Trans-activation of Herpes Simplex virus type 1 immediate early genes is specifically inhibited by human recombinant interferons

Vol. 159, No. 2, 1989 March 15, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 439-444

TRANS-ACTIVATION OF HERPES SINPLEX VIRUS TYPE 1 IHMEDIATE EARLY GENES IS SPECIFICALLY INHIBITED BY HUMAN RECOMBINANT INTERFERONS P. Ranier0

De

Stasio and Milton

Program in Microbiology, Indiana University, Received

January

31,

W. Taylor

Department Bloomington,

of Biology, IN 47405

1989

The effect of human recombinant interferons (IFNs) alpha and gamma on the transcription of Herpes Simplex virus type-l (HSV-1) immediate early (IE) genes has been studied. Human amniotic cells (WISH) were transfected with a chimaeric plasmid containing the regulatory region of the HSV-1 IE genes controlling the expression of a reporter gene (CAT). Subsequently cells were infected with live HSV-1 to trans-activate the IE sequences. When cell samples were treated with IFNa or IFN? prior to viral infection, tran0 1989 Academic Press, Inc. scription of the CAT gene was greatly inhibited.

The anti-viral the molecular mechanism of

lesser if

extent

at all,

(2,3,4) tion

inhibition

that

three

early

activators cycle

of DE and L genes occurs

and late

if

(6,7).

The

to a much

few years

Type 1 (HSV-1) replicaWe have

HSV-1 production occurs via the

groups of genes defined

(DE)

as immediate

(L) genes. All the IE

of the genes expressed later In other words no expression

IE genes are not transcribed

The IE genes of HSV-1 are activated

and through 0006-291X/89

439

at

have been barely,

and beta).

of Herpes Simplex virus

the replication properly.

involved

IFN (gamma) also inhibits

of at least delayed

as models (1).

groups in the last

by type I IFNs (alpha

The replication

has been studied

has been explored

Herpes Simplex virus

that type II

expressed

events

Several

genes are transcriptional during

(IFN)

DNA viruses

of

and the molecular

is inhibited

(IE),

of interferon

mostly using ENA viruses

elucidated.

expression early

level

reported

reported (5).

activity

a $1.50

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

BIOCHEMICAL

Vol. 159, No. 2, 1989

trans-activation

event

between nuclear

that

proteins

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is mediated

and a viral

by a complex formed

component (VP16 or Vmw65)

(8,9).

A very action

attractive is that

hypothesis

to explain

IFN acts specifically

on the expression

trans-activating

IE gene and therefore

viral

Recently

functions.

transcription blasts inhibited Panet's

group for

reporter sequence. quently

MATERIALS

that

the

in human fibro-

that

(5) confirmed

that the transcriptional inhibited

were transfected

When transfected

hybridization, reporter

cells

gene under the control infected

subsequent

HSV-1 IE genes are the results

found by

IFN type I.

1 IE genes is specifically Human WISH cells

all

inhibited

with IFNa. Our observations

in IFNI( treated

Here we report

inhibits

of the

Oberman and Panet (4) reported

of IE genes is strongly

treated

the IFN anti-Herpes

cells

trans-activation by

with

type I and type II a plasmid

containing

of HSVIFNs. a

of the HSV-1 IE promoter/enhacer were treated

with

IFN, subse-

with HSV-1, and then assayed by RNA dot-blot a remarkable

inhibition

in the transcription

of the

gene was observed. AND METHODS

1 Human amnion WISH cells and HSV-1 Strain HP were obtained f&m the American Type Culture Collection (Rockville, Md.). Cells were grown in Dulbecco's minimal essential medium (DMEW, GIBCO, Grand Island, NY) supplemented with 100 pg/ml streptomycin, 100 u/ml penicillin, 2mWglutamine and 5% fetal bovine serum (Hyclone, Logan, UT). HSV-1 was grown on African Green Monkey kidney (Vero) cells (ATE) and titered on both Vero and WISH cells. For DNA transfections Cells were grown on 6-well dishes. For viral infection 4 h prior to harvesting the cells, the medium was removed and the monolayer was rinsed twice with phosphate buffered saline (PBS). Virus was added to the monolayer at a MO1 of 10. Virus was allowed to adsorb at 37-C for 30 min., after which 3 ml of medium was added. Human recombinant IFNa and IFN$ were a gift of Genentech Inc. (San Francisco, CA). Cells were treated at 10 rig/ml of medium (1000 u/ml) 24 h after the DNA transfection. . DNA Cells were transfected by the DEAE-dextran method as described in Ausubel et al. (10) with some variations. Briefly plasmid DNA was resuspeded in warm TBS buffer (25mW Tris440

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cl pIi7.4, 137mW NaCl, 5mK KCl, 0.6W Na$iP04, 0.7mW CaC12, 0.5mW MgCl2) containing DKAK-Dextran Q.W. 500,000 (Pharmacia, Uppsala, Sweden). The DNA solution was then mixed 1:l with 2xDNKWto a final concentration of lpg/ml DNA and 5OOcrg/ml DBAK-Dextran. This mixture was quickly added onto the cell monolayer8 in a ratio of 0.3 ml/well corresponding to approx. 2ml/lO* cells. Cells were incubated in these conditions for 4h. DWSClshock was performed by removing all the medium and adding lml 10% DKSO in PBS for 2 min. at room temp. After a total of 72 h incubation in DWKWat 37-C cells were harvested blotted and fixed for RNA detection as described in Paeratakul et al. (11). Plasmids Plasmid pIEllOCAT containing a bacterial CAT gene under the con&o1 of the HSV-1 II3110 gene enhancer promoter region was kindly provided by Dr. O'Hare, Oxted, Surrey, UK (12). Plasmid pSAW-I containing a mouse APRT gene was a gift of Dr. J. Tischfield, Indianapolis, IN. Probes were radiolabeled using the oligonucleotide random-priming method (13). Briefly, the DNA (0.2 Bg) was denatured by boiling,and labeled in a reaction buffer containing 50 mWTris XC1 (pH 7.2), 10 mW else, I 0.1 ml4 DTT 50 Wg/ml BSA, '1 nW of each dCTP, dGTP, dTTP, 100 pM of alpha 32P dATP (Amersham, Arlington Heights, IL), 1 wg of hexaoligonucleotide random primer, and 2 units of K. coLi DNA polymerase I Klenow fragment (Boehringer-Wannheim, Indianapolis, IN) in a total volume of 30 ~1. The reaction was incubated at 10-C for 12 hours, after which the labeled DNA was purified by ethanol precipitation. This method routinely yields probes with specific activities of 1 x lo8 cpm/bg. The probe was denatured by boiling before use. -. The hybridization of NC filters was done according to Naniatis et al. (14) with some modifications. The membranes were first wetted in 3x SSC, and underwent a prehybridization step for 2 hours at 65-C in 3x SSC, 35% formamide, lx Denhardt's solution, 0.5 % SDS, and 100 ug/ml sonicated salmon sperm DNA. The prehybridization buffer was then removed, and the filter was hybridized with the denatured probe in the same pre-hybridization buffer adjusted to 10 mN EDTA, for 12 hours. The filter was washed sequentially at room temperature with 2x SSC, 0.5% SDS for 15 mfn, lx SSC, 0.5% SDS for 15 min, and 0.1x SSC, 0.5% SDS at 55-C until the radioactive background was low. Autoradiography was performed overnight at -70-C with an intensifying screen. RESULTS

. of Wish

Dot-Blot

Hvbr.j&zation . ected wm HSV-1 .

A system to simulate designed.

The plasmid

Oxted, Surrey, dextran

the very

early

pIEllOCAT

method (10).

pIEllOCAT

chloramphenicol

stage of HSV-1 infection

(kindly

UK) was transfected

sequence of the HSV-1 IEllO bacterial

DIECAT

into

provided WISH cells

was

by Dr. O'Hare, by the

DEAE-

contained

the IE enhacer/promoter

gene controlling

the expression

acetyl

transferase 441

(CAT) gene (12).

of a

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b.

a.

cells rpottd x ll+ lfo so . 25 . 12 .

Figure 1.

.

<&ectransfected

.

with

~Z~~OCAT and pSAK-I (mouse APRT) as described in the materials and methods section. The "control" lane corresponds to cells transfected with an unrelated plasmid (pBR322). After 24h transfection two of the samples were treated with IFNs as marked on the left. At 68h the IFN treated samples and an untreated one were infected with HSV-1 at an HOI=10 (W?W~). After 4h of viral infection cells were harvested and resuspended at lo6 cells/ml. Twofold dilutions were spotted onto NC and the cell number applied is indicated. The filters were then fixed in 1% glutaradehyde treated with proteinase K and hybridized with a 32P labeled probe as previously described (11). a. The NC filter was probed with CAT sequences. b. A replica of the same filter was probed with mouse APRT sequences.

Another

plasmid

containing

phosphoribosyl

transferase

internal

control.

infected

with

72 hours.

the mouse "housekeeping"

Cells

HSV-1 for

Cells

(APRT) gene

fixed

according

(11).

The NC filter

applied

to the RNA dot-blot

decrease

the

untreated with

labeled

genes unrelated

expression

of the

were treated

control

(Fig.

68 hours and then

with la).

transfection

period

32P-labelled experiment.

CAT gene

et al.

CAT

seguen-

A remarkable

was observed

in

the

IFNa or IFNr when compared to the A replica

of the same blot,

mouse APRT gene, showed no IFN induced to the HSV-1 IE system (Fig.

probed

inhibition

lb).

DISCUSSION The novelty

of the work we describe

summarized by the following:

of

(NC) and

method of Paeratakul

of this

as an

to nitrocellulose

was then probed with

1 shows the results

samples that

for

4 hours for a total

ces. Figure in

was co-transfected

were incubated

were harvested,

adenine-

1) it 442

in this

is the first

paper can be example of a

of

BIOCHEMICAL

Vol. 159, No. 2, 1989

single

molecular

replication

(IFNa)

I

inhibited

is the first

and type II

selectively event

plasmid

upon viral

cycle.

a protein

After

early

transport

complex (IEC=immediate

This complex interacts

sequences and activates IFNs after

the inhibition

transcription

of

the pIEllOCAT

efficiency.

the IEC is either

molecular of the viral

early

complex)

components and a to the nuclei

with the IE regulatory IE

genes (8,9).

transfection

cannot be due to secondary

the transfection that

a CAT gene

VP16 or Vmw65, which is also imported

entry.

by adding

of

containing

is formed between one (or more) cellular protein,

on the effect

report

of IFNs on the very

in the HSV-1 replication

viral

the

of the WV-1 IE sequence we were able to

study the effect

DNA to the nuclei

by IFNs during

on HSV-1 at the molecular

(IFN$

By the use of a chimaeric

under the control

(8)

specifically

of HSV-1; 2) it

both type level.

event

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

we also show that

effects

These data support

not formed or impaired

Moreover

of the IFNs on the hypothesis

in the presence

of

IFN. The possibility

that

viral

entry

and uncoating

by IFN has also been investigated. isolated treated

nuclei with

to the nuclei and Taylor,

DNA extracted

has been compared in control

IFN. No difference was detected manuscript

The significance currently

Viral

could

cells

be affected from

and cells

in the amounts of DNA transported

by molecular

hybridization

(De Stasio

in preparation).

of the molecular

inhibition

Experiments

being investigated.

we observe

are Currently

performed to analyze DNA-protein interactions whether IFN acts directly on VP16 or modifies

is being

and to establish a cellular component

of the IEC. ACKNOWLEDGMENTS

The authors providing

wish to thank the plasmids

Dr.

O'Hare and Dr. Tischfield

pIEllOCAT

for

kindly

and pSAM-I. This work was sup443

Vol. 159, No. 2, 1989

BIOCHEMICAL

ported

by U.S. Public

Health

grant

from the United

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Service

Grant AI21898,

and a

DK25498

Cancer Campaign.

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