J. gen. Virol. (1986), 67, 515-526. Printedin Great Britain
515
Key words: CM V/plasma membraneproteins/glycoproteins
Plasma Membrane Proteins and Glycoproteins Induced by Human
Cytomegalovirus Infection of Human Embryonic Fibroblasts
By G L E N D A S U L L I V A N - T A I L Y O U R A N D
H E L E N M. G A R N E T T *
Department of Microbiology, University o f the Witwatersrand, 1 Jan Smuts Avenue,
Johannesburg 2001, South Africa
(Accepted 27 November 1985)
SUMMARY
An analysis of the plasma membrane proteins of human embryonic fibroblasts
(HEF) infected with human cytomegalovirus strain AD169 (HCMV) was performed
using in vitro radioactive labelling techniques followed by PAGE. Of the 12 virusinduced proteins detected in infected cells, glycoproteins of mol. wt. 34000 (34K), 53K
to 55K, 60K to 63K, 70K to 72K, 98K to 103K and 145K to 150K and proteins of 130K
to 133K and 260K to 270K were considered significant. The 60K to 63K, 70K to 72K
and 130K to 133K components were detectable at early stages of infection (8 h),
although only the latter two were labelled by surface iodination. The others only
appeared in the membrane from 48 h to 80 h after infection. Serological studies
indicated that the 34K, 70K to 72K, 98K to 103K and 145K to 150K components may
be HCMV-specified virion constituents, as these glycoproteins reacted with antibodies
raised against virions and extracted envelope glycoproteins. Of immunological
importance was the exposure on the cell surface of the protein moieties of 70K to 72K
and 130K to 133K proteins at 8 h and 53K to 55K, 60K to 63K, 70K to 72K and 145K to
150K components at 80 to 90 h after infection. Pooled human immune sera contained
antibodies which reacted with these exposed proteins, as well as with three other virusinduced membrane components of 230K to 240K, 98K to 103K and 78K to 80K.
INTRODUCTION
It is well established that human cytomegalovirus (HCMV) infection typically results in
alterations in the morphology, social behaviour and antigenicity of human fibroblasts.
Documented changes include cell rounding and hypertrophy (Michelson-Fiske, 1977),
decreased cellular adhesiveness (Diosi et al., 1972), altered surface topography (Garnett, 1979 a),
the cell fusion phenomenon (Booth et al., 1978 ; Garnett, 1979 b) and altered antigenicity at early
stages of infection (Th6 & Langenhuysen, 1972).
These observations suggest that alterations in the protein composition of the plasma
membrane may occur during the virus infection cycle, despite previous suggestions that HCMV
does not mature at the outer cell surface (Severi et al., 1979). Indeed, Farrar & Oram (1984) have
shown that virion glycoproteins, present in a fragile outer envelope, are probably derived from
the cell plasma membrane or its progenitors in the cytoplasm.
One compositional change in the plasma membrane is the reduction in fibronectin by an early
function of HCMV (Ihara et al., 1982), a factor which may play a role in the induction of cell
rounding in virus-infected cells. In addition, insertion of HCMV-specified glycoproteins into
the plasma and microsomal membranes of HCMV-infected cells has been reported at 20 to 24 h
after infection (Stinski et al., 1979). These glycoproteins may contribute to the altered
antigenicity and social behaviour observed in HCMV-infected cells. It has also been shown that
neutralizing antibodies raised against HCMV virions react with the surfaces of infected cells
(Pereira et al., 1982b), which suggests that some glycoproteins exposed on HCMV-infected cells
are antigenically related to the envelope components of the virus.
0000-6815 © 1986 SGM
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516
G. SULLIVAN-TAILYOUR AND H. M. GARNETT
Characterization of the antigenically important HCMV-specified constituents at the infected
cell surface may contribute to a better understanding of the immune response to HCMVinfected ceils, as well as to the development of an effective vaccine for the prevention of HCMV
infection. The present paper describes a study of the effect of HCMV infection on the protein
and glycoprotein composition of the human fibroblast plasma membrane.
METHODS
Cells and virus. Primary human embryo fibroblasts (HEF) were cultured from foetal lungs and used between the
6th and 25th passages for all experiments and for the propagation of HCMV (strain AD169). Cells were cultivated
in Eagle's MEM with Hanks' salts (Gibco) supplemented with 10~ foetal calf serum (FCS) for growth and 2~o
FCS for maintenance. For experimental infection with HCMV, a m.o.i, between 2 and 5 was used. Virus titres
were determined by a tissue culture infectivity assay, where the 50~ tissue culture infectious dose was used to
estimate the titre.
Membrane isolation. Plasma membranes were isolated by a modification of the fluorescein mercuric acetate
(FMA) technique as given by Scher & Barland (1972). Since FMA inhibits enzyme activity, membrane purity was
tested by phase-contrast and electron microscopy. Membranes were solubilized in a sample buffer containing 8Mo/
o/
urea, 2/o
SDS and 2/o
2-mercaptoethanol. The sample was heated in a boiling water-bath for 5 min to complete the
protein disaggregation.
Radioactive labelling. Pulse-labelling of proteins was performed using 40 ~tCi/ml L-[2,3-3H]arginine (sp, act. 12
Ci/mmol) in arginine-free MEM for 1 h at 37 °C. Glycoproteins were metabolically labelled with 10 gCi/ml D-[23H]mannose (sp. act. 10 to 20 Ci/mmol) in glucose-reduced MEM containing 2 g/1 glucose for 18 to 20 h at 37 °C.
The tyrosine residues of the exposed plasma membrane proteins were iodinated by a modification of the
lactoperoxidase technique as given by Pena et al. (1979). The iodination reaction mixture consisted of phosphatebuffered saline (PBS) pH 7.1 containing 3 mM-glucose, 20 gg/ml lactoperoxidase (Miles Laboratories), 0.2 units/ml
glucose oxidase (Merck) and 1 mCi carrier-free Na 125I (sp. act. 17 Ci/mg). Monolayers were washed in PBS,
labelled for 20 min at room temperature and the reaction was terminated with cold (2 to 4 °C) PBS pH 7.1
supplemented with 0.8~ sodium iodide. A modification of the original galactose oxidase/sodium borohydride
method was used to label externally exposed sugar moieties (Lehto et al., 1980). Monolayers were washed in
sodium chloride phosphate (NaCI-P) buffer pH 7.2 supplemented with CaClz (140 mM-NaCI, 10 mM-NaH2PO4,
10mM-Na2HPO~, 18 mM-CaC12) and treated with 10 units/ml galactose oxidase (Type V from Dactylium
dendroides, Sigma) or 100 units/ml glucose oxidase (Merck) in NaC1-P buffer for 30 min at 37 °C. Reductive
titration was carried out with 10 ml 1 mCi/ml sodium boro[3H]hydride (sp. act. 100 mCi/mmol) at room
temperature for 20 min. All radiochemicals were purchased from New England Nuclear.
Pol.vacrylamide gel electrophoresis. The protein content of membrane samples was determined using the Bio-Rad
protein assay system (Bradford, 1976). Analytical PAGE was performed in 8~/o, 10% or 12~ polyacrylamide slab
gels in a continuous Tris-glycine buffer pH 8-5 containing SDS (Laemmli, 1970). A 4 ~ stacking gel was employed
to concentrate samples. Separated proteins were visualized by staining with 0.25~/o Coomassie Brilliant Blue
(CBB) or Kenacid blue and recorded photographically and/or densitometrically in a LKB 2202 Ultroscan laser
densitometer equipped with a helium-neon laser source of wavelength 632.8 nm.
Autoradiography and fluorography. Gels containing ~-'5I-labelled proteins were soaked in a solution of 2 ~
glycerol and 2~/o methanol before being dried and placed in contact with Fuji RX X-ray film at - 7 0 °C.
Fluorography of 3H- and ~4C-labelled components was performed following the procedure of Bonner & Laskey
(1974) using a 10°/o solution of 2,5-diphenyloxazole in dimethyl sulphoxide for impregnation. The film was
developed with Kodak DI9 developer and the radioactivity present quantified densitometrically.
Protein transJbrs and antibody treatment. Polypeptides separated on polyacrylamide gels were transferred to
sheets of 0.45 ~lm nitrocellulose (BA 85, Schleicher & Schfill) by transverse electrophoresis in a buffer composed of
25 mM-Tris, 192 mM-glycine, 20~ methanol, pH 8.3 (Ryhicki &von Wechmar, 1982). After transfer, the filters
were incubated in 2 ~ bovine serum albumin in Tris-buffered saline (TBS) containing 10 mM-Tris-HC1 pH 7.4,
200 mM-NaC1 for I h at 37 °C with agitation. Filters were then incubated with the same buffer containing a 1/25 to
1/100 dilution of antiserum, preadsorbed with normal fibroblasts for 1 to 2 h at 37 °C and then washed in multiple
changes of TBS. Four antisera were used, i.e. immune serum from patients shown to have a high titre of HCMV
antibodies by ELISA and human sera shown to be negative by the same procedure (gift from J. Alexander) and
antiserum raised in rabbits against detergent-extracted HCMV envelope glycoproteins (gift from M. F. Stinski) as
well as normal rabbit sera. Bound antibodies were detected by treatment with either goat anti-human (Sigma) or
goat anti-rabbit (Sterilab) polyvalent immunoglobulin-peroxidase conjugate for 1 to 2 h at 37 °C followed by 25
~tg/ml o-dianisidine, containing 0.01 ~ H20, for 20 to 30 min, at room temperature. Filters were rinsed and
scanned in a densitometer while still wet.
lmmunoprecipitation. Immunoprecipitation was performed essentially as described by Blanton & Tevethia
(1981). Plasma membrane proteins were labelled with 50 ~tCi/ml L-[35S]methionine (sp. act. 400 Ci/mmol) in
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HCMV-induced plasma membrane proteins
517
methionine-free MEM supplemented with 1~ FCS for 18 to 20 h at 37 °C (modified method of Hughes & August,
1981). Labelled membranes were suspended in 1 ml extraction buffer (0-02 M-Tris-HC1 pH 9.0, 3 M-NaCI, 10~
glycerol, 1 mM-CaClz, 2 mM-EDTA, 0.5~ NP40, 300 ~tg/ml phenylmethylsulphonyl fluoride, 1~ aprotinin) and
incubated for 20 min at 4 °C with sporadic agitation. The preparation was clarified by centrifugation in an
Eppendorf microfuge. The clarified membrane material (500 ~tl)was incubated with preadsorbed antiserum (10 to
20 ~1) for 2 h at 4 °C. Five antisera were used, i.e. immune serum from HCMV-positive patients, human sera
negative for HCMV, a rabbit antiserum against detergent-extracted HCMV envelope glycoproteins, a rabbit
antiserum against HCMV virions and dense bodies (Stinski et al., 1979) and normal rabbit serum. Immune
complexes were adsorbed to Staphylococcusaureus(100 ~tlofa 10~ suspension), washed in 0.05 M-Tris-HC1buffer
pH 7.4 containing 0.5 M-LiCI,0.1 M-NaCI, 1~ NP40 and solubilized in electrophoresis buffer. They were then
analysed by SDS-PAGE and fluorography.
RESULTS
Detection of virus-induced plasma membrane proteins in HCMV-infected cells
S D S - P A G E of H E F plasma membranes on l0 to 1 2 ~ gels followed by protein staining
revealed the presence of approximately 30 membrane proteins in the mol. wt. range of 15K to
300K (Fig. 1). Productive infection of H E F with H C M V at a m.o.i, of 5 resulted in the
appearance of a number of new bands (Fig. 1). The alterations in the membrane proteins were
variable and could be correlated with the development of viral c.p.e. The results of two
separations performed with uninfected and HCMV-infected cell membranes at 80 to 90 h after
infection are given in Fig. l, the cells used being derived from various foetuses.
Analysis of the plasma membrane composition at various times after H C M V infection
revealed that the proteins of molecular weights 60K to 63K, 70K to 72K and 130K to 133K could
be detected as early as 6 to 8 h after infection (Fig. 2) and continued to be present until 96 h after
infection (Fig. 3). All other proteins were only detectable at later times, i.e. 48 to 96 h after
infection (Fig. 3).
Pulse-labelling with arginine of uninfected and HCMV-infected cells at 72 to 96 h after
infection confirmed the appearance of'eight of the novel (virus-induced or -enhanced) proteins
previously detected by protein staining. The molecular weights of the novel membrane proteins
of infected cells and their frequency of detection are shown in Table 1.
Proteins exposed at the cell surface
Iodination experiments revealed that approximately 16 or 18 proteins in the mol. wt. range of
26K to 275K were exposed on uninfected HEF. However, both the number of proteins, as well
as the amount of each protein exposed, was variable, a result of the dynamic nature of the
membrane structure. Novel proteins were detectable in the HCMV-infected cell membrane at
both early and late times after infection (Fig. 4). At 8 h after infection, two novel proteins of 70K
to 72K and 130K to 133K were exposed, while at 80 to 90 h after infection four proteins of 53K to
55K, 60K to 63K, 70K to 72K and 145K to 150K, and perhaps another of 130K to 133K had
their protein moiety exposed to the cell exterior.
Identification of plasma membrane glycoproteins
Fluorography of mannose-labelled glycoproteins revealed differences in the glycoprotein
composition of uninfected and HCMV-infected cell plasma membranes. The latter contained
increased amounts of radioactive label in bands corresponding to glycoproteins of 205K to
215K, 160K to 162K, 145K to 150K and 98K to 103K, as well as novel glycoproteins of 60K to
63K and 34K (Fig. 5). Time sequence experiments revealed that the changes in five of the virusinfected cell glycoproteins 34K, 98K to 103K, 145K to 150K, 160K to 162K and 205K to 215K
could be detected as early as 24 h after infection, while the novel 63K species could only be
detected in the plasma membrane from 48 h after infection by this technique.
Radioactive labelling experiments following treatment with glucose oxidase only yielded one
glycoprotein of 145K to 150K with a detectable amount of label. There was greater exposure of
the glucosyl residues of this species in the infected cell membrane at 80 to 90 h after infection
than in the uninfected celt. No radioactive label could be detected using galactose oxidase.
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518
G. SULLIVAN-TAILYOUR
A N D H. M. G A R N E T T
E ~u
~.7
oougq~osqv
e-,.~
~o~
~
J
1
J
~N
~
omN
.~ ~ P
E
~ou~qaosqv
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HCMV-induced plasma membrane proteins
--150
130
--97.4
-
-
8
72
68
60
j
rm
72
160
<
--45
2
i
I30
30
Relative mobility
Fig, 2, Photograph and densitometer scan of plasma membrane proteins of uninfected (1) and HCMVinfected cells at 8 h after infection (2) analysed on 12% gets and stained with Kenacid blue. The
molecular weights ( x 10-3) of novel proteins are indicated.
2
8
70
t.
<
\
J62~5~1
o,l°lJ 7
k 1 /762
W
Relative mobility
Relative mobility
Fig. 3
Fig. 4
Fig. 3. Densitometer scan of the time sequence of appearance of novel plasma membrane proteins in
HCMV-infected cells. Membrane proteins of uninfected cells (1) and HCMV-infected cells at 24 h (2),
48 h (3) and 72 h (4) were analysed by S D S - P A G E using 8% gels and stained with CBB. The molecular
weights ( x 10-3) of novel proteins are indicated.
Fig. 4. Densitometer scan of 125I-labelled surface-exposed membrane proteins of uninfected (1) and
HCMV-infected H E F at 8 h (2) and 80 to 90 h (3) after infection separated on 12% gels. Cells were
labelled with 1 mCi ~: 5I/107 cells for 20 min at room temperature by the lactoperoxidase technique. The
molecular weights ( x 10-3) of virus-induced exposed proteins are indicated, the markers used being
~4C-labelled standards,
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519
G. S U L L I V A N - T A I L Y O U R
520
1
150 - -
2
3
A N D H. M. G A . R N E T T
4
-
•
215
12
-~
•
162
148
S l --
.A.314
11~5098
I
~l
1f162 ~3 ^
I
34
98
97.4 - 68--
I
63
I/
-~
/~
"
~1
~/162 63 /'~
I .^98A ~
\34
\1
34
30
Relative mobility
Fig. 5. Fluorograph and densitometer scan of [3H]mannose-labelled m e m b r a n e glycoproteins of
uninfected (1), and HCMV-infected cells at 24 h (2), 48 h (3) and 72 h (4) after infection. Cells were
labelled with 100 #Ci [3H]mannose/10 v cells in glucose-free M E M + 0.1% FCS for 18 to 20 h. Plasma
m e m b r a n e s were analysed on 10% gels and stained with Kenacid blue. The position of the molecular
weight markers ( x 10 -3) are marked on the left of the fluorograph. Note that the 145K to 150K
glycoprotein detected at 24 h is possibly masked at 48 h and 72 h by the 160K to 162K glycoprotein.
Table
Mol. wt.
( × 10-3)
2&26
32-34
42 43
53 55
60-62
70 72
78-80
92
98 103
130-133
145-150
160 162
188
205-215
210
230-240
260-270{
1.
Characteristics of the novel plasma membrane proteins and glycoproteins of HCMVinjected HEF
Detected in
m e m b r a n e by CBB
stain, frequency of
detection* and time
of appearance
+ 30°,o
.
72h
.
Protein
moiety
~
8 h
80 h
-
-
.
.
.
+ 20%
+ 50%
+ 60%
+8090%
+ 20%
+ 30%
+ 80-90%
+ 80°0
+ 30°,o
72 h
48 h
8 h
8h
72 h
72 h
24-48 h
8 h
48 72 h
+
.
+
-
+
+
+
+ 20°,o
72 h
-
-
.
+
+
Proteins labelled
with[3H]arginine
Glycoproteins
(mannoselabelled)
Glucosyl
residue
exposed
1
2
3
+
-
-
-
+
-
+
+
+
+
+
+
-
+
+
+
+
.
-}-
.
.
+
+
+
.
+
+
+
+
+
+
+
+
+
+
+
+ 80%
72 h
-
-
+
* The frequency of occurrence of the novel proteins in the infected cell m e m b r a n e s was calculated from 20
separations.
t 1, Reacts with antibodies in h u m a n serum; 2, reacts with antibodies in antiserum to virions and dense bodies;
3, reacts with antibodies in antiserum to envelope glycoproteins.
{ A protein exceeding 250K and ranging in tool. wt. up to 275K was frequently detected. It was difficult to
assign an exact molecular weight and it is possible that a group of proteins is involved.
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HCMV-induced plasma membrane proteins
(a)
521
2
(c)
Relative mobility
<
149
I
70
32
(b)
Relative mobility
Fig. 6. Membrane proteins of uninfected (l) and HCMV-infected cells at 80 to 90 h post-infection (2)
were transferred to nitrocelluloseand reacted with (a) human immune serum to HCMV and (b) rabbit
antiserum to HCMV envelope glycoproteins.The adsorbed antibodies were detected with appropriate
peroxidase-conjugatedantisera using dianisidine as substrate and the reactions visualized. The results
were photographed (a) or recorded with a laser densitometer (b). Proteins in the 60K to 64K region from
a gel were also cut out and concentrated in a 4% stacking gel before separation in the second direction
on a 15% gel, transfer to nitrocellulose and reaction with human immune serum (c).
Serological studies
The question arises as to whether the changes detected in the plasma m e m b r a n e composition
are recognized in vivo by the immune system of the host. Membrane proteins transferred to
nitrocellulose were treated with human i m m u n e serum. Seven (glyco)proteins of the H C M V infected cell m e m b r a n e sample yielded a positive reaction (Fig. 6a). However, two dark bands
in the uninfected cell membrane sample revealed that two host (glyco)proteins (60K to 63K and
43K) were able to cross-react with antibodies in the serum. HCMV-negative sera also reacted
with these proteins. Further electrophoresis of the protein(s) in the 60K to 64K region on a
second gel ( 1 5 ~ acrylamide) revealed the presence of a novel 61K species in the H C M V infected cell membrane detectable only by immune sera (Fig. 6 c). The results suggest that serum
antibodies were present to infected cell m e m b r a n e components of 145K to 150K, 98K to 103K,
78K to 80K, 61K, 53K to 55K and 47K. Such antibodies were not present in the H C M V negative serum.
Immunoprecipitation studies performed using the h u m a n i m m u n e serum immunoprecipitared a number of m e m b r a n e proteins. Comparison between the immunoprecipitates from
uninfected and HCMV-infected cell membranes suggested that proteins of tool. wt. 230K to
240K, 98K to 103K, 78K to 80K, 70K to 72K and 53K to 55K (Fig. 7a) were induced by the
virus.
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G. S U L L I V A N - T A I L Y O U R AND H. M. G A R N E T T
522
/
(b)
1
2
M
150
97-4
68
-30
(c) L
Relative mobility
Fig. 7. Densiiometer scan of plasma proteins from uninfected (1) and HCMV-infected HEF, 80 to 90 h
post-infection (2). Cells were metabolically labelled with 500 gCi [3~S]methionine/107 cells in
methionine-free MEM + 1% FCS for 18 to 20 h. Proteins were immunoprecipitated using either
human immune serum (a, b) or rabbit antiserum to HCMV virions and dense bodies (c) and separated
on 10% and 12% gels respectively before fluorography.
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HCMV-induced plasma membrane proteins
523
The combined results suggest that antibodies to virus-induced membrane antigens of 98K to
103K, 78K to 80K and 53K to 55K were present in immune serum. Antibodies to other
membrane components of 230K to 240K, 145K to 150K, 70K to 72K, 60K to 63K and 47K were
also possibly present. A comparison of the immunoprecipitates obtained from infected and
uninfected cell membranes using rabbit antiserum to HCMV virions and dense bodies revealed
that four (glyco)proteins of 145K to 150K, 98K to 103K, 70K to 72K and 24K to 26K were
possibly virion constituents (Fig. 7b). Normal rabbit sera did not precipitate these proteins.
When transfers of membrane proteins were treated with antiserum to HCMV envelope
glycoproteins, three bands were present in the virus-infected cell membrane sample only (Fig.
6b), corresponding to membrane glycoproteins of 145K to 150K, 70K to 72K and 32K to 34K. It
therefore appears that these three glycoproteins are viral envelope glycoproteins, which are
inserted into the host plasma membrane.
DISCUSSION
A study of membrane composition requires an undamaged membrane preparation
uncontaminated by cytoplasmic material. The method of Scher & Barland (1972) employed in
the current investigation has been characterized by McClure et al. (1979), who reported that
although the membrane preparation was uncontaminated, the efficiency of membrane release
was relatively poor. Membrane removal was dependent upon cell morphology, and some of the
upper membrane at the cell edges was not removed, resulting in reduced yield of glycoproteins,
high molecular weight species and proteins that can be iodinated. However, a comparison of this
membrane isolation procedure with several other techniques (Brunette & Till, 1971 ; Krah &
Crowell, 1982: Scott, 1976) revealed that it gave a superior yield of membrane proteins
detectable by PAGE. It is possible, however, that the high molecular weight components are
under-represented in the results obtained.
Following HCMV infection, 12 novel proteins could be detected in the plasma membrane of
primary cultures of HEF. Of these, proteins of 53K to 55K, 60K to 63K, 70K to 72K, 98K to
103K, 130K to 133K and 260K to 270K occurred most frequently (Table 1). Radioactive
mannose-labelling experiments revealed that virus-induced glycoproteins of 34K, 53K to 55K,
60K to 63K, 70K to 72K, 98K to 103K and 145K to 150K were present in infected cell
membranes. In addition, lectin-binding studies have suggested that a 235K to 245K membrane
glycoprotein is able to bind concanavalin A (Sullivan-Tailyour, 1984). It is possible that the
235K to 245K receptor and the 260K to 270K components are similar glycoproteins of about
250K, shown to be extremely heterogeneous by G. H. Farrar (unpublished data). These virusinduced alterations in the plasma membrane glycoproteins may involve modification of existing
host components or the appearance of new virus-specific glycoproteins, either of which may be
constituents of the HCMV virion.
An analysis of the literature revealed that some of the novel membrane constituents
correspond in molecular weight to reported protein constituents of the HCMV virion. Most
studies reported the occurrence of virion polypeptides of 33K to 35K, 53K to 54K, 60K to 62K,
70K to 72K and 100K, and the presence of 130K to 135K and 145K to 150K species has also
been documented (Ebeling et al., 1983; Gibson, 1983; Gupta et al., 1977; Siqueira-Linhares et
al., 1981; Stinski, 1976, 1977). A few workers have indicated which virion components are
glycoproteins and these are given in Table 2 for comparison with the molecular weights of the
membrane glycoproteins detected in this study.
Recently, Farrar & Oram (1984) have reported that the HCMV envelope contains proteins of
23K and 30K, as well as glycoproteins of 52K, 67K, 95K, 130K and 250K. Three other abundant
virion proteins (35K, 64K, 150K) were detected and were considered to be associated with the
nucleocapsid. Plasma membrane proteins and glycoproteins of similar sizes have been detected
in the HCMV-infected cell in this study, although there is a discrepancy as to which proteins are
in fact glycosylated. The apparent differences between those proteins reported to be
glycosylated in this study compared with other work may result from the use of different
detection methods, or may reflect variations between different strains of HCMV.
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524
G. S U L L I V A N - T A I L Y O U R
A N D H. M. G A R N E T T
Table 2. Glycoprotein components o f the H C M V virion and infected cells*
Glycoproteins of
HCMV virion
Glycoproteins reported
in infected cells
(72 h after infectionp
250e
210c
145a'b
130-132''d'~
120"
115~
145
132
120
115
Novel glycoproteins in
plasma membranet
210
160
145-150
98-103
90~'
78 80d
702
64"'d
62bx
57b.c
52% 54--55~'bd
34~
16"
90
70
64
70-72
60 63
55
53-55
34
* References: ~, Stinski (1977) using Towne; b, Gibson (1983) using AD169; c Fiala et al. (1976) using AD169;
d, Stinski (1976) using Towne; e, HCMV envelope constituents as identified by Farrar & Oram (1984) using
ADI69.
t Results obtained in this study using AD169 by metabolic labelling with 100 ~tCi [3H]mannose/10~ cells at
37 °C overnight. Labelled glycoproteins were analysed by fluorography.
Serological studies confirmed that four membrane components of 24K to 26K, 70K to 72K,
98K to 103K and 145K to 150K may be constituents of the H C M V virion and/or dense bodies,
while three glycoproteins of 32K to 34K, 70K to 72K and 145K to 150K appeared to be virus
envelope glycoproteins (Table 1). Since these results correlate well with the major virion
constituents of H C M V identified by F a r r a r & Oram (1984), it is likely that at least five virion
constituents of these molecular weights are inserted into the infected cell m e m b r a n e during virus
replication. Two further H C M V envelope glycoproteins identified by these workers are those of
52K and 250K, and this suggests that the 53K to 55K and 260K proteins detected in the current
study are also virion constituents. Stinski and his co-workers (Stinski et al., 1979) reported that
at least four virus-induced glycoproteins are synthesized and bound to the plasma membrane
between 20 h and 24 h after infection, the molecular weights given being 12K, 70K, 100K and
145K. The latter three glycoproteins may correspond to the 70K to 72K, 98K to 103K and 145K
species reported in the current investigation. Further, Pereira et al. (1982b) have shown that cell
surface glycoproteins of 130K, 100K, 60K, 55K and 34K were immunoprecipitated with
monoclonal antibodies to CMV.
It is interesting that the 32K to 34K component reacted with the antiserum to envelope
glycoproteins, but not with the antiserum to whole virions (Table 1). This supports the
suggestion of F a r r a r & Oram (1984) that this glycoprotein may be closely associated with lipid in
the virion envelope and hence is not immunogenic when present in the intact virion.
Alternatively, it m a y have an epitope in common with one of the envelope glycoproteins.
Iodination experiments revealed that the protein moieties of the 70K to 72K and 130K to
133K components were exposed on HCMV-infected cells at both early (8 h) and late (80 h) times
after infection, while proteins of 54K, 62K and 145K to 150K were only exposed at later times
(Fig. 4). In addition, the 145K to 150K species contained glucosyl residues which were
accessible to enzyme treatment. This high molecular weight glycoprotein proved difficult to
detect with protein staining, which suggests that it may be a highly glycosylated molecule.
Serological studies using a high titre HCMV-positive serum indicated that antibodies are
produced in vivo which react with membrane components of 47K, 53K to 55K, 60K to 63K, 70K
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HCMV-induced plasma membrane proteins
525
to 72K, 78K to 80K, 98K to 103K, 145K to 150K and 230K to 240K (Table 1). Novel
glycoproteins of similar molecular weights (except for the 47K species, which could be a product
resulting from altered processing of another membrane component) have been detected in the
HCMV-infected cell membrane. Of these, the 145K to 150K, 70K to 72K, 60K to 63K and 53K
to 55K components were shown to be exposed at the cell surface. It is possible that antibodies to
the apparently unexposed membrane glycoproteins were elicited following their exposure
during destruction of infected cells in vivo, or due to their presence in the envelope of the HCMV
virion. In facz, HCMV-specific antibodies to infected cell polypeptides have been reported in
human sera by a number of workers. Pereira et al. (1982a) have shown that many HCMVinfected cell polypeptides, in particular the viral glycoproteins, are highly immunogenic in
humans and that antibodies to viral glycoproteins are present in convalescent sera. The current
study has shown that several of these virus-induced proteins are found in the plasma membrane
of infected cells.
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