11mu), AccgD (YP2), or AcYM at 10 PFU/cell for 96
solution was added to the cells. After incubation for 3 hr at hr, washed, suspended in PBS and subjected to three cycles
37° C, the solution was removed. The cells were treated of freezing and thawing. Lysates prepared from each of the
infected Sf9 cells (1 × 10 6) cells were separately injected
with 5 ml of 10% dimethyl sulfoxide for 1 min and returned
to DMEM containing 10% FCS. After 72 hr post- into a mouse (Balb/c, 8 weeks old) intraperitoneally in
transfection, the transfected cells were scraped off the plates Freund’s complete adjuvant. The same lysate in Freund’s
and analyzed by IFA, immunoblot analysis, and incomplete adjuvant was injected intraperitoneally into the
hemadsorption (HAD) test. mouse on days 14 and 28. Sera from immunized mice were
Transfection and selection of recombinant baculovirus : collected 14 days after the last immunization.
Sf9 cells were co-transfected with linealized BaculoGoldTM VN assay : Virus neutralizing activity of antisera was
baculovirus (AcNPV) DNA (PharMingen, San Diego, CA) tested in a 50% plaque reduction assay performed on MDCK
and pAccgD (YP11mu) by use of Lipofectin reagent cells with or without 5% rabbit serum as a source of
(GIBCO BRL, Gaithersburg, MD). After three cycles of complement. Neutralizing titers against CHV YP11mu
plaque purification, the recombinant virus was isolated, and strain were expressed as the reciprocal antibody dilution
was designated as AccgD (YP11mu). giving 50% plaque reduction.
MAbs : MAbs 11F7, 09D1, 10C10, and 05B7 against CHV
- ROLE OF ONE N-LINKED OLIGOSACCHARIDE CHAIN ON CHV GD 1125
Expression of CHV gD (YP11mu) in COS-7 cells was
RESULTS
further confirmed by immunoblot analysis using a mixture
Cloning and sequence analysis of the gene encoding CHV of four MAbs, 11F7, 09D1, 10C10, and 05B7 against CHV
gD (YP11mu): Approximately 1.05 kbp fragment containing gD (Fig.2A). The MW of the authentic CHV gD (YP11mu)
an open reading frame (ORF) encoding gD (YP11mu) was was approximately 47–51 kDa with a minor band of
amplified from viral DNA by PCR method using two approximately 44 kDa which seems to be a precursor form
primers, CGD-UP and CGD-DOWN. This amplified of the CHV gD. These bands were smaller than those
fragment was inserted into pBluescript KS- and was (approximately 51–55 and 48 kDa) of pME-cgD (YP2)-
designated as pBS-cgD (YP11mu). Nucleotide sequence of transfected COS-7 cells. In pME-fgD-transfected cells, any
the insert fragment of pBS-cgD (YP11mu) was determined specific band was not detected (Fig. 2A lane3).
and compared with that of 1,050 bp published for CHV gD Expression of CHV gDs in insect cells: Expression of
[11]. The result showed that the nucleotide sequence for CHV gD (YP11mu) in Sf9 cells was examined by IFA using
the ORF of gD (YP11mu) was 1038 bp and lacked 12 MAbs. All of the MAbs against CHV gD and one MAb
nucleotides, AATAAAACTATT (position at 823–834 25C9 against FHV-1 gD reacted with AccgD (YP11mu)-
nucleotides), which encodes four amino acids, NKTI infected Sf9 cells as well as with AccgD (YP2)-infected Sf9
(position at 275–278 amino acids) (Fig. 1A). No other cells (data not shown).
change was found in the ORF of gD (YP11mu). Expression of CHV gD (YP11mu) was confirmed by
immunoblot analysis using an MAb 10C10 against CHV
Further PCR analysis was carried out to confirm the
gD (Fig. 2B). The MAb 10C10 detected a specific band of
deletion using two primers, CGD790F and CGD902R. The
39–44 and 41–46 kDa in AccgD (YP11mu)- and AccgD
113 bp fragment amplified by these primers contains the
(YP2)-infected Sf9 cells, respectively (Fig. 2A). When
region of mutation of gD (YP11mu). Figure 1B showed
AccgD (YP11mu)- and AccgD (YP2)-infected Sf9 cells
that the amplified fragment of only YP11mu strain was 101
were treated with 10 µg/m l of tunicamycin (TM), the both
bp and was smaller than those (113 bp) of other six CHV
MWs of the TM-treated gD (YP11mu) and gD (YP2) were
strains. These results indicate that this 12 bp deletion was
approximately 37 kDa (Fig. 2B lanes 3 and 4). Since CHV
specific for YP11mu strain.
gD consists of 345 amino acid residues with a predicted
Expression of CHV gDs in COS-7 cells: Expression of
MW of approximately 38 kDa [10], the estimated MW of
CHV gD (YP11mu) in COS-7 cells was examined by IFA
the TM-treated gDs seems to be reasonable.
using MAbs. All of the MAbs against CHV gD and one
MAb 25C9 against FHV-1 gD reacted with pME-cgD HAD and HA tests : We examined whether cells expressed
(YP11mu)-transfected COS-7 cells as well as with pME- CHV gD (YP11mu) could adsorb canine red blood cells
cgD (YP2)-transfected COS-7 cells (data not shown). (RBC) and whether the expressed CHV gD (YP11mu) could
Fig. 1. Differences in the nucleotide and amino acid sequences of gDs among CHV strains. (A) Nucleotide sequence of
heterogeneous region between CHV YP2 and YP11mu strains. Double dots show identical nucleotide sequence. Bars show
gaps. NKT showed by a box indicates potential asparagine-linked glycosylation site. Two primers, CGD790F and antisense of
CGD902R are boxed. (B) Amplification of the heterogeneous region in several CHV strains. Arrows show length of fragments.
- K. MAEDA, ET AL.
1126
Table 1. Immunogenic properties of recombinant gDs
VN titera)
Serum against
w/o C’ w C’
AccgD (YP11mu) 320 1280
AccgD (YP2) 80 2560
AcYM
- ROLE OF ONE N-LINKED OLIGOSACCHARIDE CHAIN ON CHV GD 1127
regions for HA and HAD activities and immunogenicity 12. Limcumpao, J. A., Horimoto, T., Xuan, X., Tohya, Y.,
Azetaka, M., Takahashi, E., and Mikami, T. 1991. Homolo-
would be expected to understand the initial stage of
gous and heterologous antibody responses of mice immunized
herpesvirus infection. In addition, this deletion of gD
with purified feline herpesvirus type 1 and canine herpesvirus
(YP11mu) might be a good selective marker for
glycoproteins. J. Vet. Med. Sci. 53: 423–432.
development of a live recombinant vaccine. Indeed, Xuan
13. Maeda, K., Kawaguchi, Y., Ono, M., Inoshima, Y., Miyazawa,
et al. [27] expressed rabies virus glycoprotein using the T., Tohya, Y., Kai, C., and Mikami, T. 1994. A gD homolo-
YP11mu strain as a vector and examined the biological and gous gene of feline herpesvirus type 1 encodes a hemagglutinin
immunological characteristics of the recombinant virus in (gp60). Virology 202: 1034–1038.
vitro and in vivo . 14. Maeda, K., Ono, M., Kawaguchi, Y., Niikura, M., Okazaki,
K., Yokoyama, N., Tokiyoshi, K., Tohya, Y., and Mikami, T.
ACKNOWLEDGEMENTS. This work was supported in 1996. Expression and properties of feline herpesvirus type 1
gD (hemagglutinin) by a recombinant baculovirus. Virus Res.
part by grants from the Ministry of Education, Science,
46: 75–80.
Sports and Culture, from the Ministry of Agriculture,
15. Maeda, K., Ono, M., Kawaguchi, Y., Okazaki, K., Yokoyama,
Forestry and Fisheries and by Research Aid of Inoue
N., Tokiyoshi, K., Tohya, Y., and Mikami, T. 1997. Adhe-
Foundation for Science.
sion of insect cells expressing the feline herpesvirus type 1
hemagglutinin (gD) to feline cell lines. J. Vet. Med. Sci. 59:
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