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J. gen. ViroL 0974), 22, I59-~69
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Type Specific and Type Common Antigens
in Cells Infected with Herpes Simplex Virus Type 1 and on the
Surfaces of Naked and Enveloped Particles of the Virus
By R. W. H O N E S S , * K. L. P O W E L L t, D. J. R O B I N S O N , ~
C A R O L I N E S I M AND D. H. W A T S O N §
Department of Virology, The Medical School, Birmingham, BI 5 2TJ, U.K.
(Accepted 12 September I973)
SUMMARY
Of eleven virus-specific antigens detected in cells infected with herpes simplex
virus type 1, six have been shown to be type specific, i.e. not present in cells infected
with type 2 virus. Two of the five type common antigens are also present in cells
infected with pseudorabies virus. Electron microscopic studies of immune agglutination of virus particles have shown that both naked and enveloped particles possess
type specific and type common antigenic determinants on their surface. Further,
one of the type common determinants on the naked particle surface interacted
with antiserum to pseudorabies virus.
INTRODUCTION
Previous work in this laboratory has shown that there are at least twelve virus-specific
antigens in B H K 21 cells infected with herpes simplex virus type t (Watson et al. 1966). One
of these, designated Band II, is presumably a structural antigen since virus infectivity is
neutralized by an antiserum against Band II (Watson & Wildy, 1969). This antigen is common to both type I and type 2 viruses (Sim & Watson, 1973). Another antigen presumably
corresponds to the virus-specific thymidine kinase, since after absorption with an extract of
cells infected with the kinaseless mutant of Dubbs & Kit (1964) an antiserum against
herpes-infected R K 13 cells gives only one precipitin line in immunodiffusion tests with
extracts of cells infected with wild-type virus (Buchan, Luff & Wallis, 197o). Hitherto there has
been little information about the other antigens, although one antigen is apparently common
to cells infected with either herpes simplex or pseudorabies viruses (Watson et al. 1967).
in this paper we provide further information about some of the other antigens present in
cells infected with herpes simplex type I. We have extended our previous results on antigens
common to cells infected with herpes simplex and pseudorabies viruses. We have confirmed
and extended the observation of Schneweis & Nahmias (I97I) that some of the antigens
present in cells infected with herpes virus type 1 are type common (i.e. also present in cells
infected with type 2 virus), while others are type specific (i.e. present only in cells infected
with type I virus). We shall also describe the results of studies on agglutination of purified
* Present address: Committee on Virology, Department of Microbiology, University of Chicago,
Illinois 6o637, U.S.A.
~ Present address: Department of Virology and Epidemiology, Baylor University College of Medicine,
Houston, Texas 77o25, U.S.A.
:~ Present address: Scottish Horticultural Research Institute, Invergowrie,Dundee.
§ Present address: Department of Microbiology, School of Medicine, Leeds LS2 9NL.
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R. W. H O N E S S A N D O T H E R S
preparations of naked and enveloped particles of herpes simplex virus type [. These provide
information about the location of some type specific and some type common structural
antigens. We have also identified another type common antigen present in naked particles.
This differs from Band II in that the corresponding antiserum does not neutralize virus
infectivity.
METHODS
Cells. BHK 2t cells and R K I3 cells were grown irL Eagle's medium containing calf serum
and rabbit serum, respectively (Watson et al. 1966).
Viruses. Stocks of herpes simplex virus type I strain HFEM, herpes simplex virus type 2
strains Lovelace and 3345, and pseudorabies virus strain Dekking were all grown in BHK 2I
cells as previously described (Watson et al. 1966, ~967). Stocks for preparation of immunizing antigen (see below) were grown in R K 13 cells.
Infected cell extracts. BHK 21 cells were infected in suspension at an input multiplicity of
to p.f.u./cell, and dispersed in rotating 8o oz bottles. The cells were harvested 16 h after
infection and disrupted in distilled water at a concentration of lOs cells/ml by ultrasonic
vibration. Concentrated extracts were prepared where necessary by vacuum dialysis of the
supernatant fluid obtained after centrifuging infected cell extracts at ,ooooog for I h.
Antisera. General antisera (to all herpes virus-specific proteins) were prepared by repeated
injections of freeze-dried extracts of R K 13 cells infected with herpes simplex virus type [
(Watson et al. z966), or pseudorabies virus (Buchan & Watson, I969). A general antiserum
against herpes simplex virus type 2, kindly supplied by Dr G. R. B. Skinner and Mrs M. E.
Thouless, was prepared in a similar manner.
Antiserum against Band II, a structural antigen of herpes simplex type I, was prepared as
previously described (Watson & Wildy, 1969) by lymph-node inoculation of a rabbit with
the precipitin band obtained by reaction of general antiserum with the appropriate fractions
from preparative polyacrylamide gel electrophoresis (Watson, I969). Further antisera
against Band II antigen were prepared by similar inoculation of the precipitin band obtained
using the original antiserum to Band II antigen in the reaction with an appropriate electrophoretic fraction.
Antiserum specific for type ~ virus was prepared by absorption of a general antiserum
against type z virus with an excess of extract from cells infected with type 2 virus (Sim &
Watson, 1973). The absorbed serum was centrifuged at Iooooog for ] h and the antiserum
in the supernatant fluid concentrated back to its original vol. by vacuum dialysis. This
antiserum is described as type t specific (absorbed) antiserum.
An antiserum against purified naked virus particles (see below) was prepared by lymphnode inoculation of the virus preparation after inactivation with formaldehyde. 2 × xon inactivated particles were inoculated with Freund's incomplete adjuvant using the methods
described previously (Watson & Wildy, i969). A second inoculation was made 6 weeks after
the first. The rabbit was bled ~o days after the second inoculation.
Virus purification. Purified preparations of type z virus comprising less than I ~o enveloped
particles were obtained by centrifuging in sucrose density gradients and chromatography on
calcium phosphate as previously described (Robinson & Watson, I97I). These preparations
contained rto diffusible antigens detectable in immunodiffusion tests with either general
antiserum against type z virus or antiserum against uninfected host cells. They contained
2 to 5/zg protein/lo 10 virus particles as determined by the method of Lowry et al. (I95I).
We have also now purified enveloped herpes simplex type I virus. These preparations
contained less than 1% naked particles. The procedure will be described in detail and evalu-
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Herpes virus specific antigens
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ated in another paper. Briefly, advantage
release large amounts of enveloped virus
virus was collected by precipitation with
material further purified by two cycles of
was taken of the fact that infected HEp-2 cells
particles into the medium late in infection. The
polyethylene glycol, and the concentrated virus
sedimentation in sucrose velocity gradients.
Electron microscopic analysis of immune agglutination of virus particles. Appropriate
(about 5 x io 11 particles/ml) concentrations of purified naked and enveloped virus particles
were incubated at 37 °C for I h, with 5 vol. of the serum under test. These samples were then
diluted and processed for particle counting by the loop-drop method of Watson (1962).
Grids were examined at 4oooo x and the distribution of particles, singly and in aggregates,
was counted. A particle was said to be part of an aggregate if it was separated from its
neighbour by less than half a particle diam. All samples were examined at the same relative
dilution of the original particle suspension and the particle recoveries (by comparison with a
standard calibrated latex suspension) were comparable, except when very large aggregates
were formed. In every series of determinations, the particles were also incubated with preimmune serum and a comparable concentration of bovine serum albumin. In some cases a
third control was used: general antiserum against type I virus absorbed with the homologous
antigen, freed of excess particles by sedimentation.
Immunodiffusion tests. These were done in 3-mm-thick layers of agar ([onagar no. 2) in
phosphate-buffered saline (Dulbecco & Vogt, 1954). The wells were arranged hexagonally
and were 8 mm in diam., each well being separated from neighbouring wells by 3 mm of gel
(see Figs. I to 5).
RESULTS
Serological tests
Type specific and type common antigens
Fig. I shows an agar gel immunodiffusion plate in which dilutions of general type I antiserum were tested against dilutions of type I infected cell extract in a pattern arranged so that
antiserum/antigen ratios from 27[I to I[27 were tested on the same plate. A total of I[
precipitin bands were detected by examination of the original plate with a hand lens.
Similar tests with the type specific (absorbed) antiserum showed that it gave 6 precipitin
bands with an extract of cells infected with type I virus, but none with an extract of cells
infected with type 2 virus. (Fig. 2 shows an immunodiffusion test of type specific (absorbed)
antiserum with one dilution of each infected cell extract.) Six of the I I virus-specific antigens
detected in cells infected with type I virus are therefore type specific. Presumably the other
five should be type common (i.e. present in cells infected with either type of virus). The
accuracy of this estimate was confirmed by tests of the extract of cells infected with type I
virus against general antiserum to type 2 virus (which showed 4 lines) and of the general
antiserum to type I virus with an extract of cells infected with type 2 virus (which showed
5 lines). Similar results were obtained with several batches of antisera and infected cell
extract.
Band H antigen
We have previously reported that antiserum to Band I[ gives a single precipitirL band with
extracts of cells infected with either type z (Watson & Wildy, I969) or type 2 virus (Sire &
Watson, I973). We have now found a second precipitin band by using the antiserum concentrated fivefold (Fig. 3). The additional precipitin band was not revealed in tests with
dilutions of the concentrated antiserum. Both lines represent type common antigens since
they were also detected in tests with extracts of cells infected with type 2 virus. They thus
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R. W. HONESS AND OTHERS
Fig. I. Agar gel immunodiffusion test of an extract of cells infected with herpes simplex virus
type ~ (I) with general antiserum against type I virus (A-I). Dilutions 0/3, I/9, 1/27) of both
infected cell extract and antiserum are labelled accordingly.
Fig. 2. Agar gel immunodiffusion test of extracts of cells infected with herpes simplex virus type I (I)
or type 2 virus (2) with type specific adsorbed antiserum (A-I/2) prepared by absorption of general
antiserum against type I virus with an extract of cells infected with type 2 virus.
differ f r o m the ' t h y m i d i n e kinase a n t i g e n ' which can be detected in type I infected cells
(Buchan et al. I97 o) b u t n o t in type 2 infected cells (Thouless, i972), using general a n t i s e r u m
to type ~ virus a b s o r b e d with excess o f an extract o f cells infected with the t h y m i d i n e - k i n a s e deficient m u t a n t o f type ~ virus.
B o t h precipitin b a n d s were detected in similar tests with B a n d I I antigen f r a c t i o n p r e p a r e d
b y p o l y a c r y l a m i d e gel etectrophoresis. A n t i s e r u m to B a n d I I showed no r e a c t i o n with
extracts o f uninfected cells in i m m u n o d i f f u s i o n tests.
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Herpes virus specific antigens
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Fig. 3. Agar gel immunodiffusion test of concentrated antiserum against Band lI antigen (A-IIc)
with extract of cells infected with type I virus (I). Dilutions indicated appropriately.
Antigens shared with extracts of cells infected with pseudorabies virus
We have previously shown that general antiserum to type I virus gives a precipitin band
with pseudorabies virus (Watson et al. 1967). We have now extended these results and shown
the reciprocal reaction between general antiserum to pseudorabies virus and extracts of cells
infected with type I virus. We have shown that two precipitin bands can be revealed in both
reactions, although not necessarily at the same antigen/antiserum ratio. Eight different
general antisera to type I virus have shown two precipitin bands with extracts of cells
infected with pseudorabies virus. The same precipitin bands were obtained in reactions of
these extracts with general antisera to type 2 virus.
Similar results were obtained in tests of general antisera to pseudorabies virus with extracts of cells infected with type I virus. Two different precipitin bands were again observed.
These differed from the precipitin bands given with antiserum to Band II and antiserum to
'thymidine kinase antigen' (Fig. 4). These antigens were type common, i.e. also present in
cells infected with type 2 virus. The cross-reactions were confirmed by complement fixation.
None of the antisera to pseudorabies virus showed any significant neutralizing activity
against either type I or type 2 virus. Similarly, general antisera against either type of herpes
simplex virus did not neutralize pseudorabies virus.
Capsid antigen
Antiserum to purified naked particles produced one precipitin band with extracts of cells
infected with both type I and type 2 virus but gave no reaction with extracts of cells infected
with pseudorabies virus. The precipitin band differed from Band iI antigen and thymidine
kinase antigen (Fig. 5). The antiserum gave no significant neutralization of either type I or
type 2 virus, in agreement with Dreesman et al. 0972) who also failed to observe neutralization of type ~ virus by antiserum to purified nucleocapsids.
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R. W. H O N E S S A N D O T H E R S
Fig. 4. Agar gel immunodiffusion test of extract of cells infected with type I virus 0 ) with concentrated antiserum against Band II antigen (A-IIc) and pseudorabies virus (A-PRc) and with
antiserum specific for the virus thymidine kinase antigen ( A q / T K - ) . The single precipitin line
shown in the reaction with antiserum to pseudorabies virus differs from the lines given with the
two other antisera. On the original plate an additional precipitin line was shown in this reaction this also differed from the precipitin lines revealed with the other antisera.
Fig. 5. Agar gel immunodiffusion test of extract of cells infected with type I virus (I) with antiserum
against Band II antigen (A-II) to purified naked particles (A-NV) and against the thymidine kinase
antigen (A-I/TK-). The capsid antigen precipitin band revealed in the reaction with A-NV differs
from the Band II and thymidine kinase antigens.
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Herpes virus specific antigens
Table I. Agglutination of naked particles of herpes simplex virus
type I by specific antisera
Percentage agglutinated particles calculated from
No. of single particles in
mixture with immune serum
Percentage total no.
compared with similar mixture
of particles seen in aggregates with pre-immune serum*
Antiserum
(no. of experiments in brackets)
Pre-immune (2)
Bovine serum albumin (z)
Type I general (2)
Type 2 general (2)
Band II (2)
Pseudorabies general (2)
Type ~ (absorbed) specific (2)
* Percentage agglutinated particles =
3"9
5 (NS)?
> 99
98"1
45"7
64"2
95'9
Ioo
(I
(o)
o
98"9
89"2
47"7
72"4
88-9
no. of single particles in mixture with immune serum]
serum
t NS indicates agglutination not significant. All other agglutinations are significant.
Table 2. Agglutination of enveloped particles of herpes simplex virus
type I by specific antisera
Percentage agglutinated particles calculated from
f
Antiserum
(no. of experiments in brackets)
Pre-immune (5)
Bovine serum albumin (5)
Type I general (5)
Type 2 general (5)
Band II (4)
Pseudorabies general (2)
Type ~ absorbed specific (2)
Type 2 absorbed with type I
antigen (2)
No. of single particles in
mixture with immune serum
Percentage total no.
compared with similar mixture
of particles seen in aggregates with pre-immune sermun*
14"6
17"3 (NS)t
95"1
71"9
37"4
~o'o (NS)t
68"5
7"5 (NS)~
(o)
o~
96"7
7z-o
37"3
23"5 (NS)t
86.5
o~
* Calculated as shown in footnote to Table 1.
t See footnote to Table I.
In these mixtures there were actually more single particles (although not significantly more) in mixture
with immune serum than in mixture with pre-immune serum.
Immune agglutination of naked and enveloped particles
Naked particles
Table I shows the results o f a g g l u t i n a t i o n experiments with n a k e d particles o f type I virus.
The figures q u o t e d were derived f r o m a n u m b e r o f experiments with each antiserum. The
particles were very efficiently a g g l u t i n a t e d b y general antisera to b o t h types o f virus. I n
a d d i t i o n the type I specific ( a b s o r b e d ) a n t i s e r u m also agglutinated. B o t h type specific a n d
t y p e c o m m o n antigens are therefore exposed o n the surface o f the n a k e d particles.
A n t i s e r u m to B a n d I [ also agglutinated n a k e d particles. The surface c o m p o n e n t s o f the
particles responsible for this a g g l u t i n a t i o n m a y account for some or all o f the agglutination
b y general antisera to the type 2 virus. However, these general antisera c o n t a i n type c o m m o n
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R.W.
HONESS AND
OTHERS
antibodies reacting with extracts of cells infected with pseudorabies virus (see preceding
section). Table I shows that general antiserum to pseudorabies agglutinated naked particles.
It therefore seems likely that this agglutination represents interaction(s) with type common
surface antigen(s) other than Band II antigen(s). General antisera to type 2 virus are presumably able to agglutinate by interaction with both Band I[ antigen(s) and type common
antigen(s) shared with pseudorabies virus.
Enveloped particles
Table 2 shows the result of corresponding experiments for enveloped particles of type
virus. Significant agglutination was again found with general antisera to both type I and
type 2 viruses. Previous experiments did not show agglutination of enveloped particles by
antisera to type I virus (Watson & Wildy, 1963), presumably because of the inferior antisera
then available. The agglutination by general antisera to type 2 virus was abolished by absorption with Band II antigen, and we deduce that the only virus-specific type common antigens
on the surface of enveloped virus reacting with general antisera to type 2 virus are those
which also interact with antiserum to Band II antigen. However, our failure to detect type
common antigens other than Band II in the envelope surface cannot be taken to prove
absence of such a determinant. The result may only reflect deficiencies in the antisera (cf. the
failure of Watson & Wildy (I963) to observe immune agglutination of enveloped particles).
Type specific antigens were also revealed on the envelope surface by the agglutination by
type I specific (absorbed) antiserum. As expected, general antiserum to type z virus did not
agglutinate after absorption with type I extract. The slight agglutination recorded for
antiserum to pseudorabies virus was not significant. Once again, this may not prove the
absence of the corresponding antigen.
DISCUSSION
We have extended previous reports (Schneweis & Nahmias, I97I) on type specific and
type common antigens of herpes simplex virus. The total number of virus-specific antigens
recognized is small compared to the coding potential of the virus. This may reflect not only
the insensitivity of gel imlnunodiffusion tests but also the fact that apparently single lines
may be multiple (cf. the Band II single line which can be resolved as two at certain serum•
antigen ratios). The recognition of type specific antigenic determinants should also provide
a further basis for the selection of restricted classes of virus-specific proteins for further
analysis, e.g. by polyacrylamide gel separation of polypeptides precipitated by type specific
antisera. We have already proved the value of immunological selection in providing a
criterion of virus specificity and in restricting the number of polypeptides to be studied
(Honess & Watson, I974). Such a selection is invaluable with a virus capable of coding for
a number of proteins: sufficient to challenge the resolution of the best available separation
techniques. That we should have found both type specific and type common antigens is
expected since Kieff et al. (1972) found that types I and 2 viruses shared 47 % of the base
sequences of the DNA.
Some of the other antisera studied here provide the basis for even greater selectivity.
Antiserum to Band iI antigen is plainly more complex than originally reported but is still
considerably more specific than general antiserum since it has a restricted neutralizing
activity (Sim & Watson, I973) as well as only producing two type common precipitin bands
in gel immunodiffusion. The restricted number of antigens shared with pseudorabies virus
provides another selective procedure for further work. The immunological result can be
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related to the more restricted genetic homology between pseudorabies and herpes simplex
viruses compared to the homology between the two simplex types (Bronson et al. i972g
Ludwig, Biswal & Benyesh-Melnick, 1972). One of the 'shared' antigens can also be detected
in cells infected with varicella virus (G. Nankervis, E. Gold & D. H. Watson, personal
communication). It is interesting to note here that Kirkwood, Geering & Old 0972) have
presented evidence for a 'group specific antigen' in nucleocapsids of Luck6, Burkitt and
herpes simplex viruses.
The agglutination studies have provided some information on the location of type specific
and type common antigens on the surfaces of the capsid and the envelope. These studies
show at least one type specific determinant in the envelope together with one type common
antigen which may correspond to the antigen which interacts with antiserum to Band II to
cause agglutination and neutralization of infectivity. Similarly we have recognized at least
one type specific antigen and two type common sites on the capsid surface. One of the type
common sites presumably relates to the agglutination of naked particles by antiserum to
Band II antigen and the other to agglutination by antiserum to pseudorabies viruses.
There are some problems in correlating precipitin bands in this way to antigens on particle
surfaces which may or may not react with neutralizing antibody. These are best illustrated
by reference to antiserum to Band II antigen. This we have shown previously has type specific
and type common neutralizing activities (Sim & Watson, I973), produces two type common
precipitin bands and agglutinates both naked and enveloped particles. There may, of course,
be more than one determinant involved in any or all of these activities. Conversely, at least
three determinants must be involved because the two precipitin lines are type common and
there is type specific neutralizing activity. The agglutinating and type common neutralizing
activities could then relate to the two precipitin bands. Correlation of the neutralizing
activities with precipitin bands or agglutinating antibodies is difficult because of the much
greater sensitivity of the neutralization test. Neutralizing activity of this antiserum was detected using antiserum/virus particle ratios about xooo-fold greater than those at which agglutination is observed. We can, therefore, never be entirely sure that neutralization is not due
to a small population of antibodies not detectable in other tests. There are less acute problems in correlating precipitin bands with agglutination, and supporting evidence may be
given by polyacrylamide gel electrophoresis of immune precipitates. We show elsewhere
(Honess & Watson, ~974) that the major polypeptide of Band II immune precipitates, and
therefore presumably of Band II precipitin lines, co-electrophoreses in polyacrylamide gels
with a naked particle polypeptide. It therefore seems reasonable to relate agglutination of
naked particles to a Band II precipitin line.
Accordingly, we believe the two Band II precipitin lines are related to agglutination of
naked and enveloped particles respectively. Subject to our reservations on correlations with
neutralizing activity we link type common neutralizing activity of the antiserum to Band II
with envelope agglutinating activity. There are at least two further type common structural
antigens (see Table 3). One is the capsid antigen, related to a type common precipitin band
but not to neutralizing activity, and which may or may not be on the capsid surface. The
other is a pseudorabies shared antigen which we believe is represented on the capsid surface,
since general pseudorabies antisera agglutinate naked particles but do not neutralize
infectivity or agglutinate enveloped particles. We cannot definitely correlate this structural
antigen with either of the two pseudorabies shared antigens identified in immunodiffusion
tests.
There are two type specific structural antigens corresponding to the two type specific
neutralizing activities of general antisera (Sim & Watson, I973). One can apparently be
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R. W. HONESS AND OTHERS
Table 3- Summary of antigens and precipitin bands identified
T
Function to which related
Correlated to
precipitin band?
Enveloped particle agglutination by antiserum
to Band I1 and type common neutralizing
activity of antiserum to Band II and
general antisera
Yes
structural
Type common
b
i
structural
Naked particle agglutination by antiserum
to Band II
Naked particle agglutination by general
antisera to pseudorabies
Capsid antigen
Pseudorabies shaled antigen
Yes
?
Yes
Yes
or
non-structural
T
1
t
structural
Type specific
1
non-structural
Pseudorabies shared antigen
Yes
Total number correlated precipitin bands
Number to be correlated
5
Total observed
5
First type specific neutralizing activity of
general antisera
Second type specific neutralizing activity of
general antisera (identical to similar activity
of antiserum to Band II)
0
Yes
No
?
Type specific agglutination of naked particles
Thymidine kinase
Yes
Total number correlated precipitin bands
Number to be correlated
4
Total observed
6
2
related to a precipitin band in immunodiffusion tests (K. L. Powell, A. Buchan, C. Sim &
D. H. Watson, personal communication) and we shall return to this c o m p o n e n t in a subsequent publication. The other, corresponding to the type specific neutralizing activity o f
antiserum to Band II, obviously cannot relate to a precipitin band. There must be one
further type specific structural antigen corresponding to type specific agglutination o f naked
particles which m a y or m a y not correspond to a precipitin line.
It now remains for us to consider non-structural antigens. Correlation o f precipitin bands
with some o f the virus-specific enzymes identified in cells infected with type I virus (Keir
et al. 1966; Klemperer et al. 1967; Morrison & Keir, 1968) is once again difficult, although
tbymidine kinase has been linked with one particular precipitin line (Buchan et aL 197o ).
Even here there is a slight problem; the antigen detected in immunodiffusion tests is type
specific but there seems to be limited cross-neutralization o f enzyme activity (Tbouless,
1972).
We summarize these findings in Table 3. We have identified in all seven structural antigens.
Three are type specific and we can relate one o f these to a precipitin band. F o u r are type
c o m m o n and we can relate three o f these to a precipitin band. O f the remaining five type
specific precipitin bands we have observed, one corresponds to thymidine kinase antigen,
leaving four we have been unable to correlate with any function. The remaining two type
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common precipitin bands correspond to pseudorabies shared antigens, which may or may
not be structural.
This work was supported by a research grant from the Medical Research Council. Two of
us (R.W.H., K.L.P.) are indebted to the Science Research Council for research studentships.
We gratefully acknowledge technical assistance from Mrs C. Wallis, R. Wilkinson and
A. Hall.
REFERENCES
BRONSON, D. L., GRAHAM, B. 1., LUDWIG, H.O., BENYESH-MELNICK, M. & BISWAL, N. (I972). Studies o n t h e
relatedness o f herpes viruses t h r o u g h D N A - R N A hybridization. Biochimica biophysica acta 259, 24-34.
BUCHAN, A., LUFF, S. & WALLIS, C. (I970). Failure to d e m o n s t r a t e interaction o f s u b u n i t s o f t h y m i d i n e kinase
in cells simultaneously infected with herpes virus a n d a kinaseless m u t a n t . Journal of General Virology 9,
239-142.
BUCHAN, A. & WATSON, D. H. (1969). T h e i m m u n o l o g i c a l specificity o f t h y m i d i n e kinases in cells infected by
viruses o f the herpes group. Journal of General Virology 4, 461-463.
DREESMAN, G. R., SURIANO, J. R., SWARTZ, S. K. & McCOMBS, R. M. (I972). Characteristics o f the herpes virus. I.
Purification a n d a m i n o acid c o m p o s i t i o n of nucleocapsids. Virology 50, 528-534.
DOBBS, D. R. & KIT, S. (I964). M u t a n t strains o f herpes simplex deficient in t h y m i d i n e kinase-inducing ability.
Virology 2 2 , 493-5o2.
DULBECCO, R. & VOGT, M. (I954). Plaque f o r m a t i o n a n d isolation o f pure lines with poliomyelitis viruses.
Journal of Experimental Medicine 99, 167-I82.
HONESS, P,. W. & WATSON, D.H. (I974). Herpes virus-specific polypeptides studied by polyacrylamide gel
electrophoresis of i m m u n e precipitates. Journal of General Virology 22, 171-I 8 5,
KEIR, H. M., SUBAK-SHARPE,H., SHEDDEN, W. L H., WATSON, D. H. & WILDY, P. (I966). I m m u n o l o g i c a l evidence
for a specific D N A polymerase p r o d u c e d after infection by herpes simplex virus. Virology 3 o, 154-157.
KIEFF, E., HOYER, B., BACHENHEIMER,S. & ROIZMAN, B. (1972). Genetic relatedness of type I a n d type 2 herpes
simplex viruses. Journal of Virology 9, 738-745.
KIRKWOOD, J., GEERING, G. & OLD, L. J. (I972). D e m o n s t r a t i o n o f group- a n d type-specific antigens o f herpes
viruses. In Oncogenesis and Herpes Viruses, p. 479. Edited by P. M. Biggs, (3. de-Th6 a n d L. N. Payne.
Lyons: IARC.
KLEMPERER, H. G., HAYNES, G. R., SHEDDEN, W. I. H. & WATSON, D. H. (I967). A virus-specific t h y m i d i n e kinase
in B H K Zl cells infected with herpes simplex virus. Virology 3 I, I2O-I28.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L & RANDALL, R. J. (195I). Protein m e a s u r e m e n t with the Folin
phenol reagent. Journal of Biological Chemistry 193 , 265-275.
LUDWIG, H. O., BISWAL, N. & BENYESH-MELNICK,M. (1972). Studies on the relatedness of herpes viruses t h r o u g h
D N A - D N A hydridization. Virology 49, 95 i o t .
MORRISON, J. M. & KEIR, H. M. (1968). A new DNA--exonuclease in cells infected with herpes virus: partial
purification a n d properties o f the enzyme. Journal of General Virology 3, 337-347.
ROBINSON, D. J. & WATSON,D. H. (I971). Structural proteins of herpes simplex virus. JournalofGeneralVirology
io, 1 6 3 - t 7 I .
SCIINEWEIS, K. E. & NAHMIAS, A. J. (I97I). A n t i g e n s o f herpes simplex virus type 1 a n d 2 - i m m u n o d i f f u s i o n
a n d inhibition passive h a e m a g g l u t i n a t i o n studies. Zeitschriftfiir Immunitgitsforschung und experimentelle
Therapie 141 , 471-487.
SIM, C. & WATSON, D. H. (I973). T h e role o f type specific a n d cross reacting structural antigens in the neuralization o f herpes simplex virus types I a n d 2. Journal of General Virology 19, 217-233.
THOULESS, M. E. (I972). Serological properties of thymidine kinase p r o d u c e d in cells infected with type I or
type 2 herpes virus. Journal of General Virology I7, 3o7-316.
WATSON, D. H. (I962). Particle c o u n t s o n herpes virus in p h o s p h o t u n g s t a t e negatively stained preparations in
electron microscopy. Sth International Congress for Electron Microscopy (Philadelphia, U.S.A.), vol. 2,
X 4 . Edited by S. S. Breese.
WATSON, D. H. (1969). T h e separation of herpes virus specific antigens by polyacrylamide gel electrophoresis.
Journal of General Virology 4, I 5 I - I 6 I .
WATSON, D. H., SHEDDEN, W. I. H., ELLIOT, A., TETSUKA,T., WILDY, P., BOURGAUX-RAMOISY,D. & GOLD, E. (1966).
Virus specific antigens in m a m m a l i a n cells infected with herpes simplex virus. Immunology xI, 399-418.
WATSON, D. I-I. & WILDY, P. (I963). S o m e serological properties of herpes virus particles studied with the electron microscope. Virology 2I, IOO-I I I.
WATSON, D. H. & WlLDY, P. (I969). T h e preparation of ' m o n o p r e c i p i t i n ' antisera to herpes virus specific
antigens. Journal of General Virology 4, 163-168.
WATSON, D.H., WILDY, P., HARVEY, B. A. M. & SHEDDEN, W. I. H. (1967). Serological relationships a m o n g
viruses o f the herpes group. Journal of General Virology x, 139-147.
(Received 5 July I973)
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