Download Processing of human cytomegalovirus envelope glycoproteins in

Journal of General Virology (1992), 73, 253-260. Printed in Great Britain
253
Processing of human cytomegalovirus envelope glycoproteins in and egress
of cytomegalovirus from human astrocytoma cells
Bruce Kari,* Robert Radeke and Richard Gehrz
Biomedical Research Institute, Biochemistry Division, Children's Hospital St P a u l Minnesota 55102, U.S.A.
The synthesis of human cytomegalovirus (HCMV)
envelope glycoproteins and the production of infectious HCMV in human astrocytoma and skin fibroblast
(SF) cells were analysed. HCMV envelope glycoproteins synthesized in astrocytoma cells had lower Mrs
than the same glycoproteins synthesized in SF cells
regardless of the strain of HCMV used, showing that
the differences observed were due to differences in
processing by the host cell and not the strain of HCMV
used. HCMV envelope glycoproteins synthesized in
astrocytoma cells were found to contain less galactosamine. Moreover, when synthesized in SF cells some
HCMV glycoproteins contained a protease-resistant
fragment owing to the presence of a cluster of O-linked
oligosaccharides on the polypeptide. This fragment
was not present when these HCMV glycoproteins were
synthesized in astrocytoma cells. These data suggested
that HCMV glycoproteins synthesized in astrocytoma
cells contain fewer O-linked oligosaccharides. In
contrast, other post-translational events such as proteolytic cleavage of the HCMV gB glycoprotein and the
formation of disulphide-linked complexes did occur.
The virus produced in astrocytoma ceils was capable of
infecting SF cells, suggesting that complete O-glycosylation is not needed to produce infectious HCMV.
However, astrocytoma cells were slow to release virus
into the culture medium, suggesting that a fully
functional Golgi network is needed for efficient egress
of HCMV from the host cell.
Introduction
the culture medium 9 to 11 days p.i. (Wroblewska et al.,
1981). Thus, the egress of HCMV from primary brain
cells appears to be impaired relative to that from
fibroblast cells.
By using the inhibitors monensin (Johnson & Spear,
1982) and brefeldin A (BFA) (Whealy et al., 1991) and
electron microscopy (Jones & Grose, 1988; Severi et al.,
1988), it has been established that the Golgi network is
important in the egress of herpesviruses from infected
cells. Both monensin and BFA can inhibit the processing
of herpesvirus glycoproteins in the Golgi network, but
not the assembly of enveloped particles. These enveloped
particles remained cell-associated and, with monensin,
were infectious (Johnson & Spear, 1982).
Since the Golgi network appears to be important to the
egress of herpesviruses from the host cell, it seemed
possible that brain cells might be slow to release HCMV
because of differences in HCMV glycoprotein processing in the Golgi network. To investigate this possibility,
we examined the production of infectious HCMV and
the synthesis of HCMV envelope glycoproteins in
astrocytoma cells. The HCMV envelope glycoproteins
analysed in this study are contained in disulphide-linked
glycoprotein complexes designated glycoprotein complex I (gC-I) and glycoprotein complex II (gC-II) (Gretch
Human cytomegalovirus (HCMV) is a major cause of
birth defects due to congenital infection and a
significant cause of morbidity and mortality in immunocompromised patients (Ho, 1982). In the foetus, susceptibility of the developing central nervous system to HCMV
is a primary cause of irreversible sequelae such as mental
retardation and sensorineural hearing loss (Stagno et al.,
1984). HCMV encephalitis has also been observed in
AIDS patients, in whom both glial cells and neurons are
infected (Wiley et al., 1986). Little is known about the
replication of HCMV in cells of nervous tissue origin. In
vitro models of HCMV brain cell infection have been
established to attempt to gain a better understanding of
HCMV replication in brain cells. In vitro, HCMV is
capable of replicating in primary brain cells and several
cell lines of nervous tissue origin (Wroblewska et al.,
1981; Ogura et al., 1986; Kari & Gehrz 1986; Duclos et
al., 1989; Poland et al., 1990). Primary brain cells are
capable of producing infectious HCMV; however, these
cells are slow to release infectious virus, 60 to 70% of
infectious HCMV remaining cell-associated 9 to 11 days
post-infection (p.i.). In contrast, 60 to 70% of the
infectious HCMV made in fibroblast ceils is released into
0001-0268 © 1992SGM
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254
B. K a r l R. Radeke and R. Gehrz
et al., 1988 a). gC-I complexes are c o m p o s e d of multimers
o f the H C M V gB glycoprotein (Karl et al., 1990b). This
H C M V glycoprotein was designated gB because o f its
similarity to the gB glycoprotein from herpes simplex
virus (Cranage et al., 1986). T h e gB glycoprotein is
synthesized as a large precursor glycoprotein w h i c h is
eventually cleaved into two glycoproteins in the Golgi
network o f infected fibroblasts (Gretch et al., 1988c;
Britt & Vugler, 1989; K a r l et al., 1990b). g C - I I
complexes contain two groups o f biochemically and
immunologically distinct glycoproteins (Karl et al.,
1990 a). These glycoproteins have no k n o w n h o m o l o g y to
other herpesvirus glycoproteins. T h e H C M V glycoproteins most extensively analysed in this study were those
g C - I I glycoproteins with Mrs of 47000 to 63000 (gp4763). The gp47-63 glycoproteins are encoded by the
H C M V H X L F gene family (Gretch et al., 1988b) and
contain the highest content o f O-linked oligosaccharides
o f all the glycoproteins found in g C - I and g C - I I (Karl &
Gehrz, 1988). This high content of O-linked oligosaccharides m a d e gp47-63 useful for analysing potential
differences between glycoprotein processing in the Golgi
network o f astrocytoma cell lines and skin fibroblast
(SF) cells.
until use, at which time they were frozen and thawed twice to release
intracellular virus. Virus titre (p.f.u./ml) was determined as previously
described (Lussenhop et al., 1988) using confluent monolayers of SF
ceils.
Methods
Determination of hexosamine content. Hexosamine content was
determined as previously described (Karl & Gehrz, 1988).
Cells and virus. Human SF cells were obtained from foreskins. The
astrocytoma cell lines U-373MG and Hs683 were obtained from the
ATCC. Stocks of the Toledo and Towne strains of HCMV were
obtained by growth in SF cells, as previously described (Karl et al.,
1986).
Results
Monoclonal antibodies (MAbs). MAbs 15F9 and 27B4 recognized gCII disulphide-linked glycoprotein complexes and have been previously
described (Karl et al., 1990a). Glycoprotein complexes containing the
HCMV gB glycoprotein were immunoprecipitated with MAb 41C2
(Lussenhop et al., 1988).
Immunofluorescence and immunoprecipitation of radiolabelled HCMV
glycoproteins using MAbs. Infected cells were fixed with cold acetone
and methanol (50 : 50) and processed for indirect immunofluorescence
as described (Karl et al., 1990b). Immunoprecipitations were done with
[3H]GlcN (Amersham)-labelled HCMV glycoproteins. Briefly, SF or
U-373MG ceils were scraped from culture flasks in PBS pH 7.5,
collected by centrifugation and resuspended in Tris-buffered saline
(TBS) (50 mM-Tris-HC1pH 7.5, 10 mM-NaC1,2 mM-PMSF)containing
1.0~ NP40. Cells were extracted for 1 h with constant mixing.
Insoluble material was removed by centrifugation and the supernatant
used for immunoprecipitations. Immunoprecipitations were done
using biotinylated MAbs and streptavidin-agarose as described
(Gretch et aL, 1987).
Plaque assay. Cultures were infected at a multiplicity of 3. Virus was
allowed to adsorb for 1 h before cultures were rinsed with PBS to
remove free virus. Cultures were given fresh medium and, 3 and 7 days
p.i., culture medium from infected cultures was removed, centrifuged
to remove cell debris and stored in liquid nitrogen. After removal of the
culture medium, infected cells were rinsed with PBS and then scraped
into 1 ml of PBS. The suspended cells were stored in liquid nitrogen
SDS-PAGE andfluorography. Radioactively labelled HCMV glycoproteins were separated by SDS-PAGE in 9% polyacrylamide gels
following the method of Laemmli (1970). Radioactivity in gels was
detected by fluorography using EN3HANCE (New England Nuclear).
Purification of gC-H glycoproteins, gC-II complexes from SF or
U-373MG cells were immunoaffinity-purified using biotinylated MAb
15F9 and streptavidin-agarose as described above. Immunoaffinitypurified gC-II complexes were reduced with DTT and alkylated with
iodoacetamide as described (Karl & Gehrz, 1988). Individual gC-II
glycoproteins were separated by gel filtration HPLC using coupled
TSK 3000 and 4000 columns (Toyo Soda). HPLC was done at a flow
rate of 0-3 ml/min using a buffer of 10 mM-Tris-HCl pH 7.8 containing
0.1 ~ SDS. The eluate was monitored for radioactivity.
Pronase digestion, G-50 chromatography and lectin chromatography.
Pronase digestion and G-50 column chromatography were done as
described (Karl & Gehrz, 1988). Agarose-wheatgerm lectin (WGA)
and agarose-peanut lectin (PNA) were obtained from United States
Biochemical and Helix pomatia lectin-Sepharose (HPA) was obtained
from Pharmacia. WGA and PNA affinity chromatography (Karl &
Gehrz, 1988) and HPA affinity chromatography (Lundstrom et al.,
1987a) were done as described. Briefly, glycopeptides obtained by
pronase digestion were adsorbed to 1.0 ml affinity columns and the
columns were washed with 15 column volumes of PBS. Bound
glycopeptides were eluted with the appropriate sugars as indicated in
Fig. 6.
Analysis o f infected cells for g C - H glycoproteins by
immunofluorescence, and for production and release o f
infectious H C M V
The intracellular localization o f g C - I and g C - I I
glycoproteins was determined by indirect i m m u n o fluorescence using fixed permeabilized cells. Cells were
infected with H C M V T o w n e strain at a multiplicity o f 3
and were fixed 3 days p.i. with cold acetone :methanol
(50:50, v/v). Cultures were processed for indirect
immunofluorescence using M A b s specific for g C - I I
glycoproteins. W h e n this was done, a bright spot o f
perinuclear immunofluorescence was detected in m a n y
U373, Hs683 and SF cells (Fig. 1 a, b and c). T h e staining
detected was typical of proteins localized to the Golgi
network (Donaldson et al., 1990). Similar results were
obtained with M A b s specific for gC-I (data not shown).
Production o f infectious virus was determined for
Hs683, U - 3 7 3 M G and SF cells as described in Methods.
The n u m b e r o f infectious units generated by each cell
type was determined by plaque assay using SF cells. A t 3
days p.i., 2 ~ o f the infectious virus was found in the
culture m e d i u m f r o m infected U - 3 7 3 M G cells and 23 ~o
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H C M V glycoprotein synthesis
4 2.0
4
c
3
s
3
c
7
s
7
4
1.0
"
255
~
c
3
s
c s
c
s
c
s
3
7 7
3
3
7 7
Time p.i. (days)
Fig. 1. Immunofluorescencemicrographsof HCMV Towne strain-infectedU-373MG (a) and Hs683 (b) astrocytomacells and SF cells
(c). Cells were fixed with cold acetone:methanol (50 : 50) and prepared for indirect immunofluoresenceusing the gC-II-specificMAb
27B4. Bar markers represent 25 ~tM.(d, e, f ) Virus productionin U-373MG, Hs683 and SF cells, respectively.Bars representthe mean
plus and minus the standard deviation of virus production in six separate cultures. C, Cell; S, supernatant.
by 7 days p.i. (Fig. 1d). With Hs683 cells, 2~o was found
in the culture medium at 3 days p.i., but this increased to
4 1 ~ by 7 days p.i. (Fig. 1 e). With SF cells, 4 2 ~ of the
infectious H C M V had been released from the cells at 3
days p.i. and 8 8 ~ by 7 days p.i. (Fig. l f ) . These data
show that H C M V was released most efficiently from SF
cells, less efficiently from Hs683 cells and least efficiently
from U-373MG cells. SF cells also produced more virus
in total than either astrocytoma cell line, but the virus
produced by astrocytoma cells was capable of infecting
SF cells.
Analysis of H C M V gB synthesis in astrocytoma cells
Since drugs that interfere with herpesvirus glycoprotein
processing in the Golgi network have also been shown to
inhibit their egress from the infected cell (Johnson &
Spear, 1982; Whealy et al., 1991), we compared the
H C M V glycoproteins synthesized in SF and U-373MG
cells to determine whether there was any difference in
how astrocytoma cells process H C M V glycoproteins. U373MG cells were used because they were the slowest to
release H C M V into the culture medium. Studies were
initiated by analysis of the H C M V gB glycoproteins. To
do this, infected cells were incubated with [3H]GIcN to
label glycoproteins. Infected cells were harvested and
extracted with NP40, insoluble material was removed by
centrifugation and the supernatant was used for
immunoprecipitations using a gB-specific MAb. The
MrS of the gB glycoproteins synthesized in SF and U373MG cells and the disulphide-linked complexes
formed by gB were compared by S D S - P A G E . When
isolated from SF cells the most abundant disulphidelinked complexes had M:s of 150000 and 250000 (Fig.
2a, lane 1); a minor component with an Mr of 130000
was also detected (Fig. 2a, lane 1). This component was
not isolated from extracellular virus (Kari et al., 1990b)
and may be a precursor to the higher Mr complexes.
When isolated from U-373MG cells, complexes were
obtained which had Mrs less than 150000, as were
complexes with an Mr of 250000 (Fig. 2a, lane 2). The
component with an Mr of 130000 was also detected. The
amounts of the 150000 and 250000 Mr complexes were
lower relative to the 130000 Mr component, suggesting
that these complexes were slower to form in U-373MG
cells.
After reduction of disulphide bonds in complexes
obtained from SF cells, three glycoproteins were detected
in SDS-polyacrylamide gels (Fig. 2b, lane 1). These
glycoproteins had Mrs of 52000 (gp52), 93000 to 130000
(gp93-130) and 158000 (gp158); gp158 is a precursor
which generates gp52 and gp93-130 after proteolysis
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B. Kari, R. Radeke and R. Gehrz
256
(a)
1
2
(b)
1
2
(a)
1
(b)
2
1
2
250K- 150K-130K--
158K130K93K-
130K--
130K
93K-67K--
52K-
- 90K
~'-63K
Fig. 2. U-373MGand SF cells were infected with the Towne strain of
HCMV. Glycoproteinswere labelled with [3H]GIcN. At 7 days p.i.,
infected cells were collected, extracted with 1~ NP40 in TBS, and
insoluble material was removed by centrifugation. Extracts were
immunoprecipitated with gB-specificMAb 41C2. (a) gC-I complexes
containing gB examined by SDS-PAGE under non-reducing conditions. Lane 1, gC-I complexes obtained from SF cells; lane 2, gC-I
complexes obtained from U-373MG cells. (b) gC-I complexes
examined by SDS-PAGE under reducing conditions. Lane 1, gC-I
glycoproteins synthesized in SF cells; lane 2, gC-I glycoproteins
synthesized in U-373MG cells. Mrs are indicated.
Fig. 3. U-373MGand SF cells were infected with the Toledo strain of
HCMV. Glycoproteinswere labelled with [3H]GlcN. At 7 days p.i.,
infected cells were collected, extracted with 1~ NP40 in TBS and
insoluble material was removed by centrifugation. Extracts were
immunoprecipitated with gC-II MAb 27B4. (a) gC-II complexes
examined by SDS-PAGE under non-reducingconditions. Lane 1, gCII complexesobtained from SF cells; lane 2, gC-II complexesobtained
from U-373MGcells. (b) gC-ll glycoproteinsexaminedby SDS-PAGE
after reduction of disulphide bonds. Lane I, gC-II glycoproteins
synthesized in SF cells; lane 2, gC-II glycoproteinssynthesizedin U373MG cells. Mrs are indicated.
(Kari et al., 1990b). Three glycoproteins were also
detected in SDS-polyacrylamide gels after reduction of
complexes obtained from U-373MG cells (Fig. 2b, lane
2). A 52000 Mr glycoprotein was detected, but instead of
gp93-130 a 80000 to 93000 Mr glycoprotein was
detected. Thus, although gB was synthesized, cleaved
and assembled into disulphide-linked complexes in
U-373MG cells, the Mrs of some of the glycoproteins
were different.
U-373MG cells. Another glycoprotein, which had an Mr
of 90000 when synthesized in SF cells, was observed
to have an Mr of 70000 when synthesized in U-373MG
cells (Fig. 3 b, lanes 1 and 2).
The experiments with gC-II were also done with the
Towne strain of H C M V and similar results were
obtained (data not shown), suggesting that the differences observed were due to differences in cell processing
of H C M V glycoproteins and not the strain of H C M V
used.
Analysis of gC-H synthesis in astrocytoma cells
gC-II complexes were isolated and examined as described above. Disulphide-linked gC-II complexes were
obtained from both SF and U-373MG cells (Fig. 3a,
lanes 1 and 2), but there were differences in the Mrs of the
most heavily labelled complexes. Those from SF cells
had MTs of 67000 to 93000, whereas those from U373MG cells had Mrs of 63000 to 90000 (Fig. 3a, lanes 1
and 2).
gC-II complexes were also reduced prior to SDSP A G E to examine individual glycoproteins. When
synthesized in SF cells and labelled with [3H]GIcN, the
most heavily labelled gC-II glycoproteins had MrS of
47000 to 63000 (gp47-63) (Fig. 3b, lane 1). When
synthesized in U-373MG cells, glycoproteins contained
in gC-II complexes had lower MrS than the same
glycoproteins synthesized in SF cells (Fig. 3 b, lanes 1 and
2). In particular, the glycoproteins from SF cells
designated gp47-63 had Mrs of 33000 to 50000 in
Analysis of GalN incorporation into gp47-63 synthesized
in SF and U-373MG cells
When synthesized in SF cells, gp47-63 contains a high
content of O-linked oligosaccharides (Kari & Gehrz,
1988). Furthermore, by using monensin to prevent posttranslational processing in the Golgi network of SF cells,
in which O-glycosylation occurs, the Mr of gp47-63 was
reduced by 20000 (Gretch et al., 1988b). This decrease
was similar to that observed when gp47-63 glycoproteins
were synthesized in U-373MG cells. Taken together,
these data suggest that the glycoproteins which had lower
MTs when synthesized in U-373MG ceils may have
contained fewer O-linked oligosaccharides. Owing to
this, we analysed gC-II gp47-63 for their O-linked
oligosaccharide content by comparing the hexosamine
content of gC-II glycoproteins obtained from infected SF
and U-373MG cells because the amount of GaIN present
would be an indication of the level of O-glycosylation.
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HCMV glycoprotein synthesis
1
2
iiiiiiiIiillllrllrlllllll
257
[irlllllrtltllr~tlr
(a)
58%,
-GlcN, 76%
42%,
-GAIN, 24%
Fig. 4. U-373MG and SF cultures were infected with the Toledo strain
of HCMV, and gC-II complexes were labelled with [3H]GlcN. gC-II
complexes were immunoaffinity-purified, complexes were reduced and
glycoproteins were digested with pronase. Pronase glycopeptides were
hydrolysed with 4 M-HCI to liberate hexosamines; these were separated
by TLC and detected by fluorography. Fluorograms were scanned to
determine the percentages of GIcN and GAIN, which are indicated.
Lane l, gC-II glycopeptides obtained from infected SF cells; lane 2,
gC-II glycopeptides obtained from infected U-373MG cells.
Infected cells were incubated with [3H}GlcN. Under
the conditions of these experiments, both GIcN and
GaIN are labelled because UDP-GlcNAc can be
converted into UDP-GalNAc by the action of a 4epimerase (Schachter & Roden, 1973). gC-II complexes
were immunoaffinity-purified from extracts of infected
SF or U-373MG cells. The purified gC-II complexes
were reduced and alkylated, and individual gC-II
glycoproteins were purified by gel filtration HPLC.
Purified glycoproteins were digested with pronase to
obtain water-soluble glycopeptides. These peptides were
hydrolysed with 4 M-HC1 to obtain free hexosamines,
which were separated by thin-layer chromatography.
When this was done, gC-II glycoproteins obtained from
HCMV Towne- or Toledo-infected SF cells contained
approximately twice as much radioactive GaIN as gC-II
glycoproteins obtained from HCMV Towne- or Toledoinfected U-373MG cells (Fig. 4). These data indicated
that gC-II glycoproteins synthesized in SF cells contain
more O-linked oligosaccharides than gC-II glycoproteins
synthesized in U-373MG cells.
Pronase sensitivity of gC-H group 1 glycoproteins
We have shown previously that when gp47-63 is
digested with pronase, a large fragment is obtained
which is resistant to further proteolysis (Kari & Gehrz,
1988). The resistance of this fragment to proteolysis is
due to the presence of a large number of O-linked
oligosaccharides. If fewer hydroxyamino acids were O-
Wo
VI
V
V
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12
20
II[[[([~llllll[ll~llll
30
40
50
IIIIl[llll[llltlllll_
(b)
10
- '
10
V0
V~
V
V
20
30
Fraction number
40
50
Fig. 5. gC-II group l glycoproteins were isolated from Toledo strain
HCMV-infected SF (a) and U-373MG (b) cells as described in
Methods. Glycoproteins were labelled with [3H]GIcN. Purified group 1
glycoproteins were digested with pronase and the pronase glycopeptides were separated by G-50 size exclusion chromatography. Blue
dextran was used to determine Vo of the G-50 column and [3H]GlcN
was used to determine Vt.
glycosylated when gC-II glycoproteins were synthesized
in U-373MG cells, these glycoproteins would be more
susceptible to proteolysis with pronase. To test this
hypothesis, gC-II gp47-63 glycoproteins were isolated
from infected SF cells and gp33-50 from infected
U-373MG cells, and digested with pronase. The pronase
glycopeptides were separated by size exclusion chromatography on G-50. gC-II gp47-63 glycoproteins obtained
from either HCMV Towne- or Toledo-infected SF cells
contained a pronase-resistant glycopeptide which eluted
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B. Karl R. Radeke and R. Gehrz
258
3 ~ ,,,,,, ,,i ,ll,r,i ,i]111
3 J't ...... I........ '"'~
2
0
1.5
f " "(C)
l " l " " ' l ' '111'11
(~
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iJl
1.2 ~1,11,11,1,,,i,1,,1111
(e)
0.8
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z
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nllll,ll,lllllllllllll
10
i11
1.5 ~ , , , , , , , , l l , , , l l l l l l l l
~ 1.0
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L,
~
5
o
1.0
~' '('~'1 ......... I'~'
0.5
~
] ItllillllJILalaJl~JIJ
20
Fraction number
10
20
Fig. 6. The [3H]GlcN-labelledglycopeptidesin the peaks near the Vt
of the G-50 column were collected and the glycopeptides in these
fractionswere analysedby lectinaffinitychromatography.HPA (a and
b), PNA (c and d) and WGA (e and f) columnswere used, whichbind
the glycopeptides Ser-GalNAc, Ser-GalNAc-Gal and Ser-GalNAcGal-NANA/GlcNAc, respectively. Boundglycopeptideswere eluted
with 0-1 M-GalNAc(a and b), 0.05 M-Gal(c and d) or 0.1 M-GIcNAc
(e and f). (a), (c) and (e) are the radioactivityprofilesobtained with
glycopeptidesfrom infectedSF cells; (b), (d) and (f) are the profiles
obtained with glycopeptidesfrom infected U-373MG cells.
near the void volume (V0) of the G-50 column
(representative data for H C M V Toledo are shown in Fig.
5 a). This peak contained a high content of GAIN, which
has been shown to be O-linked to the peptide backbone
(Kari & Gehrz, 1988). When gC-II glycoproteins were
obtained from HCMV Towne- or Toledo-infected
U-373MG ceils, the pronase-resistant Vo peak was
greatly diminished relative to the peak near the total
included volume (Vt) (representative data for HCMV
Toledo are shown in Fig. 5b). These data showed that
gC-II glycoproteins synthesized in U-373MG cells were
cleaved into much smaller glycopeptides by pronase.
This would occur if fewer hydroxamino acids were
glycosylated so that O-linked oligosaccharides were more
dispersed on the peptide backbone.
Lectin analysis of Vt peak obtained from infected
U-373MG and SF cells
Herpes simplex virus gC-I synthesized in C1300 neuroblastoma cells has been reported to contain O-linked
oligosaccharides consisting of the monosaccharide GalNAc O-linked to hydroxamino acids. In contrast, gC-I
synthesized in green monkey kidney cells contains Olinked oligosaccharides which are tri- and tetrasaccharides (Lundstrom et al., 1987 b). The differences in
glycosylation of gC-I in these two cell types were
characterized using lectins (Lundstrom et al., 1987a b).
Since smaller O-linked polysaccharides could account for
part of the reduction in Mr of gC-II gp47-63 glycoproteins in infected U-373MG cells, we used lectin columns
to characterize the structure of the polysaccharides
present in the Vt peaks in G-50 chromatography. The
glycopeptides in the Vt peak most probably contained
O-linked oligosaccharides because of their small Mr
and because they contained a large amount of GaIN
(data not shown). Three lectins (HPA, PNA and WGA)
were used: HPA is specific for terminal N-acetylgalactosamine (Hammarstrom et al., 1977; Goldstein & Hayes,
1978); PNA has a narrow specificity for the terminal
disaccharide sequence GaI(1-3)GalNAc (Goldstein &
Hayes, 1978, Mansson & Olofsson, 1983); W G A has
affinity for non-reducing terminal sialic acid and
GlcNAc residues (Peters et al., 1979). Analysis of gC-II
glycopeptides from SF or U-373MG cells showed that
they have very little affinity for HPA lectin (Fig. 6a and
b), suggesting that little free GalNAc O-linked to
hydroxyamino acids is present. In contrast, gC-II
glycopeptides from infected U-373MG or SF cells have
affinity for PNA and W G A lectins (Fig. 6c to f ) . The
greatest percentage of radioactive glycopeptides obtained from either type of cell bound to WGA. These
data suggest that the same oligosaccharide structures
were present in glycopeptides obtained from either type
of cell. Therefore, the Mr differences were not due to
the synthesis of smaller O-linked oligosaccharides in
U-373MG cells.
Discussion
The functions of the Golgi network in the assembly and
egress of herpesviruses have been studied by examining
the effects of drugs such as monensin and BFA on these
processes (Johnson & Spear, 1982; Whealy et al., 1991).
Although these drugs have proved useful, they interfere
with all processing events in the Golgi network. For
example, monensin prevents the late processing of
oligosaccharides on the H C M V gB glycoprotein, but also
prevents the proteolytic cleavage of gB into gp52 and
gp93-130 (Gretch et al., 1988c). Interference with
several processing events simultaneously makes interpre-
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H C M V glycoprotein synthesis
tation of results difficult. In contrast, we have observed
that the HCMV envelope glycoproteins synthesized in
U-373MG cells are completely processed with respect to
the proteolytic cleavage of gB and the assembly of
disulphide-linked glycoprotein complexes gC-I and gCII. The only apparent difference in the processing of
these HCMV glycoproteins was in the extent of Oglycosylation. We have shown previously that among
gC-I and gC-II glycoproteins, gC-II gp47-63 glycoproteins are the most heavily O-glycosylated (Kari & Gehrz,
1988). In the present study, we have shown that when
these glycoproteins are synthesized in U-373MG cells,
they have lower MrS because fewer hydroxyamino acids
are O-glycosylated. This conclusion is based on several
observations. First, the decrease in the Mr of gp47-63
synthesized in U-373MG cells as compared to SF cells is
similar to that detected when monensin was used to
prevent O-glycosylation in the Golgi network of SF cells
(Gretch et al., 1988b). Second, gC-II gp47-63 glycoproteins synthesized in U-373MG cells contain less
[3H]GalN than the same glycoproteins synthesized in SF
cells. Third, gC-II gp47-63 glycoproteins synthesized
in U-373MG cells lack a pronase-resistant fragment.
This suggests that the hydroxyamino acids which were
O-glycosylated are not clustered, but dispersed on the
peptide backbone. One function that has been associated
with oligosaccharides attached to proteins is protection
against proteolysis (Rademacher et al., 1988). This may
be one of the functions of the O-linked oligosaccharides
on gC-II gp47-63 glycoproteins.
As stated above, when processed in SF cells the
HCMV gB glycoprotein precursor is cleaved into gp93130 and gp52. When synthesized in U-373MG cells,
gp93-130 had a lower Mr; in contrast, gp52 had the same
Mr regardless of the type of cell infected. Previous
analysis of these glycoproteins has shown that gp93-130
contains detectable amounts of O-linked oligosaccharides whereas gp52 does not (Britt & Vugler, 1989; Kari et
al., 1990b). These results suggest that the decrease in Mr
of gp93-130 is in part due to lack of O-glycosylation of
this glycoprotein.
Even though several major HCMV envelope glycoproteins were not completely O-glycosylated in U-373MG
cells, the virus produced in these cells was still infectious
in SF cells. Thus, complete O-glycosylation is not needed
to produce infectious HCMV. Infectious HCMV was
also released into the culture medium from U-373MG
cells, suggesting that complete O-glycosylation is not an
absolute requirement for release of H C M V from infected
cells. However, unlike SF cells most of the infections
HCMV produced in U-373MG cells was found to be cellassociated. This result is very similar to that obtained
when monensin is used to prevent the processing of
HSV-I glycoproteins in the Golgi network of infected
259
cells, i.e. infectious virus is produced, but most of this
virus remains cell-associated (Johnson & Spear, 1982).
These results suggest that a fully functional Golgi
network is needed for complete processing of herpesvirus
glycoproteins and efficient egress of herpesviruses from
the host cell.
There are several possible reasons why HCMV
envelope glycoproteins are under-glycosylated in U373MG cells. First, there may be other factors that cause
transport failure of HCMV through the Golgi network of
U-373MG cells, causing a decrease in the O-glycosylation of the envelope glycoproteins. Alternatively, the
Golgi network of HCMV-infected U-373MG cells may
O-glycosylate glycoproteins differently from HCMVinfected SF cells. For example, the inability of U-373MG
cells to O-glycosylate HCMV glycoproteins completely
could involve a number of different enzymes needed for
O-glycosylation. The signals or amino acid sequences
that identify O-glycosylation sites are not well-defined.
However, in general the amino acid sequence of a
protein and the capacity of a cell to glycosylate a protein
determine which potential glycosylation sites are used
and the structure of the polysaccharides at these sites
(Datema et al., 1987). It is likely that accessibility of
specific serine or threonine residues to the N-acetylgalactosaminyl transferase plays a major role in determining
whether these residues become O-glycosylated. The
inability of U-373MG cells to O-glycosylate HCMV
glycoproteins completely may reflect differences in the
accessibility of serine or threonine residues to glycosyltransferases in SF and U-373MG cells. Another possibility is that the level of N-acetylgalactosaminyl transferase
activity is too low in U-373MG cells to accommodate the
synthesis of HCMV glycoproteins. It has been demonstrated that HCMV can trigger or increase the activity of
several glycosyltransferases in fibroblasts and teratocarcinoma ceils (Andrews et al., 1989). It may be that
HCMV can increase the activity of N-acetylgalactosaminyl transferase in SF cells but not in U-373MG ceils.
Without an increase in the level of enzyme activity, the
amount of HCMV glycoproteins synthesized in an
infected U-373MG cell may exceed the capacity of the
cell to glycosylate these glycoproteins fully.
We would like to thank Nancy Lussenhop and Curt Nelson for
technical assistance. This work was supported by a grant from the
Helen Lang Trust, Biomedical Research Institute of Children's
Hospital, and grant PO 1 HD 19937 from the National Institute of Child
Health and Human Development.
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