Journal
of General Virology (1997), 78, 1399–1403. Printed in Great Britain
...............................................................................................................................................................................................................
SHORT COMMUNICATION
Identification and characterization of bovine herpesvirus-1
glycoproteins E and I
Nagaoki Yoshitake, Xuenan Xuan and Haruki Otsuka
Department of Animal Resource Science, Graduate School of Agricultural Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113, Japan
To identify the products of the bovine herpesvirus1 (BHV-1) gE and gI genes, we constructed baculovirus recombinants containing the putative gE or gI
genes. These recombinant viruses synthesized
BHV-1 gE and gI with apparent molecular masses of
84 and 41 kDa, respectively. Polyclonal antibodies
against these recombinant gE or gI proteins were
produced and by using these antibodies, we
showed the presence of gE and gI with apparent
molecular masses of 94 and 45 kDa, respectively,
in purified BHV-1 virions. We also demonstrated
that like their herpes simplex virus-1 and pseudorabies virus counterparts BHV-1 gE and gI form a
complex. A gINBHV-1 mutant failed to express gI
but gE was found in the virions. On the other hand,
neither gE nor gI was found in the virions of a gEN
BHV-1 mutant. In th gEN BHV-1 mutant, gI was
produced but released into the medium without
being integrated in the virions.
The alphaherpesviruses, which include herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus
(VZV), pseudorabies virus (PRV) and bovine herpesvirus-1
(BHV-1), have a number of structurally homologous glycoproteins. Most herpesvirus glycoproteins are present in the
viral envelope and are important for virus–host interactions.
Glycoproteins gE and gI of HSV, VZV and PRV form a
functional complex (Johnson & Feenstra, 1987 ; Zuckermann et
al., 1988). The gE–gI complex of HSV-1, but not PRV, binds
the IgG Fc fragment, which may help HSV-1 to escape from
the host immune response (Bell et al., 1990 ; Frank & Friedman,
1989 ; Hanke et al., 1990 ; Johnson et al., 1988). Small plaques
produced by gE− or gI− mutants are evidence that the gE–gI
complex facilitates cell-to-cell spread of virus in cultured cells
(Dingwell et al., 1994 ; Zsak et al., 1992). In vivo, the gE–gI
complex is important for the spread of herpesviruses through
Author for correspondence : Haruki Otsuka.
Fax ­81 3 5800 3903. e-mail aotsuka!hongo.ecc.u-tokyo.ac.jp
the central nervous system of the host animal (Dingwell et al.,
1995 ; Enquist et al., 1994 ; Kritas et al., 1994 ; Mulder et al.,
1994). Deletion of the gE gene from both PRV and BHV-1
attenuates virulence of these viruses but maintains a strong
immunizing potential and gE deletion mutants of PRV and
BHV-1 are used as marker vaccines (Jacobs et al., 1993 ; Van
Engelenburg et al., 1994 ; Van Oirschot et al., 1990). The
nucleotide sequence of the unique short (US) region of BHV-1
has been determined and open reading frames (ORFs) of the gE
and gI genes deduced by comparison with the sequences of the
previously identified gE and gI genes of HSV-1 and PRV
(Leung-Tack et al., 1994).
Previously, we constructed a gE− insertion mutant of BHV1, BHV-1}TF9-3, and a gI− insertion mutant, BHV-1}TF9-7,
and found that these mutants formed smaller plaques than
those produced by their parental line, IBRV(NG)dltk (Otsuka
& Xuan, 1996). It is the purpose of this communication to
identify the products of the gE and gI genes of BHV-1 and
elucidate some of the characteristics of BHV-1 gE and gI.
The BHV-1 EcoRI C fragment was cloned at the EcoRI site
of pUC19. The ORF of the gE gene was cleaved by digesting
with Csp45I and the ORF of the gI gene was cleaved by
digesting with Tth111I ; the fragments were inserted at the
SmaI site of the baculovirus transfer vector, pBacPAK9
(Clontech). The transfer vectors containing the BHV-1 gE and
gI ORFs were used to construct a recombinant baculovirus
expressing BHV-1 gE and another recombinant expressing
BHV-1 gI was constructed by the homologous recombination
method described by Matsuura et al. (1987) ; the recombinants
were designated AcBgE and AcBgI, respectively. Sf-9 cells
were infected with AcBgE and AcBgI and total protein extracts
of these infected cells were analysed by Western blotting using
anti-BHV-1 rabbit serum. Results are shown in Fig. 1. AntiBHV-1 serum reacted strongly with a 84 kDa protein from
AcBgE-infected cells (Fig. 1 A, lane 1) and with a 41 kDa
protein from AcBgI-infected cells (Fig. 1 A, lane 3). AntiBHV-1 serum did not react with any band from wild-type
baculovirus-infected cells or uninfected Sf-9 cells. The apparent
molecular masses of gE and gI expressed by AcBgE and AcBgI
are greater than those predicted from the gE or gI ORF
sequences (61±2 or 39±9 kDa) (Leung-Tack et al., 1994),
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N. Yoshitake, X. Xuan and H. Otsuka
Fig. 1. Western blot analysis of gE and gI produced by the recombinant baculoviruses AcBgE and AcBgI and the effect of
tunicamycin treatment. (A) Sf-9 cells were infected with recombinant baculoviruses or control wild-type baculovirus. Infected
cell extracts were subjected to Western blot analysis using anti-BHV-1 rabbit serum. Lane 1, AcBgE-infected cell extract ; lane
3, AcBgI-infected cell extract ; lanes 2 and 4, wild-type AcNPV-infected cell extract. (B) Sf-9 cells were infected with AcBgE or
AcBgI with or without tunicamycin (10 µg/ml). Infected cell lysates were analysed by Western blotting using anti-BHV-1 rabbit
serum. Lane 1, AcBgE with tunicamycin ; lane 2, AcBgE without tunicamycin ; lane 3, AcBgI with tunicamycin ; lane 4, AcBgI
without tunicamycin. Molecular masses of markers are given in kDa on the right.
Fig. 2. Western blot analysis of gE and gI produced by BHV-1 and its gE− and gI− mutants and the effect of tunicamycin
treatment. (A) MDBK cells were infected with IBRV(NG)dltk, BHV-1/TF9-3 (gE− mutant) or BHV-1/TF9-7 (gI− mutant), or
mock-infected and incubated for 48 h. The infected cell lysates were prepared and analysed by Western blotting using anti-BgE
(lanes 1–3) or anti-BgI (lane 4–6). Lanes 1 and 4, IBRV(NG)dltk-infected cell lysate ; lane 2, BHV-1/TF9-3-infected cell lysate ;
lanes 3 and 6, mock-infected cell lysate ; lane 5, BHV-1/TF9-7-infected cell lysate. (B) MDBK cells were infected with
IBRV(NG)dltk with or without tunicamycin (10 µg/ml). Infected cell lysates were analysed by Western blotting using anti-BgE
(lanes 1 and 2) or anti-BgI (lanes 3 and 4) mouse serum. Lanes 1 and 3, with tunicamycin ; lanes 2 and 4, without
tunicamycin. Molecular masses of markers are given in kDa on the right.
indicating that these gE and gI proteins may be glycosylated
like gE and gI expressed by BHV-1. Western blot analysis of
tunicamycin-treated gE and gI expressed by AcBgE and AcBgI
demonstrated that 84 kDa gE and 41 kDa gI were replaced by
bands with apparent molecular masses of 80 and 40 kDa,
respectively (Fig. 1 B). This result suggests that gE and gI
expressed by baculovirus recombinants contain N-linked
sugars.
Anti-BgE and anti-BgI-sera were produced in mice by
BEAA
inoculating the extracts of Sf-9 cells infected with AcBgE or
AcBgI and were used to study gE and gI gene products in
BHV-1-infected MDBK cells and purified virions. Fig. 2 shows
Western blotting analyses of BHV-1 and gE− and gI− mutants
using the anti-BgE and anti-BgI sera. The anti-BgE serum
reacted with a protein with an apparent molecular mass of
94 kDa from the extract of MDBK cells infected with
IBRV(NG)dltk (Fig. 2 A, lane 1). However, the anti-BgE serum
did not detect any protein in the extracts of MDBK cells
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Identification of BHV-1 gE and gI
infected with the gE− insertion mutant of BHV-1 (BHV1}TF9-3) (lane 2) or mock-infected cell lysate (lane 3). The antiBgI serum reacted with the 45 kDa protein in IBRV(NG)dltkinfected cell extracts (lane 4). However, this 45 kDa protein
band was missing in the gI− mutant (BHV-1}TF9-7) (lane 5).
Therefore, it was concluded that the 45 kDa protein band
represents BHV-1 gI.
To determine if gE and gI underwent N-glycosylation,
BHV-1-infected cells were treated with tunicamycin from 0 to
48 h post-infection and the infected cell extracts were analysed
by Western blotting (Fig. 2 B, lane 1–4). The 94 kDa BHV-1expressed gE was replaced by a band with an apparent
molecular mass of 84 kDa. The 45 kDa BHV-1-expressed gI
was replaced with a band with an apparent molecular mass of
40 kDa. These results indicate that recombinant-baculovirusexpressed gE and gI contain N-linked sugars.
To examine whether BHV-1 gE and gI form a complex as
is the case in HSV and PRV (Johnson & Feenstra, 1987 ;
Zuckermann et al., 1988), BHV-1-infected MDBK cells were
labelled with [H$]glucosamine from 6 to 24 h after infection
and the extracts were immunoprecipitated with anti-BgE or
anti-BgI and analysed by SDS–PAGE. As shown in Fig. 3 (A),
anti-BgE or anti-BgI precipitated both gE and gI together from
extracts of IBRV(NG)dltk-infected cells (lanes 1 and 5),
demonstrating that gE and gI form a complex. In the gI−
mutant (BHV-1}TF9-7), where gI is not expressed, gE appeared
to be synthesized and present in the infected cell extracts
without forming a gE–gI complex (Fig. 3 A, lane 3). Furthermore, gE was detected in the purified virions of the gI−
mutant (BHV-1}TF9-7) as shown in Fig. 3 (B), lane 3. On the
other hand, gI was detected neither in the gE− mutant (BHV1}TF9-3)-infected cell extracts (Fig. 3 A, lane 6) nor in the
virions of the gE− mutant (BHV-1}TF9-3) (Fig. 3 C, lane 2).
However, gI was detected in the medium of the gE− mutantinfected cells (Fig. 3 C, lane 5).
In this study we have identified BHV-1 gE and gI, the
respective products of BHV-1 ORFs 7 and 6, as glycoproteins
with apparent molecular masses of 94 and 45 kDa, respectively.
These molecular masses are larger than those predicted
(61±2 kDa and 39±9 kDa, respectively) from the sequences of
ORF 7 and 6. Since tunicamycin treatment reduced the
Fig. 3. Interaction between gE and gI in BHV-1 and its gE− and gI−
mutants. (A) Immunoprecipitation of gE and gI from BHV-1-infected cells.
MDBK cells were infected with IBRV(NG)dltk, BHV-1/TF9-3 or BHV1/TF9-7, or mock-infected. The infected cells were incubated in glucosefree RPMI medium containing 50 µCi D-[1,6-3H]glucosamine between 6
and 24 h post-infection. The cells were harvested, extracts were prepared,
and glycoproteins were immunoprecipitated with anti-BgE or anti-BgI. The
precipitates were subjected to SDS–PAGE followed by fluorography. Lanes
1–4, immunoprecipitated with anti-BgE ; lanes 5–8, immunoprecipitated
with anti-BgI. Lanes 1 and 5, IBRV(NG)dltk-infected cell lysate ; lanes 2
and 6, BHV-1/TF9-3-infected cell lysate ; lanes 3 and 7, BHV 1/TF9-7infected cell lysate ; lanes 4 and 8, mock-infected cell lysate. (B, C)
Western blot analysis of purified virions of IBRV(NG)dltk, BHV-1/TF9-3
and BHV-1/TF9-7 and virion-free supernatants. Monolayers of MDBK cells
grown in 100 mm dishes were infected with IBRV(NG)dltk, BHV-1/TF9-3
or BHV-1/TF9-7 at about 3 p.f.u. per cell. After 48 h, the media were
centrifuged at 3000 r.p.m. for 20 min. The cell-free supernatants were
further centrifuged at 100 000 g for 2 h in a Hitachi ultracentrifuge (type
55p-72) with an RPS40T rotor. The supernatants were stored at ®80 °C.
The pellets were suspended in 1 ml PBS, layered onto a linear potassium
tartrate gradient (10–40 %) and centrifuged with the same rotor at
100 000 g for 2 h. The virion band was collected, dialysed in PBS and
stored at ®80 °C. The virus-free supernatants and the virion fractions
were analysed by Western blotting using anti-BgE (B) or anti-BgI (C). Lane
1, IBRV(NG)dltk virions ; lane 2, BHV-1/TF9-3 virions ; lane 3, BHV-1/TF97 virions ; lane 4, supernatant of purified IBRV(NG)dltk ; lane 5,
supernatant of BHV-1/TF9-3 ; lane 6, supernatant of BHV-1/TF9-7.
Molecular masses of markers are given in kDa on the right.
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N. Yoshitake, X. Xuan and H. Otsuka
apparent molecular mass of gE from 94 kDa to 84 kDa, it
would appear that gE undergoes tunicamycin-sensitive Nlinked glycosylation and some other tunicamycin-resistant
modifications such as O-linked glycosylation. On the other
hand, tunicamycin treatment reduced the molecular mass of gI
from 45 kDa to 40 kDa suggesting that gI contains only
N-linked sugars.
Like gE and gI of other alphaherpesviruses such as HSV,
VZV and PRV, these proteins in BHV-1 are involved in the
cell-to-cell spread of virus since the gE− and gI− mutants of
BHV-1 (BHV-1}TF9-3 and BHV-1}TF9-7, respectively) produced smaller plaques when re-infection of cells by virus
released in the medium was prevented. When re-infection was
allowed, the gE− and gI− mutants grew to about the same level
as the parental virus suggesting that gE and gI were not
involved in the process of virus maturation and release or
attachment and penetration of the cells.
It has been demonstrated for several alphaherpesviruses
that gE and gI can form a complex (Johnson & Feenstra, 1987 ;
Mijines et al., 1996 ; Yao et al., 1993 ; Zuckermann et al., 1988),
most likely a heterodimer (Whealy et al., 1993). gE and gI of
HSV and VZV, when individually expressed in heterologous
expression systems, were readily transported from the endoplasmic reticulum (ER) to the plasma membrane (Dubin et al.,
1990 ; Litwin et al., 1992 ; Yao et al., 1993). In PRV, export of
gE and gI from the ER was inefficient unless both proteins were
present (Whealy et al., 1993). In feline herpesvirus (FHV)infected cells, gE–gI interaction is required to allow exit of gE
from the ER (Mijines et al., 1996).
Our results indicated that gE is synthesized, processed and
transferred to the virus envelope in the gI− mutant of BHV-1.
On the other hand, gI is synthesized in the gE− mutant but
released into the medium. The apparent molecular mass of the
gI released into the medium is 45 kDa, which is about the same
as that of gI produced by the wild-type virus. The putative
amino acid sequences of gE and gI revealed that both contained
a hydrophobic anchor region at their carboxy termini (LeungTack et al., 1994). It may be that mature gI lacks the
hydrophobic anchor region at the carboxy terminus. In the gE−
mutant, the gI precursor may be synthesized and processed as
normal but then released into the medium because gI cannot be
incorporated in the viral envelope without forming a complex
with gE. Interestingly, gI produced by the baculovirus
recombinant, which may be processed differently from gI in
BHV-1-infected cells, was not released into the medium but
remained associated with the membrane of infected cells (data
not shown).
While this manuscript was in preparation, Whitbeck et al.
(1996) identified BHV-1 gE and gI. They reported the presence
of a 61±5 kDa gI precursor which is cleaved to 45 kDa and
16 kDa products. We failed to detect the 61±5 kDa gI precursor
or 16 kDa product in BHV-1-infected MDBK cells. This is
probably because the 61±5 kDa precursor is present at low
concentration in BHV-1-infected MDBK cells and that the anti-
BEAC
BgI serum we used was not sensitive enough to detect
61±5 kDa gI precursor or 16 kDa product in the Western
blotting analyses.
This work was supported by the grants from the Ministry of
Education, Science, Sports and Culture of Japan and the Itoh Memorial
Foundation.
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