A 165 kd protein of the herpes simplex virion shares a common epitope with the regulatory protein, ICP4

Vol. 163, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 30, 1969

Pages

A 165 kd PROTEIN

OF

EPITCPE

THE: HERPES SIMPLEX WITH TEE REGULATORY

Viviane Institut Received

Bibor-Hardy

and

VIRION PROTEIN,

SHAREX ICP4

Frangoise

A COMMON

Sakr

du Cancer de Montreal, 1560 east Sherbrooke Montreal, QC, Canada H2L 4Ml

July

124-130

street,

3, 1989

We have investigated the possibility that immediate-early (IE) protein lCP4 could be a part of herpes simplex virus type 1 (HSV-1) virion particle. Immunodetection with a monoclonal antibody against ICP4 reveals that a component of the virion, migrating at 165 kd, shares a common epitope with this immediate-early protein. Immunolocalization studies on purified virions indicate that the antigen can be detected only in virions without membranes, and is located outside the capsid, most probably in the tegument. Ultrastructural localizations on HSV-1 infected BHK cells extracted with a nonionic detergent confirm that the protein immunoreacting with ant: i-ICP4 is present in virions. B 1989 Academic Press, Inc.

Viral

protein

IE

175K,

polypeptides

synthesized

cells

Genetic

[ 1,2].

required

in

expression early [3-81.

is

phosphorylated

Conflicting part

of the of

Cabral

virion.

ICP4, et

microscopic enveloped

have

al.

particles.

immunodetection with

the

particle.

0006-291X/89 Cupyrighr Ail rights

the

regulatory

with the

their

on

with study

to detect protein

on

A-gold

ICP4

transcription modifications have

virus 4,

at

particle

22, or

have

we now

and

47

not

ICP4

could

been

ICP4

at

with

[17,161.

present

a to

However the

electron

nucleocapsids

antibody

be

identical

[9,16,191.

as ICP4,

33

polypeptide,

mobility

monoclonal

24 to

27

virions of

detected

111-151.

estimated

of ICP4

[21,221,

124

gene

studies

localization

determinant

$1.50

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

with

a different

same immunological

of

whether

an association

Using

for

an electrophoretic

copurifies

is

cycle,

been

0,

IClJ4

regulator

virus

HSV

five

infected

that

a virion-associated IEs

published

1 (HSV-1

post-translational

infecting

the

infectious

of

has

genes

of

as a positive

wild-type

functions:

been

in

with

polypeptides of

[ 121,

a protein virus

virion

reproducibly

virion

regulator

Immunolocalization

A polypeptide

failed

HSV

[9,10].

one

type

acts

undergoes

infected

is

demonstrated

the ICP4

13.41.

175 kd

VP4,

level,

virus

have

throughout

immediate-early

results

that

and

for

activates

ICPI,

simplex

a negative

component(s)

responsible

Vmw65,

of

protein

studies

genes

of cells

number

Certain

found

form late of

in nuclei

total

in herpes

and

polypeptide

The

[161.

and

This and

early

biochemical

transcription

ICP4 mainly

infected-cell

functional

of early

gene

[8]

a

and

or

to ICP4 evidence

is associated

or 1201, that with

Vol. 163, No. 1, 1969

BIOCHEMICAL

Materials

AND BIOPHYSKAL

and

RESEARCH COMMUNICATIONS

Methods

Cells

and Virus infection Baby hamster kidney cells (BHK-21) were grown at 37’C in a-medium (Gibco) supplemented with 15% foetal calf serum tFlow Laboratories, mycoplaema free). For viral infection, BHK cells were infected with HSV-1 strain KOS, and incubated for different lengths of time at 34-C in a-medium supplemented with 2% foetal calf serum. Virion

purification BHK cells were infected at a multiplicity of For virions production, infection (m.o.i.1 of 1 p.f.u per cell with HSV-1 and incubated for 36-48 h at 34-c. Infected cel1 were scraped off in medium with a rubber policeman and centrifuged. PeHet was resuspended in a small volume of ice-cold hypotonic Nuclei buffer (10 mM Tris-HC1 pH 7.8, 3 mM CaCl2, 1 mM EDTA, 10 mM Kick). were isolated [23] and removed by low-speed centrifugation. Enveloped virus particles were purified from cytoplasmic extracts, using Dextran TlO gradients [16]. Immunolocalization on isolated virus particles All treatments were carried out in microtiter plate wells [Zl]. Purified virions were resuspended in PBS containing 1% egg albumin. A 25911 volume of suspension was mixed with 25 ~1 of monoclonal antibody 58s against ICY4 [ZO] diluted 1:lO in PBS containing 1 mM phenylmethylsulphonide fluoride (PMSF) as a protease inhibitor. The mixture was incubated for 45 min at 37-C in a humidified environment. A 25-~1 volume of protein A-gold preparation was added directly to the antigen-antibody mixture and incubated for an additional 45 min. A 50-~1 volume of the antigen/antibody/protein A-gold suspension was then transferred to another microtiter well containing a Formvar/carbon EM grid placed on top of 1% agar, and allowed to diffuse for 30 min. Grids were washed with PBS then with water, before negative staining with 1% phosphotungstic acid pH 7.2, for 1 min. Immunolocalization in infected cells BHK cells were infected at 20 p.f.u. per cell and incubated for 2 or 5 h at 34 ‘CT. After washing in PBS, cells were resuspended in a lysing buffer containing 10 mM HEPES pH 7.4, 5 mM NaCl, 2.5 mM MgCL, 0.3 M sucrose and 1 mM PMSF. To prepare cytoskeletons, Triton X-100 (15%) was added dropwise to the cells until a final concentration of 1% [22,24]. After fixation in 1% glutaraldehyde, these residual structures were partially dehydrated in ethanol and embedded in Lowicryl K4M (JBEM Services). This resin is a water compatible medium, and allows particularly good immunostaining with a reasonable degree of ultrastructural preservation [22]. Thin sections mounted on nickel grids were successively incubated for 10 min on a drop of phosphate buffer containing 0.5% Tween 20; for 30 min on a drop of the 58s monoclonal antibody against ICP4 diluted 1:25; and finally for 1.5 min on a drop of protein A-gold complex containing 5.2 mg/ml of polyethylene glycol. The thin sections were washed thoroughly with buffer, rinsed with distilled water and dried before staining with uranyl acetate and lead citrate . Western blotting and immunological detection Proteins (50 pg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes as described previously ]23]. The nitrocelluloee membranes were successively incubated for 4 h with the monoclonal antibody 58s (diluted 1:500 in the buffer), and for 2 h with anti-mouse IgG linked to peroxidase (Boehringer-Mannheim) . The complexes were revealed with diaminobenzidine (5 mg/ml) in 55 mM Tris-HCl pH 7.4 containing 0.01% H&t. 125

Vol. 163, No. 1, 1989

BIOCHEMICAL

and

Resulta Detection

of

ICP4

in

To establish from

5-h

PAGE protein profile

cells

transferred

profile was

of

Proteins

presence

of

with

virion

ICP4

the

preparations

165 kd,

but

infected

cells

migrated (Fig.

part

from

paper. and

amido

by

Spear

infected

cells

or

antibody

immunological 1, lane

virus were

Figure

described monoclonal

slightly

of the

preparations

SDS-PAGE

one

same

1, lane

was

virion

nitrocellulose

the

(Fig.

ICP4

after

to the

virions. with

not from

to

purified polypeptide

or or

virions

similar

Diecueeion

proteins

whether

infected

and

viz-ion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

particle,

1 lane black were

58s

against

on

SDS-

C shows [16]

then

as ICP4

their

probed

for

ICP4 was

This for [ZO].

A

detected

in

B).

This

polypeptide

comigrated

with

faster

than

the

polypeptide

detected

170 kd

the

staining.

Roizman

virions

determinant

proteins

separated

VP4 at

A).

AB

C VP

P

‘$’

j P

-4

-5 -7-8 -12 -14 Zig -18 -21 -22 -23

Fig.

50 ug of proteins from either 1: Detection of ICP4 in purified virione. 5-h HSV-1 infected BHK cells (lane A) or purified HSV-1 virions (lanes B and C) were separated by SDS-PAGE and transferred to nitrocelluloee. (lanes A and B) Filters were probed with a 1:500 dilution of the 585 antiwere detected with anti-mouse IgG ICP4, and immunoreactive proteins (lane C) Virion proteins (VP) were detected by labelled with peroxidase. amido black staining.

126

in

Vol.

163,

No.

Three order

BIOCHEMICAL

1, 1989

structural

variants

of decreasing

the These

variants

differ

Immunolocalization

of the

was

find

where

used

on

the

Figure

monoclonal

antibody.

particles

were

labelling

occured

antibody,

with

monoclonal

reactions

either

0.5

these

M,

that

detect

the

ICP4

any

association

and

the that

embedding.

gold

later

viral

outside

antigen

of virions

results

from

with

virions

obtained

with

et

the all

Ql

these with

serum.

All

2E).

necessary

at the electron

As any

extracted

anti-ICP4

was

Cabral

capsid.

In

further

2D and

that

without

1271.

with

the virion

1231, or with

were

(Fig.

the

the

exonuclease

the

2C show

PBS

lamins

of with

X-190,

with

nuclear

staining

react

2B and

reacted

alkaline

the

not

Figures

tegument,

were against

explains

virus

were spite

to the virions the

(Fig.

during that

in order

[12]

al.

who

to

did

microscopic

not

level.

of detergent early

surface

infected

Since

previous

detected

the times

of infected

and

protein

(Fig.

3A and

cells.

3B),

pockets

prior

there

of

virions

virions

had

needed was

could

be

with

the

incubation

particles

was

were

morphology

After gold

were

clearly

no background

were

by

to fixation

fixation

reduced

infection,

A-gold,

cells

experiment

ultrastructural after

caused

detergent-treated

X-100

and

not BHK

in

1% Triton

treatment

reactivity,

were

HSV-1 be

with

virions

also

outside

decorated

with

3C). were

the protein during

only

extracted

cytoplasm,

infection,

nucleocapsids

level.

could

At

antibody

purified

preparations,

microscopic

cells

Inside

nucleocapsids

remaining

to remove

antigen

on these

indicating

2B).

detergent-treatment

In

particles

These

(Fig.

1% Triton

particles

the

attached

Later

gold

with

incubation

preserved.

cells.

added

staining

did

Virion

immunological

monoclonal

the

treatment

the

electron

infected

detected

b variants.

immunogold

virions

before

of

at

visualized

in and

moieties

negative

by

intact

2 M urea

contamination

preserve

58s

[9,25], or

phosphate

indirect

followed

negative.

results

adequately

an

after

antibody

that

established

described

ICP4c

in cytoekeletons

To confirm examined

and

to ICP4a

of

located,

that

the

of ICP4

ICP4b

been

correspond

location

virions

epitope

Immunolocalization

to

in

failed

detect

virions,

with

1 M or

finding

cellular

[21],

shows

against

were

treatments

The

virions

polyclonal

antibody

cases,

was

mainly

detergent-treated

have

COMMUNICATIONS

in virion

However

controls,

well

and

antigen

decorated

RESEARCH

ICP4a,

SDS-PAGE

number

2A

BIOPHYSICAL

designated

could

antigen

isolated

preparations.

virion

in [ 263.

ICP4

during

in the

post-translationally

To

of

mobility

165 kd present

AND

their

viral not

nucleocapsids labelled

reacting formation

by with and

stages. 127

could protein

anti-ICP4 must

be observed A-gold

(results

is probably be acquired

not by

inside

nuclei.

not

shown),

attached

the

virion

to at

Vol. 163, No. 1, 1989

BtOCHEMtCAL

.‘T

-

L1 / :*_

jn

200nm

‘:

Fig.

2: Negatively stained HSV-1 virione, labelled with incubation with the 58s anti-ICPI. (A) Complete Virions pretreated with 1% Triton X-100. (D) Virions Triton X-100 and 0.5 M urea. (El Virions pretreated and 2 M urea.

In

conclusion,

protein

of

that

kd

between the

the

infected

presence

of

requirement

proteins

in

abortive

infections

make

it

used likely

protein(s) a

[review depend

of

in

this the

infection However,

151. on

this

that

such

factor.

128

protein A-gold after virions. (B and C) pretreated with 1% with 1% Triton X-100

demonstrate

antibody

this

in

productive in

above

monoclonal

weight 58s

for

may

an

molecular

regulatory

cell

presented

with

antibody cells,

absolute

the

experiments reacts

monoclonal

HSV-1

The

the

165

similarity

in

AND BtOPHYSlCAL RESEARCH COMMUNICATIONS

against

and

protein study

that

virion

ICP4,

tissue

for

and

The the

fact

uniquely

protein does

or

virion

ICP4.

recognizes

virion

a

is not

trophisms,

indeed

seem

expression

ICP4 ICPJ.

to

an

be of

latent

viral or

Vol. 163, No. 1, 1989

Fig.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3: Electron micrographs of virione (V) in cytoekeletone. BHK cells were treated with 1% Triton X-100 before fixation in Lowicryl. Thin sectiona were incubated eucceeeively with ICP4 and with protein A-gold. (A and B) Virions attached decorated with gold particles. (C) Pockete of virions inside are also labelled.

2-h infected and inclusion the 58s antito cells are the cytoplasm

Acknowlecbsments: We thank Dr. M. Zweig for the monoclonal antibody 58s against ICP4, and Dr. K.L. Powell for supplying the Ql monoclonal antibody again& the alkaline exonucleaee. This work wee supported by grant8 from the NCI and the MRC of Canada. 129

Vol. 163, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1. Honess, R.W. & Roizman, B. (1974) J. Virol. 14, 8-19. 2. Honess, R.W. & Roizman, B. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 12761280. 3. Dixon, R.A.F. & Schaffer, P.A. (1980) J. Virol. 36, 189-203. 4. Watson,R. & Clements, J.B. (1980) Nature 285, 329-330. 5. DeLuca, N. & Schaffer, P.A. (19851 Mol. Cell. Biol. 5, 1997-2008. 6. Gelman, I.H. & Silverstein, S. (1987) J. Virol. 61, 3167-3172. 7. O’Hare, P. % Hayward, G.S. (1987) J. Virol. 61, 190-199. 8. Preston, C.M. (1979) J. Virol. 29, 275-284. 9. Pereira, L., Wolff, M.H., Fenwick, M. & Roizman, B. (19771 Virology 77, 733749. 10. Wilcox, K.W., Kohn, A., Sklyanskaya, E. & Roizman, B. (1980) J. Virol. 37, 431-444 11. Courtney, R.J. & Benyesh-Melnick, M. (1974) Virology 62, 539-551. P.A. & Marciano-Cabral, F. (1980) J. 12. Cabral, G.A., Courtney, R.J., Schaffer, Virol. 33, 1192-1198. 13. Hay, R.T. & Hay, J. (1980) Virology 104, 230-234. 14. Ackermann, M., Braun, D.K., Pereira, L. & Roizman, B. ( 1984) J. Virol. 52, 1089-l 18. 15. Gelman, I.H. & Silverstein, S. (1986) J. Mol. Biol. 191, 395-409. 16. Spear, P.G. & Roizman, B. (1972) J. Virol. 9, 143-159. 17. Post, L.E., Mackem, S. & Roizman, B. (1981) Cell 24, 555-565. 18. Batterson, W. & Roizman, B. (1983) J. Virol. 46, 371-377. 19. Heine, J.W., Honess, R.W., Cassai, E. & Roizman, B. (1974) J. Virol. 14, 640651. 20. Showalter, S.D., Zweig, M. & Hampar, B. (1981) Infection & Immunity 34, 684-692. 21. Hopley, J.F.A. & Doane, F.W. (1985) J. Virol. Methods 12, 135-147. 22. Bibor-Hardy, V., Dagenais, A. & Simard, R. (1985) J. Gen. Virol. 66, 897901. 23. Dagenais, A., Bibor-Hardy, V. & Simard, R. (19841 Exp. Cell Res. 155, 435447. 24. Lenk, R. & Penman, S. (1979) Cell 16, 289-301. 25. Morse, L.S., Pereira, L., Roizman, B. & Schaffer, P.A. (19781 J. Virol. 26, 389-410. 26. Wilcox, K.W., Kohn, A., Sklyanskaya, E. & Roizman, B. (19801 J. Viral. 33, 167-182 P.J., Killington, R.A. & Powell, K.L. (1983) 27. Banks, L., Purifoy, D.J.R., Hurst, J. Gen. Virol. 64, 2249-2260.

130