Journal of General Virology (1990), 71, 873-880. Printed in Great Britain
873
Structural and immunological characterization of human cytomegalovirus
gp55-116 (gB) expressed in insect cells
David E. Wells, l Leone G. Vugler 2 and William J. Britt 1,2.
1Department o f Microbiology and 2Department o f Pediatrics, University o f Alabama at Birmingham, School o f Medicine,
Birmingham, Alabama 35294
The gene encoding the major envelope glycoprotein
complex, gp55-116 (gB), of human cytomegalovirus
(HCMV) was expressed at high levels in insect cells
utilizing a recombinant baculovirus. The mature intracellular form of the insect-derived gp55-116 was a
protein of Mr 150K which contained approximately
50K of N-linked oligosaccharides. The oligosaccharide
linkages were almost exclusively endoglycosidase Hsensitive. The 150K protein was processed, presumably
by proteolytic cleavage, to yield at least one of the
previously defined cleavage products of the gp55-116.
This processing step was significantly less efficient in
insect cells than the analogous step in mammalian
cells. Finally, the insect-derived gp55-116 was highly
immunogenic in experimental animals and readily
recognized by antibodies contained within HCMVimmune human serum, suggesting that this recombinant protein warrants further study as a potential
HCMV subunit vaccine candidate.
Introduction
The envelope of HCMV contains an undetermined
number of distinct proteins, although currently only six
glycosylated species have been well described (Britt,
1984; Britt & Auger, 1985; Gretch et al., 1988; Nowak et
al., 1984; Pereira et al., 1984; Rasmussen et al., 1984;
Stinski, 1976). The major component of the envelope is a
disulphide-linked complex of proteins consisting of a
heavily glycosylated protein of Mr I16K and a less
heavily glycosylated protein of Mr 55K (Britt, 1984; Britt
& Auger, 1986; Britt & Vugler, 1989). We have termed
this the gp55-116 complex (Britt, 1984), but other
nomenclature commonly in use includes gB, gp58 and
gCI (Britt & Auger, 1985; Gretch et al., 1988; Nowak et
al., 1984). It is thought to be the HCMV homologue of
herpes simplex virus type I glycoprotein B. The primary
translation product of the gp55-116 gene is a 100K
protein which is cotranslationally glycosylated and
modified to an abundant and stable intracellular form of
Mr 150K (Britt & Auger, 1986; Britt & Vugler, 1989).
Following a series of oligosaccharide-modifying steps, a
fully glycosylated, penultimate polyprotein of Mr 170K is
cleaved to yield the two mature proteins with Mr values
116K and 55K (Britt & Vugler, 1989). Biochemical and
immunological analysis of the mature and immature
forms of these proteins has been hampered by the
inability to conveniently isolate sufficient quantities of
the proteins. It has been estimated that gp55-116
represents less than 1 ~ of the mass of the virion. To
circumvent this problem, several prokaryotic and eukar-
Human cytomegalovirus (HCMV), the largest of the
human herpesviruses, is a well recognized pathogen in
humans. Immunocompromised allograft recipients suffer severe and sometimes fatal infections with HCMV in
the post-transplant period (Ho, 1982). Recent reports
have stressed the importance of HCMV in patients with
human immunodeficiency virus infections (Drew &
Mintz, 1984). In addition, HCMV is the most common
cause of congenital viral infections with an annual
incidence of 1 ~ in the U.S.A. (Alford etal., 1980; Stagno
et al., 1983). Although only 5 to 10~ of congenitally
infected infants suffer demonstrable sequelae following
this intra-uterine infection, this represents an estimated
2000 damaged infants every year in the U.S.A. (Alford et
al., 1980; Stagno et al., 1983). Protective immune
responses against HCMV are as yet undefined; however,
numerous immune effector functions have been described following HCMV infection (Betts & Schmidt,
1981; Ho, 1982; Meyers et al., 1980; Quinnan et al.,
1982). Recently investigators have attempted to study
the virus-encoded targets of these responses (Borysiewicz
et al., 1988; Forman et al., 1985; Liu et al., 1988; Van Der
Voort et al., 1989). Several reports have focused attention
on the envelope glycoproteins because of their immunogenicity as well as their surface expression on infectious
virions and virus-infected cells (Liu et al., 1988; Van Der
Voort et al., 1989).
0000-9228 O 1990SGM
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874
D. E. Wells, L. G. Vugler and IV. J. Britt
yotic recombinant expression systems have been utilized
(Britt et al., 1988; Cranage et al., 1986; Spaete et al.,
1988). At this time we are unaware of any report which
details the efficient large scale production of this
material from a recombinant system.
The use of baculovirus-based expression systems has
been shown to be useful in the isolation of large
quantities of recombinant proteins (Fraser, 1989). Many
mammalian viral genes have been successfully expressed
in baculovirus at surprisingly high levels (Estes et al.,
1987; Hu et al., 1987; Inumaru & Roy, 1987; Matsuura et
al., 1986; Murphy et al., 1988; Zhang et al., 1988).
Unfortunately, insect cells do not always authentically
glycosylate and process the heterologous protein, resulting in aberrant forms with altered immunogenicity.
Because this result cannot be predicted in advance, we
expressed the HCMV gp55-116 gene in a baculovirus
and analysed its processing and immunogenicity. Although the insect cell-derived HCMV gp55-116 was not
glycosylated and processed authentically, it retained its
immunogenicity. This expression system should prove
invaluable for production of therapeutic and diagnostic
HCMV reagents.
Methods
Cells and virus. Spodoptera frugiperda cells, clone SF9 (SF), were
maintained in Grace's medium supplemented with 10% (v/v) foetal
bovine serum, 100 units/ml penicillin and 50 ~tg/ml gentamicin. Wildtype Autographa californica nuclear polyhedrosis virus (AcMNPV) was
obtained from Dr Max Summers, Texas A & M University, College
Station, Tx., U.S.A. Wild-type viral DNA was purified by the method
of Summers & Smith (1987). The construction of the recombinant
vaccinia virus containing the intact HCMV gp55-116 (VVCMV) has
been described previously (Britt et al., 1988).
Recombinant baculovirus construction. The baculovirus transfer vector
pAcYM1 was provided by Dr David Bishop, Oxford University,
Oxford, U.K. (Matsuura et al., 1987). An XmallI fragment of
approximately 3.4 kbp containing the intact HCMV gp55-116 gene was
released from pCMVS1 (Britt et aL, 1988), treated with the Klenow
fragment of DNA polymerase and BamHI linkers were added. The
resulting fragment was then ligated into pUC 19 by standard techniques
to yield the plasmid pXC4. After release of the HCMV gp55-116 from
pXC4 with BamHI, the 3-4 kbp fragment was isolated by gel
electrophoresis and ligated into the unique BamHI site of pAcYMl to
yield the plasmid pYMXC4.
Approximately 5 ktg of CsCl-purified pYMXC4 and 1 Ixg of purified
AcMNPV viral DNA were transfected in SF cells by previously
described procedures (Summers & Smith, 1987). Four days following
the transfection, a large number of the SF cells exhibited polyhedra as
detected by light microscopy. At this time the supernatant was
collected by centrifugation and used to infect approximately forty
30 mm tissue culture dishes containing SF cells as described (Summers
& Smith, 1987). Polyhedron-negative plaques were identified and
picked using a Pasteur pipette. Initially, screening was carried out
using a 96-well microtitre plate seeded with SF cells and infected with
individual polyhedron-negative plaques. Individual wells were inoculated with 50 Ixl of medium containing suspected recombinant
baculovirus. Following a 5 day incubation period, the plates were fixed
in absolute ethanol and recombinant-positive plaques were identified
by an immunofluorescence assay using murine monoclonal antibody 717 and fluorescein isothiocyanate-conjugated anti-mouse IgG (Britt,
1984). Plaques expressing gp55-116 were subjected to three cycles of
plaque purification prior to use. One plaque-purified virus, Bac 3.1,
was selected for further characterization.
Radiolabelling o f Bac 3.1 or wild-type-infected S F cells. Cultures of
SF9 cells were infected with either Bac 3.1 or wild-type AcMNPV at
an m.o.i, of approximately 10 to ensure uniform infection of the
monolayer. Approximately 36 h later, the infected cells were washed
extensively with TN medium without methionine and pulse-labelled
with 50 ~tCi/ml of [3sS]methionine. Pulse-chase analyses were carried
out essentially as described (Britt & Auger, 1986) with a pulse period of
5 or 10 min followed by removal of the radioactive medium and
replacement with Grace's medium. Radiolabelling with [3H]glucosamine or pH]mannose was done 36 h following infection using
approximately 50 ~tCi/ml of radioactive sugar in an 8 h pulse labelling.
Inhibition of N-linked glycosylation was accomplished by incubation of infected SF cells in medium containing 1.0 pg/ml of tunicamycin
(TM; Boehringer Mannheim) for 90 rain in TN medium without
methionine. The medium was replaced with one containing 1.0 ~tg/ml
TM and 50 ~tCi/ml of [35S]methionine and labelling was carried out for
4 to 6 h. Radioisotope incorporation was monitored by measurement of
trichloroacetic acid-precipitable counts following solubilization of
infected cell proteins.
Immunoprecipitation, S D S - P A G E and Western immunoblotting. Following radiolabelling, individual cultures were rapidly frozen at
- 7 0 °C and then thawed. Infected cell proteins were solubilized in
RIPA buffer (1.0% Nonidet P40, 1.0% deoxycholate, 0-1% SDS,
0-05 M-Tris-HC1, 0.15 M-NaCI pH 7.4) at 4 °C for 20 min. Immunoprecipitation with murine monoclonal antibodies, collection of immune
complexes with Staphylococcus aureus Cowan I strain and analysis by
SDS-PAGE in 7-5% acrylamide gels have been previously described
(Britt, 1984; Britt & Auger, 1986). SDS-PAGE and estimation of
migration using Mr standards was carried out as described by Britt
(1984).
Western immunoblotting was done as described by Britt & Auger
(1986). Samples for Western immunoblotting were prepared by
immediate solubilizaZtion of infected cell pellets in disruption buffer
(2 % SDS, 5 % 2-mercaptoethanol, 0-15 M-Tris-HCI pH 8.0) followed by
heating to 100 °C for 3 min.
Endoglycosidase treatment. Endoglycosidase H (Endo H, endo-fl-Nacetylglucosaminidase H; Boehringer Mannheim) treatment of immune-precipitated protein was performed as described previously
(Britt & Vugler, 1989).
Neutralization assays. Neutralizing activity was assayed by a rapid
(16 h) immunofluorescence assay (Andreoni et al., 1989). Briefly, 0-2 ml
of mouse serum diluted in medium was added to 0-2 ml of titrated virus
containing 10% (v/v) guinea-pig complement. Following a 60 min
incubation at 37 °C, 0. l ml of the mixture was added to replicate wells
of a 96-well microtitre tissue culture plate containing SF cells. After an
approximate 2 h adsorption period, the inoculum was removed, the
monolayers were washed once with medium, fresh medium was added
and the plate was incubated overnight at 37 °C. The following day the
medium was removed, the monolayer washed once with phosphatebuffered saline (PBS, pH 7-4) and fixed in absolute ethanol. Following
fixation, the monolayers were stained with monoclonal antibody P6327 (Andreoni et al., 1989), which is reactive with the major HCMV
immediate protein, pp72, and the number of antigen-positive cells was
quantified by immunofluorescence. Results are expressed as the mean
percentage reduction of fluorescent nuclei (infectivity). The standard
error of the mean of this assay is at most 10%, with usual values of 5 %.
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H C M V gp55-116 expression in insect cells
Results
875
1 2
Expression of HCMV gp56-116 in insect cells
Protein expression of recombinant baculovirus-infected
cells was monitored by immunofluorescence using
H C M V gp55-116 specific monoclonal antibodies. Cells
infected with recombinant virus Bac 3.1, which contained the intact gene encoding H C M V gp55-116,
exhibited bright immunofluorescence indicaling expression of this H C M V protein in insect cells (data not
shown). We next examined the expression of H C M V
gp55-116 in insect cells by subjecting equivalent quantities of [3SS]methionine-radiolabelled infected cell proteins from recombinant virus 3.1- or wild-type
AcMNPV-infected cells to S D S - P A G E . Bac 3.1infected cells contained a protein of estimated Mr 150K
which was not seen in wild-type-infected cells (Fig. 1).
The level of expression was significant as it accounted for
an estimated 10 % of the infected cells' [35S]methionine.
radiolabelled proteins (Fig. 1). Immunological characterization of the insect cell-derived gp55-116 was carried out
by immunoblotting of wild-type- and 3.1-infected cells
using H C M V gp55-specific monoclonal antibody 7-17
(Britt & Auger, 1986). As controls, VVCMV (Britt et al.,
1988) or recombinant vaccinia virus-infected ceils
expressing the human immunodeficiency virus gag-pol
gene products (VVHIV) were included in the experiments. Monoclonal antibody 7-17 recognized a protein of
approximate Mr 150K (gpl50) expressed by Bac 3.1 and
a more broadly migrating species of Mr 150K to 170K in
VVCMV-infected cells (Fig. 2). Faster migrating species
of M r 55K (gp55) and Mr 52K (gp52) were seen in
VVCMV-infected and Bac 3.1-infected cells, respectively (Fig. 2). As previously reported, monoclonal antibody
7-17 recognized a single species of Mr 55K in gradientpurified H C M V virions (Fig. 2). These results were
consistent with earlier studies and indicated that at least
two of the four previously defined intracellular forms of
the gp55-16 complex were present in Bac 3.1-infected
insect cells (Britt & Auger, 1986). The increased rate of
migration of the gp52 in baculovirus-infected cells, as
compared to the migration of gp55 in VVCMV-infected
cells, suggested that post-translational modifications of
this protein may be different in insect cells compared to
mammalian cells. Furthermore, the relative amount of
gp52 compared to the higher Mr form, gpl50, in Bac 3.1infected insect cells was much reduced as compared to
near equivalent amounts of the two forms seen in
VVCMV-infected cells, suggesting that cleavage of the
precursor protein was less efficient within insect cells.
Processing of the H C M V gp55-116 (gB) in insect cells
The synthesis of the H C M V gp55-116 in insect cells was
studied by pulse--chase analysis followed by immune
-gpl50
Fig. 1. EquivalentTCA-precipitablecounts of (lane 1) wild-type-and
(lane 2) recombinant Bac 3.1-infected cell proteins analysed by SDSPAGE. Migration was estimated using Mr standards as described in
Methods.
1
2
3
4
5
Fig. 2. Immunoblotanalysis of HCMV gp55-116 expressionin insect
ceils. Equivalent numbers (10s) of recombinant 3. l-infected SF cells
(lane 5) and VVCMV-infectedHeLa cells (lane 3) were subjected to
immunoblot analysis using monoclonal antibody 7-17 to detect the
HCMV-specific proteins. As controls, equivalent numbers of wildtype-infectedSF cells(lane 4) and VVHIV-infectedHeLa cells(lane 2)
were also included in the analysis. Purified HCMV virions (lane 1)
were included as an internal marker for migration of the authentic
gp55.
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876
(a)
D. E. Wells, L. G. Vugler and W. J. Britt
1 2
3
4 5 6 7 8
(b)
1 2
3 4 5 6 7 8
--gpl50
-- gp52
Fig. 3. Pulse-chase analysis of Bac 3.1-mfected cells (a) Bac 3 .1- or (b)
wild-type (wt)-infected cultures were pulse-labelled with [3sS]methionine for 10 min and chased for the following time periods: lane 1, 0 rain;
2, 10 rain; 3, 20 min; 4, 40 rain; 5, 80 min; 6, 160 rain; 7,320 min; 8, 18
h Infected cell proteins were precipitated with antibody 7-17 and
analysed by SDS-PAGE.
(a)
(b)
i
I
1
precipitation and SDS-PAGE. Monoclonal antibody 717 precipitated a protein of estimated Mr 150K following
a 10 min pulse labelling (Fig. 3). During the ensuing
chase periods there was a slight but perceptible increase
in the Mr of this form suggesting some type of posttranslational modification(s) (Fig. 3). Approximately
80 min into the chase period, the gp52 was detected (Fig.
3, lane 5) and this species increased in quantity during
the next two chase intervals. In addition, 160 min into
the chase period a faint but detectable, broadly migrating form of estimated Mr 80K to 100K was also seen (Fig.
3, lane 6). This could possibly represent the insect cell
form of the second component of the mature complex,
gpll6. A prolonged chase period of 18 h revealed the
gpl50 form and trace quantities of the gp52 form (Fig. 3,
lane 8). These results suggested that the approximate Mr
150K protein initially seen was modified, possibly by
oligosaccharide trimming, until a slightly faster migrating form, gpl50, was generated. In addition, the
previously recognized gp52 was apparently produced by
a post-translational event, most likely by proteolytic
cleavage of the gpl50.
The carbohydrate modification of insect cell-derived
gp55-116 was investigated next. The predominant
2
I
3
I
4
(a)
Control
1
2
TM-treated
3
4
(b)
Control
1
TM-treated
2
200K~
150
gpl50~
gpl50 w
97K
68K--
45K --
%@i
25K~
Fig. 4
Fig. 5
Fig. 4. SDS-PAGE analysis of HCMV gp55-116 precipitated from recombinant baculovirus-infected insect cells. Equivalent TCAprecipitable counts of (a) wild-type- or (b) recombinant virus (Bac 3.1)-infected cell proteins radiolabelled with either [3H]glucosamine
(lanes 1 and 3) or [3H]mannose (lanes 2 and 4) were immune-precipitated with monoclonal antibody 7-17 and analysed by SDS-PAGE.
Fig. 5. Effect of TM on synthesis of HCMV gp55-116 in insect cells. (a) Equivalent TCA-precipitable counts of recombinant virus(lanes 1 and 3) or wild-type- (lanes 2 and 4) infected [3sS]methionine-radiolabelled cell proteins from cultures treated with TM or left
untreated (control) were subjected to SDS-PAGE. The arrow indicates the unique species present in the TM-treated cultures. (b)
Infected cell proteins from Bac 3.1-infected cultures treated with TM or left untreated (control) were immune-precipitated with
antibody 7-17 and analysed by SDS-PAGE.
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H C M V gp55-116 expression in insect cells
(a)
Control
l
2
3
4 5
6
877
Table 1. lmmunogenicity of insect-derived gp55-116
7
8
9
Reduction in infectivity* (~)
at serum dilutions of
m gpl50
(b)
1:100
1:200
1:400
1
2
3
4
5
Control
57
68
91
65
80
0
52
62
70
52
60
0
40
60
67
60
62
0
* Reduction of input virus (neutralization) was determined using a
16 h fluorescent nuclei reduction assay as described in Methods.
Results are expressed as percentage reduction at indicated serum
dilution.
t Individual mice were given 106 Bac 3.l-infected SF ceils
emulsified in complete Freund's adjuvant followed 10 days later with a
similar amount of cells emulsified in incomplete Freund's adjuvant.
Mice were bled 7 days later and serum was assayed as above. Control
mice were immunized with a prokaryotic cell-derived HCMV DNAbinding protein emulsified in complete Freund's adjuvant and assayed
as experimental mice (Britt et al., 1988).
Endo H-treated
1 2 3 45678
Mouset
9
ml00K
Fig. 6. Endo H sensitivity of HCMV gp55-116 protein synthesized in
insect ceils. A pulse-chase analysisof Bac 3.1-infected cellswas carried
out as described in Fig. 3, with chase times as follows: lane 1, 0 min; 2,
5min; 3, 10min; 4, 20min; 5, 40min; 6, 80min; 7, 160min; 8,
320 rain; 9, 24 h. Immune-precipitatedproteins were treated with 0-005
units of Endo H or left untreated (control) and analysed by SDSPAGE.
intracellular form, gp150, contained both glucosamine
and mannose as evidenced by immunoprecipitation of
this form following radiolabelling of infected cell
proteins with either [3H]glucosamine or [3H]mannose
(Fig. 4). We failed to detect incorporation of either
radiolabelled sugar into the gp52, most likely owing to
the low carbohydrate content of this cleavage product
and inefficiency of cleavage of the gpl50 in insect cells
(Britt, 1984; Britt & Vugler, 1989). To determine the
extent of glycosylation, Bac 3.1-infected cells were
radiolabelled with [35S]methionine in the presence of
T M and analysed by S D S - P A G E . A protein of
estimated Mr 100K could be seen in cultures incubated in
T M and this species was specifically immunoprecipitated with monoclonal antibody 7-17 along with minor,
more slowly migrating proteins which were probably
products of incomplete inhibition of N-linked glycosylation by T M (Fig. 5). Thus, the insect cell-derived gpl50
precursor contained some 50K of N-linked sugars, a
finding similar to that reported in m a m m a l i a n cells (Britt
& Vugler, 1989). The structure of the carbohydrate
linkages present in gp 150 were next characterized using
Endo H. Following a pulse-chase experiment with
similar chase intervals as in Fig. 3, half of the immuneprecipitated sample was treated with Endo H and the
other half left untreated. The samples were then analysed
by S D S - P A G E . During all chase intervals the gpl50
appeared to be sensitive to Endo H, with the size being
reduced to approximately Mr 100K following Endo H
treatment (Fig. 6). Interestingly, at 80 min into the chase
period the migration of the Endo H-treated form was
retarded slightly as compared to that seen in previous
chase intervals (Fig. 6). This decreased migration was
also seen in the subsequent chase intervals and in the
migration of species detected following a 24 h chase (Fig.
6), suggesting that at these time points some oligosaccharide side-chains were Endo H-resistant. In this
experiment, the Endo H sensitivity of the cleavage
product gp52 could not be determined as only trace
amounts of this protein were present.
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878
D. E. Wells, L. G. Vugler and W. J. Britt
(a)
1 2
(b)
3
4 5
6
1 2
3 4
5 6
--gpl50
Fig. 7. Reactivity of human anti-HCMV serum with recombinant
baculovirus-derived HCMV gp55-116 (gB). [35S]Methionine-labelled
(a) wild-type baculovirus or (b) recombinant Bac 3.1-infected cell
proteins were immune-precipitated with serum from a seronegative
individual (lane 1), monoclonal antibody 7-17 (lane 2) or serum
specimens from individuals with seroimmunity to HCMV (lanes 3 to
6). Precipitated proteins were analysed by SDS-PAGE as described in
Methods.
Immunogenicity of insect cell-derived H C M V p55-116
The immunogenicity of the insect cell-derived gp55-116
was investigated initially in vivo by injecting Bac 3.1infected cells into mice. After two immunizations all
animals developed significant quantities of neutralizing
antibodies indicating the immunogenicity of this insect
cell-derived protein (Table 1). The reactivity of human
HCMV-immune serum against the insect cell-derived
gp55-116 was next analysed by immune precipitation
and SDS-PAGE. Serum from a seronegative individual
failed' to precipitate the insect cell-derived gpl50,
whereas serum specimens from four seroimmune individuals precipitated the gp150 (Fig. 7). Thus, insect cellderived gpl50 appeared to be immunogenic in experimental animals and, perhaps more importantly, was
readily recognized by antibodies found in individuals
with naturally acquired immunity to HCMV.
Discussion
The use of a recombinant baculovirus allowed significant
levels of expression of the HCMV gp55- I 16 (gB) gene in
insect cells. Although we have not carried out an
analytical purification of the insect cell-derived gp55-
116, our initial experiments have suggested that nearly
10% of the total [35S]methionine-radiolabelled infected
cell protein was gp55-116. This level of expression was
significantly higher than we have previously obtained
using a recombinant vaccinia virus containing the
HCMV gp55-116 gene (Britt et al., 1988). Furthermore,
after additional modifications of the vaccinia virus
transfer vector in which the HCMV gp55-116 gene was
placed under control of the stronger pl 1 late promoter
(W. J. Britt & E. B. Stephens, unpublished results), we
could still demonstrate at least 10-fold more antigenically
reactive intracellular gpl50 in insect cells than in cells
infected with the recombinant vaccinia virus. Thus, it
appeared that expression of the gp55-116 in insect cells
might offer a useful system for obtaining sufficient
quantities of this protein for future biochemical and
immunological studies. Preliminary experiments have
shown that approximately 1 to 2 mg of antigenically
reactive gp55-116 was obtained by passage of a detergent-solubilized lysate from 108 infected SF cells over an
immunoaffinity matrix (data not shown).
Maturation of the intracellular precursor of gp55-116
within insect cells involved a processing step, possibly
trimming of carbohydrate side-chains, which yielded a
fully modified form of estimated Mr 150K. In contrast,
processing of the gp55-116 intracellular precursor,
gpl50, within mammalian cells involved additional
carbohydrate modification to yield a final, fully glycosylated form of estimated Mr 170K (Britt & Vugler, 1989).
In both insect cells and mammalian cells, these fully
glycosylated forms were then cleaved into the mature
smaller Mr polypeptides. In agreement with previous
studies, we found that glycosylation of the HCMV gp55116 within insect cells was not authentic (Hurwitz, 1988;
Kuroda et al., 1986, 1987). Our results suggested that the
vast majority of the oligosaccharide side-chains on the
gp 150 contained Endo H-sensitive linkages, whereas the
majority of oligosaccharides present on the gp 170 found
in mammalian cells contained Endo H-resistant, complex sugars (Britt & Vugler, 1989). The demonstration of
a small number of Endo H-resistant linkages on the
insect cell-derived gpl50 raised the possibility that
previously described glycoprotein processing pathways
unique to insect cells were operative (Hsieh & Robbins,
1984). We could not demonstrate endoglycosidase Dsensitive sugar linkages on either the gpl50 or gp52
expressed in insect cells (data not shown), suggesting that
the insect cell analogue of mammalian complex oligosaccharide linkages, mannose3, (N-acetylglucosamine)2,
were not present on the HCMV gp55-116 expressed in
insect cells (Hsieh & Robbins, 1984). In addition,
preliminary experiments also suggested that only Endo
H-sensitive carbohydrate modifications were present on
the gp52, providing additional evidence for the presence
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H C M V gp55-116 expression in insect cells
of only one type of oligosaccharide side-chains on the
gp55-116 molecules (data not shown). Conversely,
because we have not characterized the second component of the gp55-116 complex, gp116, following expression of the gp55-116 gene in insect cells, we cannot
completely rule out the possibility that this form may
contain significant quantities of Endo H-resistant
sugars. This possibility must be considered in the light of
previous studies in HCMV-infected mammalian cells
which indicated that 20 to 30 ~ of the mass of this protein
consisted of complex oligosaccharides (Britt & Vugler,
1989). Our results, however, make this possibility
unlikely as we could not detect significant quantities of
Endo H-resistant oligosaccharides on fully processed
gpl50, the penultimate form within insect ceils. Finally,
resistance to either Endo H or Endo D might have
resulted from aberrant folding of the insect-derived
protein, limiting accessibility to the respective enzyme,
and not the actual absence of the specific carbohydrate
linkage.
Although some controversy exists over the proteolytic
processing of mammalian viral polyproteins expressed in
insect cells, our results indicated that cleavage of the
gp 150 precursor, into one of the smaller forms (gp52) and
probably the second (gp116), did occur in insect cells. At
this time we cannot determine whether this cleavage
occurred in a homologous site as defined previously in
the gp55-116 of HCMV strain Towne (Spaete et al, 1988).
Because anti-gp55 antibodies recognized only one lower
Mr form, gp52, it seems likely that cleavage of the
precursor occurred at a single site, and not randomly
along the molecule. The availability of significant
quantities of gpl50 and gp52 should allow us to address
this question directly in the future. Although we could
demonstrate cleavage of the gpl50 precursor, this
processing step was much less efficient in insect cells
than that observed in mammalian cells. We estimated
that the cleavage product, gp52, represented at most 10
of the intracellular gpl50 in insect cells, whereas the
vaccinia recombinant virus-derived cleavage product,
gp55, represented approximately 50~ of its intracellular
precursor (Fig. 2). The reason for the inefficiency in the
cleavage of the precursor protein in insect cells is
unknown. Previous studies utilizing recombinant baculovirus-expressed viral glycoproteins have yielded conflicting results. In some cases, endoproteolytic cleavage
has been documented, such as in fowl influenza virus
haemagglutinin (Kuroda et al., 1986, 1987), but not in the
human immunodeficiency virus gpl60 (Rusche et al.,
1987) or the haemagglutinin of human influenza virus
(Possee, 1986). As yet, there are no explanations for these
discrepant findings, although at least one report suggested that many of the proteolytic enzymes responsible for
protein maturation in mammalian cells were not present
879
in insect cells (Lebacq-Verheyden et al., 1988). An
alternative and more simplistic explanation of our results
would be that the enzymic activity responsible for
cleavage of the precursor was saturable and unable to
process efficiently the large amount of protein present. It
was also of interest that cleavage of the gp 150 precursor
into the smaller Mr form occurred within a similar postlabelling chase interval as that previously shown for
cleavage of the gp55-116 precursor, gpl70, in HCMVinfected human fibroblast cells (Britt & Auger, 1986).
This finding suggested the kinetics of intracellular
transport were similar in both cell systems. In addition,
we have been able to demonstrate the gp55-116 on the
surface of infected insect cells by two different techniques (data not shown), providing additional evidence
that the intracellular transport of this glycoprotein was
similar in both insect and mammalian cells. Thus, it was
unlikely that inefficient cleavage of the intracellular
precursor was a result of aberrant or delayed intracellular
transport.
Authentic processing and glycosylation of the HCMV
gp55-116 in insect cells was apparently not required for
immunogenicity. Immunization of mice with Bac 3.1infected SF cells led to the production of high levels of
neutralizing antibodies. The level of neutralizing antibodies generated in these mice was comparable to the levels
previously reported in mice immunized with a replicating vaccinia :gp55-116 recombinant virus (Britt et al.,
1988). In addition, the baculovirus-derived gp55-116 was
readily recognized by human anti-gp55-116 antibodies
produced following naturally acquired HCMV. Thus, it
appeared the insect cell-derived gp55-116 was antigenically authentic. Because of the high level of expression of
this HCMV protein in insect cells and its apparent
antigenicity, this recombinant-derived product might be
an important component of a future non-replicating
vaccine to limit the morbidity of HCMV infections.
The authors wish to thank Nina Reynoldsfor preparation of the
manuscript.This workwas supportedby NICHDgrant HD10699and
grants from the March of Dimes and United Cerebral Palsy
Foundation.
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