Ackermann, M., Braun, D. K., Pereira, L. & Roizman

ncbi.nlm.nih.gov

Ackermann, M., Braun, D. K., Pereira, L. & Roizman

JOURNAL OF VIROLOGY, Oct. 1984, p. 108-118

0022-538X/84/100108-11$02.00/0

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.

108

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

adenosyl polyribosylation.

(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

report.

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

indicated (chase).

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

114

254^ s

2:4

I|

0- OI

27

HI11 13

-'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).

b

a


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.

RESULTS

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

A4

HSV-I

HSV-2

HSV-

R506013 _HSV-2

RHICT

J. VIROL.

-HSy-I

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

locations.


VOL. 52, 1984

35S 335s H 1083

. 5

*b11

40

p : g A A H i'., . r

0 ft

H 1113

A4,444

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

HSV-1 specific.

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

b

a

27

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

specific.

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

'4 d

6w

:4

22

A

_

H 1113

C N

27 27

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.

5 #AN

11

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

295

1

.*.V

::

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

c

J. VIROL.

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

band.

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

Chaps.

(iii) ICP27 was poorly soluble in buffers containing NP-40

or Chaps.

(iv) All three a polypeptides appeared to retain their

solubility in solution D after solubilization in buffers containing

SDS.

(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

(30).

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

9 .

,i,

0

a

qmI

a

35 S

Cell Lysates

4

*o 27

AB

5W

11

-4}

250

21

a

Supernatant Fluids

4 -*

27

CHARACTERIZATION OF HSV-1 a PROTEINS 113

AB

7r W

* s _M

40

..

Solubilized Pellets

3Ss AB

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

a-1

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

follows:

(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.

4,

a

2T


114 ACKERMANN ET AL.

I

5

I 1

I

25

29

a

aU' U

a

.M 4

_O 6

W-

I^

VW

01-

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

gels.

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

IIp

018

qii

25.

29

11

sa.,9 ig'

.=

t; s

,, s

_

.... :.

S

.. .S.,

*::

2 3

.

;- 4

4 .. 6..

-27 '

J. VIROL.

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

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

follows:

(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.

DISCUSSION

The salient features of the results reported here are as

follows:

(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

not affected.

(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

separation.

A

Bla seL

tsLB2; Ab

o _

5 4 3 2 1

27 54321

Acid

4 p

4, i

27

O_

C, I

(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.

ACKNOWLEDGMENTS

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.

Base

tsLB2 I" S

O 54 321

-W 3431 "*

9...

INJO

A4:Ij

4 F,

1;- . IX

4

0

27

u)

4N V

m

-j 0 us

l.w

.,

J. VIROL.

4

. 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.

0

49%,


VOL. 52, 1984

0s0

Ba se

4 p

Base

p

32p

._w 4

7 6543

54j;4i

10 .*f * .. _

_2mm,7

5 4321

.

AV

4. 4

8 7 85t41

5441 .R-

M.

Acid

Acid

Pulse

A ..

Chase

ci

CHARACTERIZATION OF HSV-1 a PROTEINS 117

21

Ab

,. 0 ; .s., f

.: 27

sows.

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.

LITERATURE CITED

_ww 27

5432

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Base 7.0-43

Base

p

S.9543

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Acid

Acid

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