JOURNAL OF VIROLOGY, Oct. 1984, p. 108-118
Copyright © 1984, American Society for Microbiology
Vol. 52, No. 1
Characterization of Herpes Simplex Virus 1 a Proteins 0, 4, and 27
with Monoclonal Antibodies
MATHIAS ACKERMANN,' DANIEL K. BRAUN,' LENORE PEREIRA,2 AND BERNARD ROIZMAN1*
The Marjorie B. Kovier Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois 606371; and Viral and
Rickettsial Disease Laboratory, California Department of Public Health, Berkeley, California 947042
Received 19 April 1984/Accepted 29 June 1984
Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes
simples virus (HSV) 1 and 2 and by HSV-1 x HSV-2 recombinants indicated that monoclonal antibody H1183
reacted with HSV-1 a ICPO, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 a
ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described
(D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these a proteins. The
results were as follows: (i) a ICPO, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. (ii)
ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICPO was considerably more soluble
than ICP4 and ICP27. (iii) ICPO, ICP4, and ICP27 were readily partially purified by immunoaffinity
chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl
sulfate. (iv) ICPO and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h)
or late (18 to 20 h) postinfection. A fraction, but not all, 32p that was incorporated early was chased in the
presence of unlabeled phosphate. (v) ICPO, ICP4, and ICP27 labeled with either 32p or [35S]methionine yielded
multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when
the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at
the origin when the direction of migration was reversed.
a Genes (9), the earliest genes expressed by herpes
simples virus 1 (HSV-1), specify five proteins designated
infected cell polypeptides (ICP): ICPO, ICP4, ICP22, ICP27,
and ICP47 (1, 8-11, 14-17, 19, 24, 28, 29). Of these genes,
two have been investigated in some detail. a4 is known to be
essential for the transition from a ICP to ICP specified by i
and y genes which are expressed later in infection. Temperature-sensitive
mutants in a4 gene at the nonpermissive
temperature accumulate a ICP; the transition to P ICP does
not ensue (5, 13, 27). Less is known of the function of a22
gene. Mutants engineered to contain deletions in a22 gene
cannot be differentiated from wild-type parents in continuous
lines of human (e.g., HEp-2) and simian (e.g., Vero) cell
lines (22). However, these mutants grow poorly in continuous
lines of rodent cells; they yield relatively little virus and
do not spread effectively from cell to cell in confluent,
resting cultures of diploid human cell strains (A. Sears and
B. Roizman, manuscript in preparation). Little is known of
the function of other a genes inasmuch as attempts to
introduce conditional lethal mutations into aO, a27, and a47
have to date not been successful.
The paucity of information on the function of most of the a
genes is matched by the lack of knowledge on the properties
of a proteins. Most of the available information is based on
the use of radiolabeled amino acids and phosphate to identify
the ICP by their electrophoretic migration in denaturing gels
(8, 9, 19, 21, 30). Although ICP4 is readily differentiable from
other host and viral proteins by its size and characteristic
pattern of migration in denaturing gels, the other a ICP,
especially ICPO, are not. The latter protein comigrates with a
i polypeptide (,B ICP8) and is not readily differentiable from
it. In this study, we report on the identification of monoclonal
antibodies to a ICPO and a ICP27. These and the
monoclonal antibody to ICP4, similar to the one identified in
* Corresponding author.
a previous study concerned with methodology for characterization
of monoclonal antibodies to poorly soluble proteins
(2), have been used in this study for preliminary characterization
of the properties of ICP4, ICPO, and ICP27.
The following points are relevant to the studies described
in this report.
(i) HSV-1 ICP4 forms several bands upon electrophoresis
in denaturing polyacrylamide gels (19, 21). The most rapidly
migrating band, ICP4a, was detected in the cytoplasm of
infected cells, and its electrophoretic mobility in polyacrylamide
gels cross-linked with (N,N'-diallyltartardiamide)
(DATD) corresponds to a protein with a molecular weight of
163,000. The more slowly migrating forms accumlating in
nuclei of infected cells have apparent molecular weights of
165,000 (ICP4b) and 170,000 (ICP4c) (19). All three forms
become labeled in the presence of 32p in the medium (30).
The 32p can be chased on or off ICP4a and ICP4c throughout
the reproductive cycle. In a recent report it has been
indicated that ICP4c accepts polyribosylation in isolated
nuclei (23). Whether this activity actually occurs in the
infected cell and whether it is related to the phosphorylation
by Pi is presently unclear. ICP4 has been reported to bind to
infected cell chromatin and DNA. Freeman and Powell (7)
have reported that purified ICP4 binds to DNA in the
presence but not in the absence of host proteins. The HSV-2
ICP4 migrates more slowly than the corresponding HSV-1
polypeptides (19, 21).
(ii) In denaturing polyacrylamide gels, HSV-1 ICP27 forms
two bands, with electrophoretic mobilities corresponding to
molecular weights of 56,500 for ICP27a and 58,000 for
ICP27b (19). ICP27 is phosphorylated; its phosphate, like
that of ICP4, can be chased on and off throughout infection
(30), but these proteins have not been reported to accept
(iii) ICPO has an apparent molecular weight of 128,000 in
gels cross-linked with DATD (19) and only 108,000 in gels
VOL. 52, 1984
crosslinked with N,N'-methylenebisacrylamide (18). Because
it comigrates with ICP8 in gels cross-linked with
DATD, its properties have not been investigated in detail.
MATERIALS AND METHODS
Virus strains. The isolation and properties of HSV-1(F)
and HSV-2(G) (6, 12, 25) and mutant HSV-1(HFEM)tsLB2
(hereafter referred to as tsLB2) (13) have been reported
elsewhere. The relevant property of tsLB2 is that cells
infected and maintained at the nonpermissive temperature
(39°C) express mainly a genes and accumulate large amounts
of a proteins. The HSV-1 x HSV-2 intertypic recombinants
RH1G7 (4), A4D (19), and RSOBG13 (26) have been described
previously. All virus strains were propagated in
either HEp-2 cells or Vero cells as indicated throughout this
Media, buffers, and solutions. Maintenance medium consisted
of mixture 199 supplemented with 1% calf serum.
Disruption buffer consisted of 0.05 M Tris (pH 7.0)-8.5%
(wt/vol) sucrose-5% (vol/vol) 3-mercaptoethanol-2% (vol/vol)
sodium dodecyl sulfate (SDS). Hypotonic buffer consisted of
1.6 mM MgC12-6 mM KCl-10 mM Tris (pH 8)-i mM
dithiothreitol-0.5% (vol/vol) Nonidet P-40 (NP-40). In studies
on the solubility of viral proteins, the solutions were as
follows: solution A, phosphate-buffered saline (PBS; 8.2 mM
Na2HPO4, 1.5 mM KH2PO4, 0.14 M NaCl, and 2.5 mM KCl)
supplemented with 1% (vol/vol) NP-40-1% sodium deoxycholate-10-5
M N-a-ptosyl-L-lysine chloromethyl ketone
(TLCK)-L-1-tosylamide-2-phenylethyl chloromethyl ketone
(TPCK); solution B, PBS modified to contain 1% 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulfonatezwitterionic
detergent Chaps (Serva, Heidelberg, Federal Republic
of Germany), 0.5 M NaCl, and l0-5 M TLCK and 10-5 M
TPCK; solution C, 0.05 M Tris (pH 7.0), 5% (vol/vol) IPmercaptoethanol,
2% (wt/vol) SDS, 10-5 M TLCK, and 10-5
M TPCK; solution D, PBS containing 1% n-octyl-P-D-glucopyranoside
(Sigma Chemical Co., St. Louis, Mo.). The ureaampholine
solution consisted of 0.5% L-alpha phosphatidyl
choline-dipalmitoyl-9 M urea-4% NP-40-4% ampholines
(pH 3.5 to 9.5; LKB, Gaithersburg, Md.).
Monoclonal antibodies. The hybridomas H640, H1083, and
H1113 were derived from BALB/C mice immunized with
lysates of cells infected with tsLB2 and maintained at the
nonpermissive temperature. The procedures for fusion and
selection of the hybridoma cells producing antibodies against
HSV antigens were done as described previously (20).
Monoclonal antibody H640 to ICP4 was similar to one
(H432) described in a preceding report (2).
Infection and labeling of cells with radioactive precursors.
Monolayer cultures containing ca. 2 x 107 cells were exposed
to 20 PFU of virus per cell. After 1 h of adsorption, the
inoculum was replaced with maintenance medium. At various
times postinfection, the cells were labeled in 10 ml of
medium per culture. For labeling with [35S]methionine, the
cells were overlaid with methionine-free maintenance medium
supplemented with 200 ,uCi (1.260 Ci/mmol) of
[35S]methionine (New England Nuclear Corp., Boston,
Mass.). Labeling with 32p was done as described by Wilcox
et al. (30). The cells were preincubated in phosphate-free
medium for 3 h before the addition of 32p (carrier free; New
England Nuclear). After 2 or 3 h of radiolabeling, as indicated,
the cells were either harvested (pulse) or washed three
times with maintenance medium and further incubated, as
Preparation of labeled protein extracts. The cells were
washed three times with PBS, scraped off, and pelleted by
CHARACTERIZATION OF HSV-1 a PROTEINS 109
centrifugation at 1,000 rpm for 5 min. Total cell, nuclear, and
cytoplasmic extracts were prepared as described previously
(2). Preparation of soluble and insoluble proteins. The tsLB2or
HSV-1(F)-infected cells in 2 x 107 cell monolayer cultures
were labeled and harvested, as indicated. The cell pellets
were suspended in 2 ml of solution A or solution B or
solubilized in solution C. In the latter case, SDS was
removed by extensive dialysis against PBS and equilibration
by dialysis against solution D. The cells suspended in these
solutions were disrupted by sonication. After centrifugation
at 25,000 rpm for 1 h at 4°C in a type 25 Beckman rotor,
samples of the supernatant fluids and pellets were solubilized
in disruption buffer and subjected to electrophoresis in
denaturing polyacrylamide gels.
Purification of a ICP by immunoaffinity chromatography.
35s H 10833
*VSV-1 MCCK 5v 2 H'Y-7 MOCK !5V-2
-'V-1 K^ w 4tV-2
1 2 3 4 5 6 7 8 9
FIG. 1. Autoradiographic images and immune reactivity of polypeptides
from infected and mock-infected cells electrophoretically
separated in denaturing gels and then transferred to nitrocellulose
sheets. Replicate cultures of HEp-2 cells were labeled with
[35S]methionine (35S) from 8 to 24 h postinfection and infected with
HSV-1(F) or HSV-2(G) or mock infected (mock). Cell lysates were
subjected to electrophoresis in denaturing polyacrylamide gels and
transferred to nitrocellulose as described in the text. Numbers in
figure indicate ICP designation. Lanes 1 through 3, autoradiographic
images of electrophoretically separated polypeptides in lysates of
infected and mock-infected cells; lanes 4 through 6, reaction of
H1083 only with a polypeptide band which comigrates in denaturing
polyacrylamide with ICPO and ICP8 HSV-1; lanes 7 through 9,
reaction of H1113 with one band formed by electrophoretically
separated polypeptides of HSV-1(F) and two polypeptide bands in
electrophoretically separated lysates of HSV-2(G). The electrophoretic
mobility of these proteins corresponds to those of ICP27 of
HSV-1(F) and HSV-2(G).
110 ACKERMANN ET AL.
Monoclonal antibodies were purified from ascites fluid by
binding to protein A-Sepharose C1-4B beads (Sigma). The
beads were washed three times with PBS containing 3%
bovine serum albumin (BSA), each time followed by washing
with solution D before incubation with infected cell
lysates contained in 0.5 ml of solution D. After 3 h of shaking
at 10°C, the mixture was centrifuged for 2 min in an
Eppendorf centrifuge. The supernatant fluid containing infected
cell constituents that were nonreactive with the
monoclonal antibodies was collected, and SDS, f-mercaptoethanol,
Tris (pH 7), and sucrose were added in quantities
corresponding to their respective concentrations in the disruption
buffer. The Sepharose bead pellet was washed three
times with cold solution D buffer and once with 100 mM
NaCl-50 mM Tris (pH 7.4). Proteins were removed from the
antigen-antibody protein A-Sepharose bead complex by the
addition of disruption buffer. The solubilized material was
boiled for 2 min before electrophoresis.
One- and two-dimensional polyacrylamide electrophoresis
and reaction of monoclonal antibodies to polypeptides transferred
to nitrocellulose. The polypeptides were separated on
9.25% (vol/vol) polyacrylamide gels containing SDS and
cross-linked with DATD as described by Morse et al. (19).
Two-dimensional electrophoresis was performed as described
previously (3). Briefly, the cells were solubilized in
an urea-ampholine solution. The polypeptides were separated
by nonequilibrium pH gradient electrophoresis for a total
of 4,000 V * h when run from the acid to the base and for
10,000 V * h when run from the base to the acid. After
equilibration in solution containing SDS, the tube gel was
placed horizontally over a stacking gel for electrophoresis in
the second dimension. This was done essentially the same
way as for the one-dimensional separations. The stacking gel
was molded to contain wells for polypeptide samples to be
separated in one dimension in the same slab gel as that used
for the second dimension. After electrophoresis, the polypeptides
were transferred electrically to a nitrocellulose
sheet and reacted with monoclonal antibodies as previously
described (2). Briefly, the nitrocellulose sheet was incubated
for 1 h at 37°C in PBS containing 3% (wt/vol) BSA and for 2 h
at the same temperature with ascites fluid diluted 1:50 in 1%
BSA. The unbound antibodies were removed by washing
with PBS containing 1% BSA. The nitrocellulose sheet was
then incubated in PBS containing 1% BSA and 5 pl per 3 ml
of horseradish peroxidase-coupled rabbit anti-mouse
immunoglobulin G (Miles Laboratories, Inc., Elkhart, Ind.).
After washing with PBS, the nitrocellulose sheet was transferred
to a substrate solution containing 0.02% (wt/vol)
ortho-dianisidine dihydrochloride and 0.06% H202. Autoradiographic
images of the labeled bands on the nitrocellulose
sheet were made on Kodak XS-1 film.
Reactivity of monoclonal antibodies H1083 and H1113 with
HSV-1, HSV-2, and mock-infected cell polypeptides. In preliminary
studies, we characterized several monoclonal antibodies
which appeared to react with a proteins. To ascertain
whether the monoclonal antibodies selected for these studies
actually reacted with ot proteins, it was necessary to map the
genes specifying the proteins with which these monoclonal
antibodies reacted. The purpose of this series of experiments
was to assess the type specificity of the monoclonal antibodies
selected for these studies. Lysates of HEp-2 cells harvested
20 h after mock infection or infection with HSV-1(F)
or HSV-2(G) were subjected to electrophoresis in denaturing
polyacrylamide gels and then transferred to a nitrocellulose
sheet as described above. The results (Fig. 1) are as follows:
(i) Monoclonal antibody H1083 reacted with a polypeptide
band (molecular weight, 128,000) of HSV-1(F) corresponding
in electrophoretic mobility to ICPO or ICP8. The reaction
was type specific in that H1083 did not react with lysates of
HSV-2(G) or mock-infected cells.
(ii) Monoclonal antibody H1113 reacted with a polypeptide
band (molecular weight, 56,500) of HSV-1(F) corresponding
in electrophoretic mobility to ICP27a (19). H1113
also reacted with two bands formed by electrophoretically
separated, transferred polypeptides contained in lysates of
HSV-2(G)-infected cells and corresponding to proteins with
molecular weights of 58,000 and 59,000. The monoclonal
antibody did not react with lysates of mock-infected cells.
Identification by mapping of the genes specifying the antigens
reactive with H1083 and H1113. The purpose of this
series of experiments was to verify the identity of the
antigens reacting with monoclonal antibodies H1083 and
H1113. The map locations of HSV-1 and HSV-2 DNA
sequences present in the HSV-1 x HSV-2 recombinants
used in these experiments are summarized in Fig. 2. HEp-2
cells were infected with each recombinant as described
above. The infected cells were labeled with [35S]methionine
from 1 to 4 h postinfection and then incubated in medium
lacking radioactive precursors until 20 h postinfection. The
results (Fig. 3) are as follows:
(i) H1083 reacted with a polypeptide band comigrating
with ICPO or ICP8 produced by the HSV-1 x HSV-2
recombinant RH1G7. ICP8 of this recombinant is coded by
HSV-2 DNA sequences, whereas the two gene copies for
ICPO are located in HSV-1 DNA sequences (4). Inasmuch as
H1083, an HSV-1-specific monoclonal antibody, reacted
with this recombinant, it must react with ICPO rather than
ICP8. The reaction of monoclonal antibody H1083 with
lysates of cells infected with the other recombinants is
consistent with this conclusion. Thus, H1083 reacted with a
polypeptide band present in lysates of cells infected with
recombinant R5OBG13, indicating that the coding region of
the reacting polypeptide mapped in the HSV-1 DNA sequences
restricted to the L component of the DNA (26). The
reaction of H1083 with the recombinant A4D was very weak.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0. 0.6 0.6 1.0
0 2T 0 4 22 4T 4
N . _HSV-2
FIG. 2. Map locations of ICP and summary of sequence arrangement
of recombinant viruses RH1G7 (4), R5OBG13 (20) and A4D
(19), used in this study. Map coordinates and a schematic diagram of
the HSV genome in the prototype arrangement are shown at the top
of the firgure. The top and bottom lines to the right of the
designation of the recombinants represent HSV-1 and HSV-2 sequences,
respectively. The heavy lines identify the sequences
present in each recombinant. The numbers around the double lines
identify at genes and the gene specifying ICP8 mapping at those
VOL. 52, 1984
35S 335s H 1083
p : g A A H i'., . r
2 3 4 6 9 10
FIG. 3. Autoradiographic images and reactivity of electrophoretically
separated polypeptides from lysates of cells infected with
HSV-1(F), HSV-2(G), and HSV-1 x HSV-2 recombinants with
monoclonal antibodies H1083 and H1113. Numbers in figure indicate
ICP designation. Lane 1, autoradiographic images of polypeptides
from HSV-1(F)-infected cells labeled 18 to 24 h postinfection
and used as markers; lanes 2 through 4, autoradiographic images of
electrophoretically separated polypeptides in lysates of cells infected
with HSV-1 x HSV-2 recombinants RH1G7, R5OBG13 (R5OBG),
and A4D, respectively, incubated in medium containing [35S]methionine
(35S) from 1 to 4 h postinfection and in medium lacking
radioactive precursors until 20 h postinfection; lanes 5 through 10,
reaction of the electrophoretically separated, transferred polypeptides
specified by the recombinant viruses with the monoclonal
antibodies H1083 (lanes 5 through 7) and H1113 (lanes 8 through 10).
Since in this particular recombinant one ICPO gene is coded
for by HSV-2 DNA, whereas the second ICPO gene is coded
for by HSV-1 DNA, the weak reaction is consistent with the
hypothesis that the infected cells produce ICPO of both
HSV-1 and HSV-2.
(ii) H1113 reacted with R50BG13, forming a band characteristic
of HSV-1 and indicating that the coding gene is
located on the L component of the DNA. In contrast, H1113
reacted with two bands formed by electrophoretically separated,
transferred polypeptides contained in lysates of cells
infected with recombinant A4D. These bands are characteristic
of HSV-2-infected cells, indicating that the gene specifying
the reactive polypeptide mapped between coordinate
0.7 and the L-S component junction, i.e., within a region
known to contain the domain of ICP27 gene (1, 14, 15, 18,
19). As expected, the reaction of H1113 with RH1G7 was
Compartmentalization of ICPO and ICP27. The purpose of
these experiments was to determine the site of accumulation
of the viral polypeptides identified by H1083 and H1113. In
these experiments, HEp-2 cells infected with tsLB2 were
CHARACTERIZATION OF HSV-1 a PROTEINS 111
labeled at 39°C with [35S]methionine from 1 to 3 h postinfection
and then maintained in medium free of label at the same
temperature until 20 h postinfection. The proteins contained
in nuclear and cytoplasmic fractions of these cells were then
electrophoretically separated in denaturing gels, electrically
transferred to nitrocellulose sheets, and reacted with monoclonal
antibodies. Both ICPO and ICP27 identified by H1083
and H1113, respectively, accumulated in the nucleus (Fig.
4). The observations that the monoclonal antibodies reacted
with polypeptides produced by tsLB2 incubated at the
nonpermissive temperature and that these polypeptide bands
comigrated with bands labeled early in infection are consistent
with the conclusion that H1083 and H1113 are a protein
Patterns of accumulation of a ICP4, ICPO, and ICP27. The
purpose of these experiments was to determine the patterns
of accumulation of a ICP4, ICPO, and ICP27. The monoclonal
antibodies used in these studies were H1083 and H1113,
which were described above, and H640, a monoclonal
antibody that is reactive with ICP4. HSV-1(F)-infected
35S H 1083
C N C N
1 2 3 4 5 6
FIG. 4. Autoradiographic images and immune reactivity of electrophoretically
separated polypeptides from lysates of HEp-2 cells
infected with tsLB2 and maintained at 39°C in medium containing
[35S]methionine (35S) from 1 to 3 h postinfection and in medium
lacking radioactive precursors until 20 h postinfection. Numbers in
figure indicate ICP designation. Lanes 1 and 2, autoradiographic
images of tsLB2 labeled with [35S]methionine; lanes 3 through 6.
photographs of antibody staining. Lanes 1, 3, and 5 contain the
cytoplasmic (C) fractions, whereas lanes 2, 4, and 6 display polypeptides
contained in the nuclear (N) fractions. The electrically transferred
polypeptides were reacted with H1083 and H 1113 monoclonal
antibodies as described in the text.
112 ACKERMANN ET AL.
Pul se Pul se Chase Chase Mock (Chase
1B -24h 1 4 h 4 lOh 4 - 24h 4 -24h
3b Ab 35S Ab 35S Ab 35S Ab 45S
4 a_4 c 4
0 de Cs
27 27 327
2 :3 4 5 6 7 8 9
FIG. 5. Autoradiographic images and immune reactivity of electrophoretically
separated, transferred polypeptides from lysates of
HEp-2 cells infected with HSV-1(F). In the experiments illustrated
here, each lane contained the lysates of ca. 105 cells. Numbers in
figure indicate ICP designation. Lane 1, HSV-1(F)-infected cell
polypeptides labeled with [35S]methionine (35S) from 18 to 24 h
postinfection; lane 2, electrophoretically separated, transferred
polypeptides harvested 4 h postinfection and reacted with a mixture
of antibodies (Ab) to ICP4, ICPO, and ICP27; the polypeptides were
labeled 1 to 4 h postinfection. Lane 3, autoradiographic image of
polypeptides in lane 2; lane 4, same as in lane 2, except that the
infected cells were harvested at 10 h; the infected cells were
incubated from 1 to 4 h postinfection in medium containing
[35S]methionine and in medium lacking radioactive precursors thereafter.
Lane 5, autoradiographic image of polypeptides shown in lane
5; lane 6, same as in lane 2, except that the infected cells were
incubated from 1 to 4 h in medium containing [35S]methionine and in
medium lacking radioactive precursors until they were harvested at
24 h postinfection. Lane 7, autoradiographic image of electrophoretically
separated polypeptides shown in lane 6; lane 8, reactivity of
electrophoretically separated, transferred polypeptides from mockinfected
cells with the mixture of monoclonal antibody to ICPO,
ICP4, and ICP27; lane 9, autoradiographic image of the polypeptides
contained in lane 8.
HEp-2 and Vero cells, respectively, were labeled with
[35S]methionine from 1 to 4 h postinfection and maintained at
34°C in medium lacking radioactive precursors. Replicate
cultures were harvested at 4 h (pulse), 10 h (chase), and 24 h
(chase) postinfection, solubilized, subjected to electrophoresis
in denaturing gels, transferred to a nitrocellulose sheet,
and reacted with a mixture of monoclonal antibodies to at
polypeptides ICP4, ICPO, and ICP27. The reactivity of the
antibody with polypeptides contained in ca. 105 cells per lane
is shown in Fig. 5. The results are as follows:
(i) The predominant form of ICP4 in lysates of cells
harvested at 4 h was the ICP4a form. At 10 and 24 h, the
predominant forms were ICP4b and ICP4c. Because some I
proteins were labeled during the pulse (e.g., ICP6), we could
not determine whether the labeled polypeptide bands migrating
at the position of ICPO were predominantly ICP0,
predominantly ICP8, or both ICP0 and ICP8. The predominant
labeled band formed by lysates of cells harvested at 4 h
postinfection comigrated with the bands formed by those
harvested at 10 and 24 h postinfection.
(ii) The a polypeptides ICP4, ICPO, and ICP27 reacted
with monoclonal antibodies throughout the experiment (24
h); moreover, the increasing amounts of polypeptides that
reacted at 10 and 24 h postinfection indicated that cessation
of et protein synthesis is not instantaneous.
(iii) H640 reacts with early (ICP4a) as well as with
processed (ICP4b, ICP4c) forms of ICP4. An additional band
reactive with H1113, and therefore a processed form of
ICP27, was apparent in electrophoretically separated lysates
of cells infected for 24 h. On the basis of its reactivity with
H1083, ICP0 contained in lysates of infected cells harvested
at 4 h postinfection formed a relatively sharp band, whereas
ICP0 contained in lysates of cells infected for 10 and 24 h
formed a broader main band and a slower migrating satellite
The results obtained with lysates of infected Vero cells are
essentially the same as those obtained with lysates of
infected HEp-2 cells (data not shown).
Solubilization of ICP4, ICPO, and ICP27. Since at polypeptides
appear to be poorly soluble in isotonic solutions (8, 30),
we investigated the solubility of ICP4, ICPO, and ICP27 in
solutions containing a nonionic detergent (NP-40), a zwitterionic
detergent (Chaps), and a negatively charged denaturing
detergent (SDS) to determine a suitable solubilization procedure
for the purification of these polypeptides. In these
experiments, HEp-2 cells infected with tsLB2 and maintained
at 39°C were labeled with [35S]methionine either
between 1 and 3 h or 18 and 20 h postinfection. Solubilization
of harvested cells in solutions A, B and C-D was done as
described above. However, because complete solubilization
of the cell pellet was not achieved, the soluble (supernatant
fluid) and insoluble (pellet) fractions were not readily comparable.
The results (Fig. 6) indicate the following:
(i) ICP0 was soluble in all three buffers. Buffers containing
NP-40 were more efficient than those containing Chaps.
(ii) ICP4 was not soluble in PBS containing either NP-40 or
(iii) ICP27 was poorly soluble in buffers containing NP-40
(iv) All three a polypeptides appeared to retain their
solubility in solution D after solubilization in buffers containing
(v) Cell lysates harvested at different times after infection
did not differ substantively with respect to the solubility of
ICP0, ICP4, and ICP27. However, the autoradiographic data
suggest that the ICP4 harvested early was slightly more
soluble than that harvested late. It is noteworthy that in
previous studies it has been shown that newly made, cytoplasmic
ICP4 is slightly more soluble than the nuclear ICP4
Purification of ICPO and ICP27. The purpose of this series
of experiments was to purify by affinity chromatography
ICPO, ICP4, and ICP27. HEp-2 cells infected with tsLB2 and
maintained at 39°C in medium containing [35S]methionine
VOL. 52, 1984
CHARACTERIZATION OF HSV-1 a PROTEINS 113
* s _M
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 18 19 20 21 22 23 24 2 5 26 27 28 29 30
FIG. 6. Autoradiographic images and immunoperoxidase reactions. HEp-2 cells were infected with HSV-1(F) or tsLB2 or mock-infected
and incubated at 39°C in medium containing [35S]methionine (35S) as indicated below. After harvesting, the cell lysates were separated on
SDS-polyacrylamide gels, transferred to nitrocellulose, and reacted with an artificial mixture of monoclonal antibodies (AB) H640, H1083,
and H1113. Numbers in figure indicate ICP designation. Lanes 6, 16, and 26, immunoperoxidase reaction of HSV-1-infected cells labeled
from 18 to 24 h postinfection; lanes 1, 11, 21, autoradiographic images of polypeptides in lanes 6, 16, and 26; lanes 7, 17, and 27,
immunoperoxidase reaction of mock-infected cells; lanes 2, 12, and 22, autoradiographic images of lanes 7, 17, and 27; lane 8, antibody
reaction of lysates of tsLB2-infected cells, labeled from 1 to 4 h postinfection and maintained in medium free of radioactive precursors until 20
h postinfection and solubilized in solution A containing NP-40; lane 3, autoradiographic image of polypeptides shown in lane 8; lane 9,
antibody reaction of replicate cell lysate as in lane 8 but solubilized with solution B containing Chaps; lane 4, autoradiographic image of
polypeptides shown in lane 9; lane 10, antibody reaction of replicate cell lysate as in lanes 8 and 9 but solubilized with solution C containing
SDS and equilibrated with solution D containing n-octyl-p-D-glucopyranoside; lane 5, autoradiographic image of polypeptides shown in lane
10; lane 18, same material as in lane 8; before electrophoresis, the solublized cell lysates were centrifugated for 1 h at 72,000 x g; the
immunoperoxidase reaction of a sample of the supernate fluid is shown. Lane 13, autoradiographic image of lane 18; lane 19, same material as
in lane 9, centrifuged exactly as the material in lane 18; the immunoperoxidase reaction of a sample of the supernate fluid is shown. Lane 14,
autoradiographic image of lane 19; lane 20, same material as in lane 10, centrifuged as described for the material in lane 18; the
immunoperoxidase reaction of a sample of the supernate is shown. Lane 15, autoradiographic picture of lane 20; lane 28, same material as in
lane 8; solubilized pellet from lane 18; immunoperoxidase reaction is shown. Lane 23, autoradiographic image of lane 28; lane 29, solubilized
pellet from lane 19; immunoperoxidase reaction is shown. Lane 24, autoradiographic pictpre of lane 29;. lane 30, immunoperoxidase reaction
of the solubilized pellet from lane 20; lane 25, autoradiographic image of lane 30.
either from 1 to 3 h or from 18 to 24 h were harvested
immediately after labeling, pooled, and solubilized in solution
C. The solution was then dialyzed, equilibrated in
solution D, and clarified by centrifugation as described
above. The soluble polypeptides contained in the supernatant
fluid were then reacted with affinity columns containing
bound monoclonal antibody. The results obtained on immunoaffinity
chromatography of ICPO are shown in Fig. 7,
whereas those of ICP4 and ICP27 are shown in Fig. 8.
Phosphorylation of ICPO and ICP27. Previous studies on
the phosphorylation of a proteins involved autoradiography
of electrophoretically separated ICP labeled in infected cells
overlaid with medium containing 32pj. Although such studies
have clearly demonstrated that ICP4 becomes labeled largely
because phosphate label comigrated with the various
forms of ICP4 (21, 30), the phosphorylation of ICP27 was
deduced solely on the basis of comigration of the 14C-labeled
amino acid and the 32P-labeled polypeptide bands. Because
ICPO and ICP8 comigrate, the discrimination between these
polypeptides hinged solely on the conditions under which
they were labeled. The purpose of this series of experiments
was to establish whether ICPO and ICP27 are phosphorylated
by purifying the specific polypeptides with immunoaffinity
columns from lysates of infected cells maintained in
medium containing 32pj. HSV-1(F) was grown in HEp-2 cells
at 34°C. Three batches of antigen were prepared and labeled
with 32p. The first batch consisted of lysates of cells labeled
from 1 to 3 h and harvested at 3 h postinfection. The second
batch consisted of lysates of cells incubated in medium
containing 32p from 1 to 3 h and in medium containing
nonradioactive phosphate until harvested at 20 h postinfection.
The third batch consisted of lysates of cells labeled
from 18 h until they were harvested at 20 h postinfection.
ICPO and ICP27 were then purified from the three batches of
infected cells as described above. The results (Fig. 9) are as
(i) ICPO was phosphorylated more extensively at early (1
to 3 h) than late (18 to 20 h) times postinfection. Furthermore,
most of the radioactivity was chased by incubation in
medium containing nonradioactive phosphate.
114 ACKERMANN ET AL.
1 2 3 4 5 6
FIG. 7. Autoradiographic images of electrophoretically separated
polypeptides from crude lysates and by-products of purification
of ICPO. Numbers in figure are ICP designations. Lane 1, autoradiographic
image of HSV-1(F) labeled from 18 to 20 h postinfection
(this lane serves as a marker); lane 2, autoradiographic image of a
portion of starting material; it consisted of a lysate of 4 x 107 HEp-2
cells infected with tsLB2 incubated at 39°C in the presence of
[35S]methionine from 1 to 4 h and in medium lacking radioactive
precursors from 4 to 20 h postinfection. The lysate was solubilized in
buffer C and dialyzed against PBS and equilibrated by dialysis
against buffer D as described in the text; this lane represents the
contents of approximately 200,000 cells. The volumes of materials
subjvcted to electrophoresis in lanes 3 through 6 were calculated to
represent the same number of infected cells as that shwon in lane 2.
Lane 3, the pellet fraction obtained after centrifugation of the
solubilized lysate (lane 2) in a Beckman type 25 rotor for 1 h at
75,000 x g; lane 4, the supernatant fraction obtained after the
centrifugation described above; it represents the starting material
for immunoaffinity chromatography; lane 5, ICPO eluted from the
column with disruption buffer; lane 6, material that did not react
with antibody in the column.
(ii) ICP27 was more extensively phosphorylated. As in the
case of ICPO, phosphorylation was readily demonstrable in
polypeptides labeled early, late, or labeled early and then
incubated in unlabeled medium until late in infection. As in
the case of ICPO, the amount of ICP27 labeled during the late
(18 to 20 h) pulse was lower than that recovered after an
early (1 to 3 h) pulse but higher than that recovered after
incubation in medium containing nonradioactive phosphate.
Two-dimensional separation of ICP4, ICPO, and ICP27. In
previous studies, it has been shown that ICP4 and ICP27
form three and two bands, respectively, on electrophoresis
in denaturing polyacrylamide gels (10, 21, 30). However, the
extent of heterogeneity of the polypeptides in each band was
not known. Nothing is known of the homogeneity of ICP0,
and in previous studies on ICP35 (3), it has been shown that
polypeptides within a given band may be highly heterogeneous.
The purpose of this set of experiments was to
determine the extent of heterogeneity of HSV-1 ICP0,
ICP27, and ICP4 by two-dimensional separation in polyacrylamide
In the first series of experiments, HEp-2 cells were
infected with tsLB2 and maintained at 39°C in medium
containing [35S]methionine from 1 to 3 h after infection and
in medium devoid of labeled precursors until 20 h postinfection.
The cell lysates were electrophoretically separated in
two-dimensional gels and then transferred to nitrocellulose
sheets. Samples of lysates of (i) mock-infected HEp-2 cells,
(ii) cells infected with HSV-1(F) and labeled from 18 to 24 h
postinfection, and (iii) portions of lysates of tsLB2-infected
4 .. 6..
FIG. 8. Autoradiographic images of electrophoretically separated
polypeptides from crude lysates and by-products of purification
of ICP4 and ICP27. Numbers in figure indicate ICP designation.
Lane 1, autoradiographic image of HSV-1(F)-infected HEp-2 cells
labeled with [35S]methionine 18 to 24 h postinfection (this lane
serves as a marker); lane 2, autoradiographic picture of mockinfected
HEp-2 cells labeled the same way as in lane 1; HEp-2 cells
were infected with HSV-1(F), labeled with [35S]methionine from 1 to
4 h, and maintained in medium free of label until 24 h postinfection.
Harvested cells were solubilized in solution C, dialyzed and equilibrated
with solution D, and freed from insoluble material by
centrifugation as described in the text. Lane 3, the supernatant used
as starting material for immunoaffinity chromatography; lane 4,
ICP4 eluted from the columns with disruption buffer; lane 5,
material that did not react with H640 in the column; lane 6, ICP27
eluted from the column; material in lane 7 not reacting with H1113 in
the column; lanes 3 through 7, the contents of ca. 200,000 cells each,
as calculated by the volumes of materials subjected to electrophoresis.
VOL. 52, 1984
3 S 32P-ICP O 32 .PcP 27
- - -
2 3 d 5 6 7
FIG. 9. Autoradiographic images of ICPO and ICP27 purified
from lysates of cells labeled with 32P and electrophoretically separated
in denaturing gels. Numbers in figure indicate ICP designations.
Lane 1, polypeptides separated by electrophoresis in denaturing
gels from lysates of HEp-2 cells infected with HSV-1(F) and
labeled from 18 to 24 h postinfection with [35S]methionine; lane 2
through 4, ICPO purified from 32P-labeled HSV-1(F)-infected cells,
whereas lanes 5 through 7 contain ICP27 purified from the same
material. The HEp-2 cells were incubated in medium containing 32p
from 1 to 3 h and harvested immediately (lanes 2 and 5), incubated
further in medium containing unlabeled phosphate until 20 h (lanes 3
and 6), or labeled from 18 to 20 h postinfection and harvested at that
time (lanes 4 and 7).
cells subjected to two-dimensional analysis were subjected
to electrophoresis on the same gel, but in the second
dimension only. After the transfer, the nitrocellulose sheet
was reacted with an artificial mixture of monoclonal antibodies
H640, H1083, and H1113 that were reactive with ICP4,
ICPO, and ICP27, respectively. The results (Fig. 10) are as
(i) Consistent with previous attempts (unpublished data),
ICP4 precipitated and did not migrate in the first dimension
when the direction of electrophoresis was from acid to base.
The precipitated ICP4 was detected both by autoradiography
and monoclonal antibody staining.
(ii) ICPO formed a series of at least five spots detected by
[35S]methionine radioactivity and reactivity with monoclonal
antibody. The polypeptides in these spots appeared to differ
in charge rather than apparent molecular weight.
(iii) ICP27 made in cells infected with tsLB2 formed a
single band (ICP27a) when subjected to electrophoresis in
denaturing gels in the second dimension. The image produced
by antibody staining consisted of a relatively weak,
broad band that was heterogeneous with respect to charge.
The corresponding autoradiographic image appears to consist
of five discrete spots differing in charge rather than
apparent molecular weight.
CHARACTERIZATION OF HSV-1 a PROTEINS 115
The purpose of the second series of experiments was
twofold. First, it was of interest to determine whether ICP4
would remain soluble if the direction of electrofocusing were
reversed, inasmuch as ICP4 precipitated at the origin when
the direction was from acid to base. The second objective
was to determine whether all or a portion of the polypeptide
spots detected by autoradiography become labeled with 32p.
In this series of experiments, replicate cultures of HEp-2
cells were infected with HSV-1(F) and incubated at 37°C in
medium containing 32p from 1 to 4 hand harvested at that
time or incubated in medium containing unlabeled phosphate
from 4 h until harvested at 20 h postinfection. The cells were
lysed in the urea-ampholine buffer as described above, and
the lysate was subjected to electrofocusing for 10,000 V * h
from base to acid before denaturation and electrophoresis in
the second dimension. The separated polypeptides were
transferred electrically to a nitrocellulose sheet and reacted
with a mixture of monoclonal antibodies to ICP4, ICPO, and
ICP27 (H640, H1083, H1113, respectively). The results (Fig.
11) are as follows:
(i) At least a portion of the ICP4 migrated in the first
dimension when the direction of migration was from base to
acid. The procedure was not entirely satisfactory, inasmuch
as a significant portion of ICP4 precipitated at the origin.
(ii) The predominant form of ICP4 at 4 h postinfection was
ICP4a. In addition to the ICP4 precipitated at the origin, we
detected five spots (spots 3 through 7) by both monoclonal
antibody staining and autoradiography. The polypeptides in
these spots differed in charge rather than in apparent molecular
weight. After the chase, antibody staining revealed
several additional spots differing in charge (spots 1, 2, and 8)
and in electrophoretic mobility (spots 2b, 3b, 4b, Sb, and 6b).
The number of actual ICP4 species may be grossly underestimated
because of the limitations of the detection method and
because the properties of the ICP4 precipitated at the origin
are not known. Autoradiographic images revealed that the
label was retained in spots 3 through 7 and appeared in spots
1, 2, 8, 2b, 3b, 4b, and Sb but was particularly strongly in
spots 4, 4b, 5, Sb, and 6. Especially noteworthy was the
absence of significant 32p label in spot 6b.
(iii) ICP0 and ICP27 each formed spots differing in charge.
Moreover, these ICP increased in amount after the chase, as
measured by antibody staining. The increased amount of
ICPO detected at 20 h postinfection rather than during
processing may account for the detection of spot 1 in lysates
of cells harvested at 20 h but not at 4 h postinfection. With
that exception, there was no evidence for a shift in electrophoretic
mobility for either ICPO or ICP27 at 20 h as
compared with 4 h postinfection. The 32P autoradiography
supported this conclusion, inasmuch as there was no apparent
change in the location or number of radioactive ICPO and
ICP27 spots, even though the total amount of radioactivity
retained by these polypeptides at 20 H was reduced relative
to that seen at 4 h postinfection.
The salient features of the results reported here are as
(i) We identified and characterized monoclonal antibodies
to a ICPO and ICP27. These and the previously identified
monoclonal antibody to ICP4 were identified by their reactivity
with polypeptides of characteristic electrophoretic
mobility separated in denaturing gels and transferred to
nitrocellulose sheets. Potential ambiguities were eliminated
by mapping the location of the gene specifying these antigens
with the aid of HSV-1 x HSV-2 recombinants.
116 ACKERMANN ET AL.
(ii) An important characteristic of the three a. proteins
studied with the aid of the monoclonal antibodies is their
poor solubility in solutions lacking strong denaturing agents.
ICP27 and the processed forms of ICP4 were particularly
insoluble, whereas the newly made ICP4 and particularly
ICPO were more soluble. The significance of this property of
ICPO, ICP4, and ICP27 and its relation to their function is
not known. The poor solubility of ICP4 observed in this
study is consistent with that previously reported (8, 30) and
raises questions regarding reports that ICP4 binds DNA in
the presence of uninfected cell proteins (7). We should note
that we failed to bind single-stranded or double-stranded
cloned viral DNA to ICP4 that was electrophoretically
separated in denaturing gels and electrically transferred to
nitrocellulose sheets (data not shown). These experiments
were done in the presence and absence of host proteins from
lysates of uninfected cells (unpublished data). The significance
of our failures to demonstrate direct binding of DNA
should be interpreted with caution, inasmuch as denaturation
with SDS could damage the capacity to bind DNA,
even though the capacity to bind monoclonal antibodies was
(iii) In previous reports it has been shown that posttranslational
modifications of ICP4 alter its apparent molecular
weight, as measured by the electrophoretic mobility of the
precursors and products in denaturing gels (19, 21, 30).
ICP27 was also shown to form more than one band in
denaturing gels (18, 21), although this property was more
readily demonstrable for HSV-2 rather than HSV-1 ICP27.
Here we report that all three a proteins are also modified
with respect to net charge since each species formed multiple
spots of equivalent apparent molecular weight on twodimensional
5 4 3 2 1
(iv) Consistent with previous reports, ICP4, ICPO, and
ICP27 became labeled in cells incubated in medium containing
32P at any time after infection. The results reported here
are also consistent with previous conclusions that at least a
portion of the phosphate is replaced, i.e., that it cycles on
and off (30). By comparing lysates of cells labeled with 32p
early in infection with those of cells maintained in unlabeled
medium thereafter, it was not possible to detect major
differences in either the electrophoretic mobility or distribution
of 32P among the spots formed by ICPO and ICP27
separated in two dimensions. These results suggest that all
species of ICPO and ICP27 detected in this study contain
stable phosphate, as well as phosphate which cycles on and
off. The situation appears to be somewhat different with
ICP4; in this instance, spots lacking labeled phosphate were
observed after a chase in medium lacking labeled phosphate.
With the possible exception of ICP4, the function of at
proteins is unknown. They are of interest chiefly because of
their potential role in the establishment or maintenance of
viral latency and in the regulation of viral and host gene
expression. The relevance of the properties of the a proteins
reported to date to their function is unknown; these properties,
however, may be useful markers of their synthesis,
structure, and, when known, functional competence.
The studies carried out at the University of Chicago were aided by
Public Health Service grants CA-08494 and CA-19264 from the
National Cancer Institute and grant MV-2R from the American
Cancer Society. The studies carried out at the California State
Health Laboratories were aided by grant PCM-820-9749 from the
National Science Foundation and by Public Health Service grant Al
19257 from the National Institute of Allergy and Infectious Diseases.
M.A. is a fellow of the Swiss Nationalfond.
tsLB2 I" S
O 54 321
-W 3431 "*
1;- . IX
-j 0 us
. 2 9
FIG. 10. Autoradiographic images and immune reactivity of ICP separated in two dimensions. HEp-2 cells were infected with tsLB2 and
maintained at 39°C. Lysates of cells were labeled with [35S]methionine (35S) from 1 to 3 h and harvested 20 h after infection in two dimensions.
The direction of migration in the first dimension was from acid to base. (A) Polypeptides that reacted with a mixture of monoclonal antibodies
to 1CP4, ICPO, and ICP27 after transfer to a nitrocellulose sheet. (B) Autoradiographic image of (A). Lysates of cells labeled with
[35S]methionine for 2 h before harvesting at 20 h after infection with HSV-1(F) or mock infection were electrophoretically separated
concurrently in the second dimension only to serve as markers.
VOL. 52, 1984
10 .*f * .. _
8 7 85t41
CHARACTERIZATION OF HSV-1 a PROTEINS 117
,. 0 ; .s., f
FIG. 11. Autoradiographic images and immune reactivity of infected cell polypeptides separated in two dimensions. HEp-2 cells were
infected with HSV-1(F) and labeled from 1 to 4 h postinfection with 32p. At 4 h, one culture was harvested (pulse), and a replicate culture was
washed and further incubated in medium containing unlabeled phosphate until 20 h postinfection (chase). Infected cells were lysated in ureaampholine
solution and subjected to two-dimensional separation. The first dimension was run from the base to the acid for 10,000 V * h. (A)
Autoradiographic images of 32p pulse-labeled and transferred polypeptides. (B) Images of the corresponding immunoperoxidase reactions by
the mixture of monoclonal antibodies to ICP4, ICPO, and ICP27. (C) Autoradiographic images of polypeptides labeled from 1 to 4 h
postinfection with 32P and chased in nonradioactive medium until 20 h postinfection. (D) Corresponding immunoperoxidase reactions. The
three lanes to the left in (A) through (D) contain lysates of HSV-1(F) labeled with [35S]methionine from 18 to 24 h postinfection, mock-infected
HEp-2 cells, and HSV-1 labeled from 1 to 4 h postinfection, chased until 20 h postinfection, and separated in the second dimension only. P, ax
polypeptides that precipitated at the origin of the electrophoretic separation in the first dimension.
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