Download Glycoproteins with Type Common and Type Specific Antigenic Sites

J. gen. Virot. (198o), 48, 297-3Io
Printed in Great Britain
297
Glycoproteins with Type Common and Type Specific Antigenic
Sites Excreted from Cells Infected with Herpes Simplex
Virus Types 1 and 2
By R. E. R A N D A L L , *
R. A. K I L L I N G T O N
AND D. H. W A T S O N
Department o f Microbiology, University o f Leeds, Leeds LS2 9JT
(Accepted 7 January 1980)
SUMMARY
Agar immunodiffusion tests demonstrated that BHK 2I cells infected with
either HSV I or HSV 2 release only a few HSV-specified antigens into the extracellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking
experiments showed that the majority of antigens/(including the Band II common
antigen) involved as target sites in antibody-mediated virus neutralization are
present in the ICRP of both HSV I and HSV 2. SDS-PAGE identified six regions
of virus-specified proteins in the ICRP from both HSV I- and HSV 2-infected
BHK cells. All these specifically released proteins are glycosylated, although to
varying degrees. The SDS-PAGE profiles of HSV I and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated
protein with an estimated tool. wt. of 5oooo to 540oo in HSV I 1CRP and 52ooo
to 5600o in HSV z ICRP. Immune precipitation demonstrated that these two
proteins contain the Band II antigenic site. Similar studies showed that the major
type t specific antigenic site, which is involved as a target site in the neutralization
of virus infectivity, is located in the highest mol. wt. glycoprotein region of
HSV I ICRP and has a similar mobility to the VP7/8 region of purified enveloped
virus.
INTRODUCTION
Antigenic analysis o f HSV I and HSV 2 has shown the presence of both type common,
i.e. cross reacting, and type specific antigenic sites involved in the interaction of virus with
neutralizing antibody (Sim & Watson, t 973). These antigenic sites have been characterized
further in that an antiserum has been raised in rabbits to the type common Band II antigen
of an HSV I (HFEM) virus isolate (Watson & Wildy, i969). This antiserum to Band II,
however, still retains some type I specific neutralizing activity after absorption with excess
type 2 antigen which removes the predominant type common neutralizing antibody (Sim &
Watson, I973). Antiserum to the major glycoprotein region (VP7/8) of HSV I purified
enveloped particles also has type I specific neutralizing activity (Powell et al. I974). However, it was shown that the type I specific activity of this antiserum differed from the type I
specific activity found in antiserum to Band II (Halliburton et al. I977). A number of
workers have shown that cells infected with herpes viruses release only a few virus-specified
antigens into the extracellular fluid (Ben-Porat & Kaplan, I97o; Pennington & McCrae,
I977; Norrild & Vestegaard, I979). Furthermore, Kaplan et al. (I975) claimed that the
virus-specified proteins released by herpes simplex virus (HSV)-infected cells elicit mainly
a type specific immune response in rabbits.
* Present address: National Institute for Medical Research, Mill Hill, London NW7 IAA.
oo22-I3317/8o/oooo-3968 $o2.oo
• 198o
SGM
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298
R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON
In this paper we have studied the proteins released by cells infected with HSV using a
variety of antisera in immunodiffusion, neutralization blocking and immune precipitation
tests.
METHODS
Cells and virus. BHK 2I cells were grown as described by Watson et al. 0966), in the
Glasgow modification of Eagle's medium containing IO °/o (v/v) tryptose phosphate broth
and 5 % (v/v) calf serum (ETC). Two HSV t strains, H F E M (Watson et al. 1966) and MPI7
(Brown et al. I973), and one HSV 2 strain, HG52 (Timbury, 197I ), were used. MPI7 and
HG52 are non-syncytial, while H F E M is syncytial in plaque morphology on B H K cells.
Production and radioactive labelling of polypeptides released from infected cells. Monolayers of BHK 21 cell cultures in 8o oz Winchester bottles were infected at an input multiplicity of 5 to to p.f.u./cell. After adsorption for I h at 37 °C, the inoculum was decanted,
the monolayer washed and fresh ETC at 37 °C was added. At 4"5 h after addition ofinoculum
(p.i.), the ETC was discarded and the monolayer washed with Glasgow modified Eagle's
medium (GMEM) lacking calf serum and tryptose phosphate broth. The cells were then
incubated for a further 2o h with radioactively labelled or unlabelled G M E M (5o ml). The
medium was collected and centrifuged at I6OOO rev/min for I h. The supernatant fluid was
concentrated to approx. IO to 15 ml by polyethylene glycol dialysis and then to 2 to 3 ml by
vacuum dialysis. The resulting concentrate was centrifuged at IOOOOOg for I h in a Christ
Omega Il ultracentrifuge and dialysed extensively against phosphate buffered saline (PBS).
This fraction is termed the infected cell released polypeptide (ICRP) fraction. The average
number of BHK 2I cells per confluent monolayer in an 80 oz Winchester bottle was estimated at 5 x io 8 cells. Thus there are approx. 2 × Io 8 cell equivalents per I ml of ICRP
fraction.
The cells from such an experiment were resuspended in 5 ml of PBS and disrupted with
an ultrasonic probe (M.S.E.). This suspension of disrupted cells was termed the total cell
antigenic fraction.
Labelling medium contained either 35S-methionine (2/~Ci/ml in G M E M with I/5th the
normal concentration of methionine) or x4C-glucosamine (sp. act. 52 mCi/mmol, o'33/~Ci/
ml in G M E M with I/2oth the normal concentration of glucose).
Purification of radioactively labelled enveloped virus particles. Purified radioactively
labelled virus was prepared from the cytoplasm of H F E M infected radioactively labelled
(3~S-methionine) BHK 2t cells by sedimentation through Dextran T~o gradients as described by Killington et al. (t977).
Antisera. HSV general antiserum was prepared by repeated immunization of rabbits with
extracts of HSV 1 (HFEM) or HSV 2 (3345) infected RK13 cells (Watson et al. I966).
Type t and type 2 specific antisera were prepared by absorption of general antiserum with
excess heterologous antigen (Sim & Watson, ~973); that is, I ml of general antiserum was
incubated for 24 h at 4 °C with extract of Io 9 cells infected with heterotogous virus, heatinactivated for 3o min at 56 °C, centrifuged at IOOOOOg for I h in a Christ Omega Ultracentrifuge and the supernatant fluid concentrated to its original volume by vacuum dialysis.
Antiserum to Band I1 (HSV l) was prepared by inoculation of rabbit foot pads with the
appropriate precipitin line from agar-gel immunodiffusion plates as described by Watson &
Wildy (I969). Antiserum to the VP7/8 region of HSV I virus (HFEM) was prepared by
immunization of rabbits with polyacrylamide gel slices containing the appropriate polypeptide region as described by Powell et al. (I974).
Neutralization blocking experiments. The antigenicity of the ICRP was studied by their
ability to absorb the neutralizing activity of the above antisera. Undiluted antisera (40 #1)
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Antigens excreted by HSV-infected cells
299
were incubated overnight at 4 °C with increasing amounts (zo, 8o or I6o #1, i.e. 4 × Ioe,
16 x Io 6 or 32 × Io ~ cell equivalents) of I C R P and made to the same vol. (2oo #1) with mock
ICRP. HSV I and HSV 2 general antisera were also diluted to I / 5 and I / I o concentration
of the original antisera (as indicated in parentheses in the Results) and absorbed with ICRP.
The absorbed antisera were heat-inactivated for 3o rain at 56 °C and tested for residual
neutralizing activity in a plaque reduction test in which the indicator viruses, H F E M (syn)
and HG52 (syn+), were artificially mixed prior to the test. The virus (approx. 2 × lo 4 p.f.u./
ml each) were incubated for 2 h with an equal vol. ofantisera. Surviving virus was assayed by
the suspension method of Russell 0962). Survival of infectious virus after incubation with
immune sera (v) is expressed as a percentage of that surviving incubation with pre-immune
sera (Vo), i.e., V/Vo × Ioo.
Agar gel immunodiffusion tests were performed in 2 to 3 m m layers of 1 % agar in the
manner of Watson et al. 0966).
Immune precipitation. Immune precipitates were obtained by incubating antisera with
HSV I or HSV 2 I C R P fractions in appropriate proportions (usually o'z ml of I C R P
fraction with o.2 ml antisera) in highly tapered glass tubes for I8 to 24 h at 4 °C (Killington
et al. I978). The tubes were centrifuged at 4ooo rev/min for I5 rain, the supernatant fluid
removed and the pellet carefully rinsed three times with cold PBS. The final pellet was
resuspended in a small volume (o-Iz ml) of disruption mixture (o'o5 ~-tris-HCl, pH 7"0,
5 % sucrose, 2 % SDS and 5 % mercaptoethanol) by disruption with a M.S.E. ultrasonic
probe. The amount of radioactivity present in the resuspended pellet was determined by
T C A precipitation on a glass fibre disc and counting in a Beckman LS-23o scintillation
counter. An appropriate volume of disrupted precipitate was then electrophoresed on an
SDS-polyacrylamide gel.
Polyaerylamide gel eleetrophoresis. Polypeptides were separated by SDS polyacrylamide
slab gel electrophoresis, followed by fixation, staining and autoradiography (Halliburton
et al. I977). HSV polypeptides of known tool. wt. (e.g. VPs 5, 16, I9c and 2z; Heine et al.
~974) were used as calibration protein markers for the estimation of the tool. wt. of unknown
polypeptides.
RESULTS
Immunodiffusion tests on polypeptides released from infected cells
HSV I and HSV 2 total cell antigen and I C R P fractions were prepared as described in
Methods and reacted against HSV I and HSV z general antisera in agar gel immunodiffusion plates (Fig. 0- Although the I C R P fractions are three times more concentrated in
terms o f cell equivalents than the total cell antigen fractions, it is clear that certain antigens
in the total cell fractions are either not present or are in undetectable amounts in the
I C R P fractions. HSV I and HSV z I C R P share one major type common antigen as detected
in agar immunodiffusion (Fig. I c) and this precipitin line was shown to have a line of
identity when the I C R P were reacted with antiserum to Band II. In fact the type common
Band II antigen is the predominant component of HSV z ICRP, although two minor
antigens can be detected when HSV 2 I C R P are reacted against homologous antiserum
(Fig. I b). The Band lI antigen is also a major constituent of HSV I ICRP. However, two
other antigens are easily detected by an immunodiffusion test. Both of these give better
precipitin lines when reacted against homologous rather than heterologous antiserum
(Fig. Ta and c). Also, two further minor precipitin lines are usually detected when HSV l
I C R P react against homologous general antiserum.
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300
R . E . R A N D A L L , R. A. K I L L I N G T O N AND D. H. W A T S O N
iN
0
\
Fig. ~. HSV i and HSV 2 total cell antigen (T1 and T2 respectively) and ICRP (rl and I~) reacted in
immunodiffusion tests with HSV [ and HSV z general antiserum (g-a/st and g-a/sz respectively).
The Band II type common immune precipitin line is arrowed.
Electrophoretic characterization of polypeptides released by infected cells
A c o m p a r i s o n was m a d e o f the e l e c t r o p h o r e t i c b e h a v i o u r o f the I C R P with those o f
infected cell p o l y p e p t i d e s (Fig. 2). B H K ceils were infected with H F E M and labelled f r o m
4"5 to z4 h p.i. with either 85S-methionine or 14C-glucosamine, a n d t o t a l cell p r o t e i n s were
e l e c t r o p h o r e s e d on a 9"25 % S D S p o l y a c r y l a m i d e gel. H F E M I C R P a n d proteins released
f r o m uninfected cells ( m o c k I C R P ) labelled with 14C-glucosamine o r aSS-methionine were
also co-electrophoresed. In a d d i t i o n , H F E M I C R P labelled with 14C-glucosamine or 35SDownloaded from www.microbiologyresearch.org by
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Antigens excreted by HSV-infected cells
I
HSVI ICRP
HSV 1 total cell protein I
(
3sS-met ) (
(
x4C-gluc
3oi
I MocklCRP i
)
3ss met
,,ff
HSV l
HSV 1
ICP
ICRP
8
9
10
21
26
•
39
40
i
rl
precipitated by
excess HSV 1 g-a/s
Fig. 2. Autoradiograms of SDS-polyacrylamide slab gels showing the polypeptides of HSV i
(HFEM)-infected BHK cells (slots [ and 2) and the polypeptides released (ICRP) by these and mockinfected BHK cells (slots 5 and 6 respectively). The precipitates formed when the ICRP were
reacted with excess homologous general antiserum were also disrupted and subjected to SDS
electrophoresis (slots 3 and 4). The cells were labelled with x4C-glucosamine or asS-methionine
from 4"5 to 24 h p.i.N.B. No mock ICRP was precipitated by HSV general antisera (g-a/s).
m e t h i o n i n e p r e c i p i t a t e d w i t h excess H S V I general a n t i s e r u m were d i s s o c i a t e d a n d electrop h o r e s e d o n the S D S - p o l y a c r y l a m i d e gel.
general antisera.
It is possible to i d e n t i f y six b r o a d mol. wt. r e g i o n s o f p r o t e i n s (labelled a to f) t h a t are
specifically released b y H S V I - i n f e c t e d B H K cells (Fig. 2 a n d 3). A l l six r e g i o n s label w i t h
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R. E. R A N D A L L ,
302
R. A. K I L L I N G T O N
AND
HSV 1
ICRP
HSV 1
VP
D. H. W A T S O N
HSV 2
ICRP
5
a al ~
87
8.5
,
a2
~
1
1314
2
16
19 c
22
3
bl
b b2
m
O
c
5
d
23
HFEM
purif,
virus
MPI 7
bet
HFEM
horn
MP17
horn
HG52
her
HFEM
het
MPI7
het
HG52
horn
Fig. 3. Autoradiograms o f SDS-polyacrylamide slab gels o f 35S-methionine labelled polypeptides o f
HSV I ( H F E M and M P I 7 ) and HSV 2 (HG52) I C R P immune precipitated with homologous (horn)
or heterologous (bet) general antisera at an antiserum to antigen ratio o f I : I (v/v). Polypeptides
were labelled from 4"5 to 24 h p.i. H F E M purified (purif.) virus particles, labelled with 35Smethionine, were also co-electrophoresed to give marker virion polypeptides.
14C-glucosamine (Fig. 2), although some proteins are more highly glycosylated than others,
e.g. band b is poorly glycosylated compared with bands a and c. Many of these regions are
complex, with more than one component being identified in certain regions. For instance,
regions a and b of MPI7 ICRP can be seen to contain a minimum of two components
(Fig. 3, slot 4)In tool. wt. estimations of H F E M ICRP, region a has an estimated mol. wt. between
I24ooo and ~38ooo and corresponds to the major glycoprotein (VP7/8) region of H F E M
purified virus (Fig. 3). Region b has an estimated mol. wt. between 59ooo and 65ooo and is
analogous in size to a glycoprotein region (VPI7/t8) of H F E M purified virus (Fig. 3). The
other four regions do not co-migrate with any glycoproteins as yet identified in purified
HSV I virus. Their estimated mol. wt. are 50000 to 54000 (c), 43000 to 45000 (d), 30000 to
35000 (e) and 2IOOO to 22000 (f).
When similar experiments were carried out by infecting various cell lines (Chang conjunctival, rabbit kidney, baby hamster kidney and HEp2) with HFEM, it was shown that,
while a similar pattern of | C R P was obtained, the mobility of the high mol. wt. region on
SDS-PAGE varied. This demonstrates that the host cell plays a role in the post translational processing of these proteins (Fig. 4)HSV 2 ICRP were studied following their immune precipitation with homologous and
heterologous general antisera. Fig. 3 shows the SDS-PAGE analyses of the immune preDownloaded from www.microbiologyresearch.org by
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Antigens excreted by HSV-infected cells
HSV 1
ICRP
303
HSV 1
VP
14
BHK
Chang
HEp2
RK
conj.
Fig. 4. Autoradiogram of an SDS-polyacrylamide gel showing the 3~S-metbionine labelled region a
of HSV I (HFEM) ICRP from BHK 2I, Chang conjunctival, HEp2 and rabbit kidney (RK) infected cells, immune precipitated with homologous general antiserum.
cipitates formed when HSV 2 ICRP, labelled with 35S-methionine, were precipitated with
excess homologous and heterologous general antisera. Six protein regions (labelled from I
to 6) released from HG5z infected ceils are identified in this manner and, like HSV t ICRP,
all six regions are glycosylated. Region I has an estimated tool. wt. of between 86ooo and
IOOOOO. Regions 2 and 3 have an estimated mol. wt. of 7oooo to 76ooo and 66ooo to 69ooo
respectively. Unlike region b of H F E M ICRP, they label efficiently with 14C-glucosamine.
Band 4 has an estimated mol. wt. of 52ooo to 56ooo compared with 5oooo to 54ooo for
region c of HSV I ICRP and, like region c, is efficiently labelled with ~4C-glucosamine.
Although not so easily distinguishable in HG52 ICRP, two diffuse regions (5 and 6) can be
detected which co-migrate in the region of d and e of HSV I ICRP. The two bands labelled
x and y in Fig. 3 have not been detected in other immune precipitates of HG5z ICRP.
The antigenic nature of both HSV I and HSV 2 ICRP were characterized further by their
immune precipitation with antisera of differing specificities, and by their ability to block the
neutralizing activities of these antisera.
Immune precipitation of H S V ICRP
HS V general an tisera
Although all HSV I ICRP could be precipitated with excess heterologous general antiserum (Fig. 6), a number of ICRP were more efficiently precipitated than others at limiting
antiserum concentration. Thus regions a2 and c are precipitated more efficiently than a a and
b by heterologous general antiserum (Fig. 3; compare slots 3 and 6 and 4 and 7)- This
pattern of precipitation could not be achieved by simple dilution of homologous general
antiserum, region al being precipitated relatively more efficiently than b or c in the latter
case. Precipitation of HSV z ICRP by homologous and heterologous general antisera produced similar profiles (Fig. 3, compare slots 5 and 8).
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304
R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON
Antiserum to Band II (HSV l)
Antiserum to Band II contains all the antibodies important in the cross neutralization of
HSV, as well as some of the antibodies involved in type ~ specific neutralization. Fig. 5
shows the precipitation of ICRP by antiserum to Band II. Regions c, d and e of HSV 1
ICRP are precipitated by this antiserum (although d cannot be detected in the photograph),
whereas regions a and b are not. A threefold increase in the antiserum : antigen ratio did not
alter the pattern of precipitation. Similarly, region 4 of HSV z ICRP is clearly precipitated
by the antiserum, whereas regions I, z and 3 are not. Although it is very difficult to detect
regions 5 and 6 of HSV z ICRP (they cannot be seen in Fig. 5), a number of experiments
have shown that these proteins are also precipitated by antiserum to Band II.
Antiserum to VP7/8 (HSV ~)
Antiserum to VP7/8, as stated earlier, is prepared by the inoculation of the high mol. wt.
glycoprotein region of H F E M purified virus into rabbits. The resulting serum is type 1
specific in neutralization activity. This antiserum has not previously been used successfully,
either in reacting to give precipitin lines in an agar gel immunodiffusion test or in immune
precipitation with HSV ~ soluble antigen fractions. However, when used in immune precipitation with H F E M ICRP, the high tool. wt. glycoprotein region a is precipitated.
Increasing the ratio of antiserum: antigen fails to precipitate any of the other regions
identified in HSV I ICRP (Fig. 6). It is not clear whether region a2 is precipitated by this
antiserum, since, when present, Band al usually obscures a2.
Type I specific antiserum
Type r specific antiserum (which contains all the type specific antibodies present in HSV I
general antiserum) was also used in the immune precipitation of HSV I ICRP. Fig. 6 shows
that the only HSV I ICRP precipitated by this antiserum was region a. However, in this
experiment it was not clear whether the plateau level for precipitation had been reached.
Thus an increased antiserum:antigen ratio may result in precipitation of further HSV I
ICRP regions.
Neutralization (serum) blocking tests on proteins released by HSV-infected cells
Antisera used in the immune precipitation experiments were also tested for their ability
to neutralize HSV, both before and after absorption with HSV I or HSV 2 ICRP. Absorption of antiserum to VP7/8, type I-specific, antiserum to Band II, HSV I general ( r / I o dilution) and HSV z general antisera with HSV I ICRP completely removed the ability of these
antisera to neutralize HSV I. Thus in a plaque reduction test the unabsorbed antisera
reduced HSV I infectivity to < 2% survival, while after adsorption of 4o/~1 of these antisera with approx. I6 × lO6 cell equivalents of HSV t ICRP (see Methods), there was > 8o %
(not significantly different from Ioo%) survival.
Absorption of 4o/~1 of HSV I general (I/5 dilution) and antiserum to Band II with
approx. 4 × Io6 cell equivalents of HSV 2 ICRP reduced the ability o f these antisera to
neutralize HSV z virus from I"7 and I8 % survival, respectively, to IOO% survival. However, absorption of HSV z general antiserum 0 / 5 dilution) with I6 × 1o6 cell equivalents of
HSV z ICRP, only reduced the neutralizing activity of this antiserum from 8 % to 61%
survival of HSV z, and similarly of type ~ specific antiserum from 6 % to 78 % survival.
Doubling the amount of blocking antigen used to absorb these two antisera did not alter
the results significantly (6I % and 84% survival respectively).
In these experiments it was possible to absorb out the cross neutralizing activity of HSV
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Antigens excreted by HSV-infected cells
I
HSV 2 ICRP
1
HSV I ICRP
3o5
[
precipitated by
pre I.
a/s to
pre I.
a/s to
a/s
Bll
a/s
BII
~
C
d
Fig. 5. Autoradiogram of an SDS 9'25 ~ acrylamide gel of asS-methionine labelled HSV ~ (HFEM)
and HSV 2 (HG52) ICRP precipitated by antiserum to Band II (a/s to Band II) and pre-lmmune
(pre I.) antiserum).
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306
R. E. R A N D A L L ,
R. A. K I L L I N G T O N
AND
D. H. W A T S O N
HSV-1
ICRP
<
d
Ratio of
antisera
to antigen
hom
het
het
het
1:1
6:1
3:1
1:1
1
6:1
anti VP 7/8 sera
3:1
1:1
I type 1
specific
1:1
pre I.
1:1
Fig. 6. Autoradiograms of SDS 9'25 ~?/oacrylamide gels of H S V I ( H F E M ) I C R P precipitated with
increasing amounts of heterologous (het) general antiserum and anti VP7/8 antiserum. The precipitates formed by reaction o f H F E M I C R P with homologous (horn) general antiserum, and type I
specific antiserum, at an antiserum to antigen ratio of I : I, are also shown.
general antiserum with a smaller a m o u n t of I C R P than that required to absorb out the type
specific neutralizing activity. Thus, incomplete absorption of HSV I general antiserum with
HSV I I C R P produced an absorbed antiserum which still reduced HSV t infectivity to 33 O~/o
of its original titre in a plaque reduction test, but failed to neutralize HSV 2 0 o 4 ~,') survival).
The original unabsorbed antiserum reduced both HSV I and HSV z infective virus to < I °/o
survival in the same experiment. Similar results were obtained by absorption o f H S V 2
general antiserum with HSV 2 I C R P , i.e. an incompletely absorbed antiserum still neutralized HSV 2 (28 ~'o survival) but failed to neutralize HSV I.
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Antigens excreted by HSV-infected cells
3o7
DISCUSSION
Only a small number of HSV-specified antigens present in infected cells are released into
the culture medium (Fig. i). SDS-PAGE of proteins released from HSV I, HSV 2 and uninfected (mock) cells, and analysis of precipitates formed when these ICRP were reacted
with HSV general antisera identified six protein regions present in both HSV ~ (regions
a to f) and HSV z (regions I to 6) ICRP that were not present in mock ]CRP (Fig. z and 3).
Region a of HSV 1 ICRP has a similar electrophoretic mobility on S D S - P A G E to the
VP7/8 region of HSV I infected cells and purified virus. Similarly, region I of HSV 2 ICRP
may be equivalent to the glycoprotein with a tool. wt. of between 80o0o and 9oooo found
in HSV 2 purified virus particles (Cassai et ctl. I975). However, we did not identify infected
cell counterparts of other ICRP. Thus, no major glycoprotein was identified in the infected
cell which co-migrated with glycoprotein c of HSV I ICRP (Fig. z) or 4 of HSV z ICRP.
Such ICRP are probably processed forms or breakdown products of their counterparts in
the infected cells.
The SDS-PAGE patterns of proteins released from cells infected with either HSV I
strain H F E M or MPI 7 are very similar but there are slight differences; for example, the
MPI7 region a~, migrates faster than the a2 region of H F E M ICRP (Fig. 3, slot 6 and 7)However, there are a number of marked differences between HSV I and HSV 2 ICRP. Thus
in HSV I ICRP there is no protein with a similar tool. wt. to region r of HSV 2, and these
in turn have no high tool. wt. glycoprotein which co-migrates with region a of HSV I ICRP.
This latter case is surprising, as in both HSV z-infected cells and purified virus there is a
glycoprotein region of similar tool. wt. to the VP7/8 region of HSV I (Cassai et al. I975).
The major region of equivalence between HSV I and HSV z ICRP is in regions c and 4
respectively. These proteins are both highly glycosylated, have a similar mol. wt. and behave
analogously in their precipitation with a variety of antisera.
Comparison of proteins released from BHK 2I, HEp2, Chang conjunctival and rabbit
kidney cells infected with HSV I show that the host cell influences the processing or glycosyIation of virus-specified proteins. Thus, the electrophoretic mobility of the high tool. wt.
glycoprotein (region a) depends on the cell line infected (Fig. 4)- Keller et al. (I97O) also
showed that the host cell affects the degree of glycosylation of HSV proteins. They demonstrated differences in the glycosylation of membrane proteins in African green monkey
(Vero) and human (HEpz) cells infected with the HSV i strains F or MP. Whether the host
cell can also alter the antigenicity of these virus proteins by modifying the processing of
glycoproteins has, unfortunately, not been studied.
The precipitation of HSV I and HSV 2 ICRP by a variety of antisera and their ability to
absorb out the neutralizing activity of these antisera demonstrated that HSV I and HSV 2
ICRP contain the Band II common antigen and the majority, if not all, of the type specific
antigens involved in antibody mediated virus neutralization. The apparent inability of
HSV 2 ICRP to remove all the neutralizing activity of HSV 2 general antiserum against
HSV 2 virus may be because the HSV z VP7/8 region is not represented in HSV 2 ICRP.
However, some of the ICRP may be breakdown products of this region. Similarly, regions
d and e o f H S V I ICRP and regions 5 and 6 o f H S V 2 ICRP may be breakdown products of
regions c and 4, respectively. Thus no antisera used in these experiments precipitated
regions c and 4 independently of regions d and e, and 5 and 6 respectively. Another possibility is that these proteins are found in small aggregates in the ICRP preparations and that
the minor low mol. wt. components are host proteins. However, regions a, b and c o f H S V t
ICRP behave independently of each other in their precipitation with various antisera,
demonstrating that they are distinct HSV antigens and physically independent in the ICRP
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R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON
fraction. For similar reasons it is unlikely that regions T, 2 or 3 are antigenically related to
the low mol. wt. regions of HSV 2, although they could be related to one another.
These results also strongly suggest that the main type specific antigenic sites of HSV I
ICRP are to be found in the high tool. wt. glycoprotein region al and the main type common
antigenic sites in the low tool. wt. glycoprotein region c. This conclusion is based on the fact
that region al is inefficiently precipitated by heterologous general antiserum but preferentially
by type I specific and anti-VP7/8 sera. On the other hand region c (and region 4 of HSV 2
ICRP) is comparatively efficiently precipitated by heterologous general antiserum and
antiserum to Band II. It is therefore likely that the main type I specific antigen, important in
neutralization, will also be found in the high mol. wt. glycoprotein region and the type
common in the low tool. wt. glycoprotein regions. However, as all ICRP are precipitated by
heterologous general antisera, even those proteins with type specific antigenic determinants
must have some type common antigenic sites as well. Presumably, the type specific determinants are located on the outside of the envelope, while most type common determinants are
embedded within the envelope. If all these type common determinants were exposed on the
surface of the virus particle there would probably be more type common antigens than the
Band II antigens involved as target sites for heterologous antibody mediated virus neutralization.
The results presented here are therefore in general agreement with those of Kaplan et al.
(r975) and Norrild & Vestergaard 0979), who also showed that only a few virus proteins
are released into the extracellular fluid of cells infected with HSV. These authors showed
that the type specific antigenic determinant is the major constituent of proteins released by
HSV-infected rabbit kidney and HEp2 cells respectively. However, our results clearly
show that the type common Band II antigen is a major constituent of polypeptides released
by both HSV I- and HSV z-infected BHK cells. Certainly the major type specific antigen is
present in a soluble form in higher concentrations in the ICRP fractions than in total cell
antigen fraction, probably being intimately associated with cellular membranes in the latter
case. In contrast, the type common Band II antigen is present in a soluble form in both the
ICRP and total cell antigen fractions (Fig. I). Norrild & Vestegaard (1979), however, did
detect a type common antigenic determinant in proteins released from HSV-infected HEp2
cells. Also, in the SDS polyacrylamide gel profiles of released polypeptides from HSV
infected rabbit kidney cells presented by Chen et al. (I978), it is likely that their released virus
polypeptide C is equivalent to our HSV I ICRP region c and HSV 2 ICRP region 4, and
contains the Band II type common antigenic site.
Cohen et al. 0978), studying the antigen CP-I, which was shown to be equivalent to the
Band II antigen in that it contained the cross-reacting antigenic determinants (Watson &
Wildy, I969; Cohen et al. I972; Powell & Watson, I975), indicated that a protein (gp52)
with a mol. wt. of 52ooo and CP-I antigenic activity was the probable precursor of the low
tool. wt. glycoprotein, gP59, found in purified enveloped particles. Cohen et al. (I978)
suggested that gp52 corresponded to Spear's glycoprotein pD (Spear, I976); gp52 and pD
are also probably antigenically equivalent to a glycoprotein with an estimated tool. wt. of
47ooo which was precipitated from the soluble antigen fraction of HSV x-infected BHK
cells by antiserum to Band II (Honess & Watson, I974). However, Cohen et al. 0978)
identified another glycoprotein in membranes of HSV-infected KB cells which also had
CP-I antigenic activity and a tool. wt. of 52ooo. They could not rule out that this glycoprotein was a breakdown product or a further processed form of gP59 and it is this latter
possibility that the work presented here would favour. Thus we would like to suggest that
glycoprotein c of HSV r ICRP and 4 of HSV 2 ICRP are equivalent to the glycoprotein
identified by Cohen et al. (r978) in the plasma membrane of HSV-infected KB cells. It
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Antigens excreted by HSV-infected cells
309
seems unlikely that the highly glycosylated ICRP c or 4 could be a precursor of the gP59
identified in purified enveloped particles.
The ICRPs contain most of the antigens important in eliciting neutralizing antibodies to
HSV infectivity and therefore they may provide a source of virus glycoproteins free from
infective virus with which to test the potential of subunit vaccines in preventing primary
HSV infections. If subunit vaccines prove efficient in protecting against primary HSV
infections, similar subunit vaccines may prove successful in immunization against other
important herpes infections such as those caused by cytomegalovirus and Epstein-Barr virus.
We are indebted to the Medical Research Council for the award of a project grant in
support of this work (DHW) and to the Science Research Council for a research studentship
(RER).
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