Coronavirus transmissible gastroenteritis virus-mediated induction of IFNα-mRNA in porcine leukocytes requires prior synthesis of soluble proteins

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Coronavirus transmissible gastroenteritis virus-mediated

induction of IFNα-mRNA in porcine leukocytes requires

prior synthesis of soluble proteins

B Charley, L Lavenant, F Lefèvre

To cite this version:

Original

article

Coronavirus

transmissible

_{gastroenteritis}

virus-mediated induction

of

IFNα-mRNA

in

_porcine

leukocytes

requires prior synthesis

of soluble

_proteins

B

_Charley

L Lavenant F Lefèvre

INRA, Laboratoire de

Virologie

et

_Immunologie

Moléculaires, 78350

Jouy-en-Josas,

France

(Received 21

September

1993;

accepted

29 October 1993)

Summary ―

We studied the

expression

of interferon

alpha (IFNa)-mRNA

in

_porcine

non-adherent

peripheral

blood mononuclear cells

(PBMC)

after induction

by

the coronavirus transmissible

gastro-enteritis virus

(TGEV).

We found that _protein

synthesis

inhibition by

cycloheximide

(CHX) blocked IFNa-mRNA

expression,

except when PBMC were

_preincubated

with a conditioned medium as a

po-tential source of

cytokines.

These data indicate that IFN<*-mRNA induction by TGEV

_requires

de

novo

_synthesis

of

proteins.

Moreover,

they

suggest that IFNa-mRNA induction in

porcine leukocytes

by

TGEV involves mechanisms identical to those described for the _herpes

_simplex

virus in humans. In addition,

experiments performed

with a TGEV mutant, dm 49-4,

previously

characterized for its low

ability

to induce IFNa, showed that addition of a conditioned medium could not normalize its

IFNn-inducing ability.

Therefore, the defect of the dm49-4 mutant may be at the level of the final

trig-gering signal

to PBMC.

coronavirus I transmissible

_{gastroenteritis}

virus (TGEV) I interferon

alpha

I _leucocytes I por-cine

Résumé &horbar; L’induction, par le coronavirus de la

gastroentérite

transmissible, des ARN

mes-sagers de I’IFN(T dans les leucocytes du porc nécessite une

_{synthèse préalable}

de

protéines

solubles. Nous avons étudié

l’expression

des ARN messagers (ARNm) de l’interféron

alpha

(IFNa)

dans des cellules mononucléées non adhérentes du sang de porc,

après

induction par le

coronavi-rus de la

gastroentérite

transmissible

_(GET).

Nous avons observé que l’inhibition des

synthèses

pro-téiques par la

cycloheximide

(CHX)

bloquait l’expression

des ARNm de l’IFNa, sauf si les leucocytes

sont préincubés

avec des milieux conditionnés, sources

_potentielles

de

cytokines.

Ces résultats

indi-quent donc que l’induction des ARNm de /7FNa par le virus GET nécessite une

_{synthèse préalable}

de

protéines.

De

_plus,

ils

suggèrent

que l’induction des ARNm de l’IFNa chez le porc par le virus GET

implique

des mécanismes

identiques

à ceux décrits chez l’homme avec le virus de

l’herpès

simplex.

Par ailleurs, l’étude d’un mutant du virus GET, dm49-4, caractérisé par sa faible

aptitude

à

*

induire l’IFNa, a montré que l’addition de milieu conditionné ne _{permet pas} de lui restaurer un

pou-voir inducteur d’IFNa normal. Cela

_suggère

_que le défaut d’inductibilité du mutant dm49-4 se situe à l’é Jpe finale du processus d’induction.

coronavirus 1

gastroentérite

transmissible

_(GET)

1 interféron et 1

leucocytes

/ porc

INTRODUCTION

Transmissible

_{gastroenteritis}

virus

_(TGEV)

is a coronavirus which induces acute diar-rhoea in newborn

_piglets;

_{during early}

in-fection,

an intense interferon

_{alpha (IFNa)}

production

is observed in the serum and in several organs of infected animals

(La

Bonnardibre and

_{Laude, 1981). ). ln}

vitro studies have shown that 2

_types

of

_porcine

leukocytes

could

_synthesize

IFNA in

re-sponse to TGEV:

_lung

alveolar

macro-phages

can secrete IFNec

_following

expo-sure to infectious TGEV but not to inactivated virus

(Laude

et

_al,

_1984).

On the

_contrary,

a

_{subpopulation}

of blood mononuclear cells

_synthesizes

IFNA

fol-lowing

a short contact with inactivated

TGEV or TGEV-infected

glutaraldehyde-fixed cell

_{monolayers (Charley}

and

Laude,

1988).

These

data,

and others obtained with different viruses

_(Capobianchi

et

al,

1985;

Lebon et

al,

1982),

indicate that IFNa

synthesis

can be induced

_by

viruses

following

2 distinct mechanisms: viral in-fection of

_{IFNa-producting}

cells

(ie

mono-cytes

or

_macrophages)

or membrane inter-actions between a viral external structure and an

_{infrequent subpopulation}

of

non-adherent,

non-T,

non-B cells referred to as

natural interferon

_{producing (NIP)}

cells

_(Ito

et

al, 1981;

reviewed

_{by Charley}

and

Laude, 1992).

Several studies have

_suggested

that the viral

_component

involved in the induc-tion of IFNa

_synthesis

in NIP cells was an

external

_{glycoprotein (Lebon,}

1985;

Ca-pobianchi

et

al,

1992).

In the case of

TGEV,

we have shown that IFNa induction

was blocked in vitro

_by

monoclonal anti-bodies directed to the N-terminal domain of the viral

_glycoprotein

M

_{(gM) (Charley}

and

_{Laude, 1988).}

Moreover,

in 2 TGEV

mutants that were selected and character-ized for their reduced

_ability

to induce

IFNa,

an amino-acid substitution inside the

gM

external domain was assumed to gen-erate the defective induction

(Laude

et

al,

1992).

One of these mutants,

designated

dm49-4,

was shown to have a

non-glycosylated

M

_protein.

These data illus-trate the role of TGEV

_gM

in the induction of IFNA in

_porcine

NIP cells. In addition to the interaction between a viral

_glycoprotein

and NIP

cells,

IFNa induction appears to involve

_complex

_{mechanisms; thus,}

human IFNa induction

_by

the

_{herpes simplex}

virus

type

1

_(HSV)

_may

_require

cell

_cooperation

and

_production

_{of accessory}

_cytokines,

such as interleukin 3

(IL3)

and

granulo-cyte-macrophage colony-stimulating

factor

(GM-CSF) (Cederblad

and

Alm, 1990;

Ce-berblad et

al,

1991

Moreover,

the induc-tion of IFNa-mRNA in HSV-stimulated

hu-man NIP cells was shown to be blocked

_by

the

_{protein synthesis}

inhibitor

cyclohexi-mide

_{(CHX), except}

when a source of

cyto-kines was

_provided

to CHX-treated cells

(Cederblad

et

al,

1991

These

data

sug-gest

therefore that de novo

_synthesis

of

proteins, presumably cytokines

such as

CSFs,

is needed for the induction of IFNa-mRNA

_by

HSV. In this

_study,

we examine

whether induction _{of IFNa-mRNA in}

por-cine

_leukocytes

_by

TGEV also

_requires

de

novo

_{protein synthesis.}

In

addition,

we de-termined whether the reduced

_ability

of the TGEV mutant dm49-4 to induce IFNa

leuko-cyte-conditioned

medium as a source of

cytokines.

MATERIALS AND METHODS

Virus

_production

and

_purification

Two TGEV strains, the

_high-passage

Purdue 115, and the mutant virus dm49-4 characterized

by

a

_{non-glycosylated}

M

protein (Laude

et al, 1992), were used as virus sources. The proce-dures for virus

propagation

in the

pig kidney

cell line PD5 and titration of

infectivity

in the swine testis

(ST)

cell line have been reported (Laude

et al, 1986). Virions were

_{purified by}

_1-step rate zonal

_{centrifugation,}

_essentially as

_previously

described

_(Laude

et _{al, 1986).} The

_quantities

of virus in

suspensions

were calculated from UV absorbance values. Viral

preparations

were

_fully

inactivated

by

UV irradiation (2 successive cy-cles of 90-s treatment) and checked for the

ab-sence of residual

_infectivity

before use.

PBMC

Non-adherent

porcine

peripheral blood

mononu-clear cells

(PBMC)

were obtained from

hepari-nized blood by Ficoll

density centrifugation

on

MSL

_(density

1.077, Eurobio,

Paris)

followed

by

adherent cell

_depletion

on tissue culture flasks

as

_already

described _(Nowacki and

Charley,

1993). PBMC were

_suspended

in RPMI-1640 medium

supplemented

with 10% fetal calf ser-um

_{(RPMI-10%FCS)}

and antibiotics and

kept

overnight

at 4°C before use.

Conditioned medium

Conditioned medium

preparations

were ob-tained from

_overnight

cultures of PBMC at 37°C: cells were incubated at 5 x 106/ml in

RPMI-10%FCS, either in the presence of TGEV-infected

glutaraldehyde-fixed

cell monolayers

prepared

as

_previously

described

(Charley

and

Laude, 1988), or with

_{Spodoptera frugiperda}

(Sf9)

insect cells

(2

x

10

6

/ml)

_{kindly provided by}

M Godet. Cell _supernatants were filter-sterilized

and

_kept

at -20°C. Conditioned medium was

used at a concentration of 25% in RPMI-10%FCS

_{during preincubation}

of PBMC for IFN-induction.

IFN induction

PBMC were induced to

_produce

IFNa

by

incuba-tion with TGEV as follows: 108cells were

resus-pended

in 5 ml RPMI without serum and pre-incubated at 37°C for 4 h in medium

supple-mented with 25% conditioned medium, as de-scribed _by Cederblad et al (1991). PBMC were

then induced for 5 h with 800 _ng

_purified

virions

(unless

otherwise

_stated),

with or without 0.4

pg/

ml CHX

(Sigma,

St Louis, MI). Cells were col-lected for mRNA analysis and supernatants were

_assayed

for IFN

activity.

Anti-porcine

IFNa antiserum

A rabbit antiserum _{(No 9181)} directed to the

re-combinant

_porcine

IFNa _(Lef6vre et al, 1990),

kindly

provided by C de Vaureix, was used at a

final dilution of 1/1 000.

IFN

_biossay

Serial 1:3 dilutions of _supernatants from induced PBMC were

_assayed

for IFN on bovine MDBK cells

_using

vesicular stomatitis virus as a

chal-lenge

(La Bonnardi6re and _{Laude, 1981).} Our internal standard

porcine

IFNa was included in each assay. This standard was calibrated on

MDBK cells with the human international

refer-ence IFN B69/19 (NIH, Bethesda, MD,

USA).

In

our results, 1 U is

equivalent

to 1 IU of human IFN.

Northern blot

_analysis

Total RNA was isolated

by

the

_guanidium

thio-cyanate method

(Chomczynski

and Sacchi, 1987) from 108PBMC

following

incubation with viruses as _{indicated, and 20 pg} of each

_sample

Electrophoresis

was carried out on a _{1 %} aga-rose

_gel

which was blotted onto

_{nylon (Hybond}

N+,

Amersham).

The IFNa

probe

used was a

0.85 kb

_{EcoR1-Hpa1 genomic fragment}

contain-ing

the _complete

_coding

_sequence of

_porcine

IFNa1 gene (Lefbvre et al, 1990).

Hybridization

at 65°C and washes were

_performed

as

previ-ously

described

_(Laude

et al,

1992).

A

_positive

control for

_{hybridization}

consisted of an actin

probe.

RESULTS

Induction of IFNa-mRNA

_by

TGEV

requires

de novo

_{protein synthesis}

Northern blot

_analysis

of total RNA extrat-ed from PBMC showextrat-ed the presence of IFNa-mRNA in PBMC induced for 5 h with the Purdue 115 strain of

virus,

but not in

PBMC incubated in control medium

_(lanes

1 and

2,

fig 1

). In

the presence of the pro-tein

_synthesis

inhibitor

CHX,

a

dose-dependent

inhibition of the

_expression

of IFNa-mRNA was observed

_(lanes

3-8,

fig

1

thus,

at a dose of 0.4

_{pg/ml (lane 4),}

a

complete

inhibition of IFNa-mRNA was

ob-served,

whereas the

_positive

control actin mRNA was not altered. In the

_following

ex-periments,

CHX was used at 0.4

_pg/ml.

These data indicate that induction of IFNA -mRNA in

_porcine

PBMC

by

TGEV

_requires

prior protein synthesis.

Effects of PBMC

_supernatants

on IFNa-mRNA induction

_by

Purdue 115 TGEV _{in the presence of CHX}

In order to determine whether soluble fac-tors

_present

in PBMC

_supernatants

could circumvent the

_{CHX-dependent}

inhibition of IFNa-mRNA

_expression,

cells were

pre-incubated for 4 _{h in the presence of} condi-tioned medium before the addition of

TGEV and CHX.

_Figures

2 and 3 show that when PBMC were

_preincubated

with control medium

_{(RPMI 1640)}

before

_being

treated with TGEV and

CHX,

no IFNa-mRNA could be observed

_(lanes

3;

nega-tive and

_positive

controls for IFNa-mRNA

are shown in lanes 1 and

2,

_{respectively).}

On the

_contrary,

when PBMC were

prein-cubated with 3 different conditioned

media,

IFNa-mRNA was

_expressed

_{in the} pres-ence of

CHX;

lanes 4 and 5 in

_figure

2,

and lane 4 in

_figure

3 show the

_positive

ef-fects of

_supernatants

obtained from PBMC stimulated

_by

Purdue 115 virus-infected

cells,

by

dm49-4 virus-infected

cells,

or

_by

Sf9 non-infected

_{cells, respectively.}

These data indicate that soluble factors

_present

in various PBMC

_supernatants

allow the

procine

PBMC,

even when de novo

_protein

synthesis

is blocked

_by

CHX.

_Although

the conditioned-medium

_preparations

tested contained

IFN

A

,

their effect may not be due to IFNA

itself,

since its neutralization

by specific

antibodies did not block IFNA -mRNA

_{expression (fig}

3,

lanes 5 and

_6).

Effects of PBMC

_supernatants

on IFNa-mRNA induction

_by

dm49-4 mutant virus

To determine whether the addition of

con-ditioned medium

_prior

to induction of

PBMC

_by

the mutant virus dm49-4 could restore a normal IFNa-mRNA

_expression,

induction was

_performed

with this virus in the presence of

CHX, following

incubation with conditioned medium

_prepared

from Purdue 115-stimulated

PBMC,

as in

_figure

2. Lanes 1 and 4 in

_figure

4 are

_negative

controls

_showing

the absence of

Purdue 115 and dm49-4.

However,

the level of mRNA is lower after induction with dm49-4

_{(lane 3) compared}

with Purdue

115 (lane 2).

DISCUSSION

Our

_present

results show that induction of IFNa-mRNA in

_porcine

PBMC

by

TGEV

was inhibited

_by

CHX. These data

confirm,

in a different animal

_species

and for

an-other

virus,

the results

_{published by}

Ce-derblad

_{et al (1991 ) showing}

that induction of IFNa-mRNA in human NIP cells

_by

HSV

required

a

_prior

de novo

_protein

_synthesis.

Therefore,

the need for

_protein

_synthesis

prior

to IFNA _gene

_{transcription}

_{may be} a

general

feature for the induction of IFNa in NIP cells

_by

different viruses.

In studies

_designed

to

_analyze

the

com-plex

mechanisms involved in IFNa

induc-tion in NIP

cells,

Cederblad and Alm

(1990) suggested

the existence of cell

cooperations

and the

_{participation}

of solu-ble factors such as

_cytokines.

Thus,

IL3 and GM-CSF were found to increase and

accelerate HSV-induced IFNa

_production

(Cederblad

and

Alm,

1991).

In

addition,

conditioned

_media,

in which such

cyto-kines may be

_present,

as well as recombi-nant IL3 or

GM-CSF,

were shown to cir-cumvent the need for

_{protein synthesis}

in IFNa-mRNA

_{expression by}

CHX-treated

human

_leukocytes

_(Cederblad

et al,

1991

).

It was

_suggested

that NIP cells need

_help

to

_synthesize

IFNa;

accessory

cytokines,

either

_{synthesized prior}

to IFNA _gene

ex-pression

or

_provided

to CHX-treated

PBMC,

may activate cell adhesion mole-cules involved in the NIP cell interaction with viral structures

_(Cederblad

et

al,

1993).

In the

_{present report,}

we show that accessory

cytokines

may also be involved in IFNA induction

_{by TGEV,}

since

preincu-bation of PBMC with conditioned

medium

allowed the

_expression

of IFNa-mRNA

_by

CHX-treated

_porcine

PBMC

_(fig

2,

lanes 4 and

5;

fig

3,

lane

_4).

This effect

_persists

in the presence of

neutralizing anti-porcine

IFNa antibodies

_(fig

3,

lane

₆₎

_{and may} therefore be

_independent

of IFNa itself. In

our

_experiments,

active conditioned

medi-um were

_{prepared by incubating}

PBMC with Sf9 insect cells

_previously

shown to induce

_porcine

IFNA

_{(Etor6 et al, personal}

communication),

or with TGEV-infected cell

_monolayers.

IFNa-mRNA

_expression

by

CHX-treated

_porcine

PBMC was not due to conditioned medium alone but to

vi-rus induction

_(fig

4,

lanes 2 and

_3).

In a

_{previous study,}

we selected and

characterized 2 TGEV mutants for their low

_ability

to induce

IFNa,

compared

with the Purdue 115

strain,

both at the mRNA and

_protein

levels

_(Laude

et

_{al, 1992).}

second the actual

_{triggering signal}

provid-ed

_by

the virus itself.

_Experiments

were

conducted to determine whether addition of active conditioned medium could

nor-malize dm49-4

_{IFNa-inducing}

_properties.

Results

_(fig

₄₎

showed that

_{preincubation}

of PBMC with a conditioned medium al-lowed some

_expression

of IFNa-mRNA

_by

dm49-4

(lane 3),

but this

_expression

re-mained lower than that with the Purdue 115 virus. These results

_suggest

that the defect of the dm49-4 mutant is at the level of the final

_{triggering signal}

to NIP

cells,

possibly

due to

_inadequate

interactions be-tween dm49-4

_{non-glycosylated}

_protein

M

(Laude

et al,

1992)

and a

_{putative receptor}

(Lebon, 1985)

on NIP cells. In order to

un-derstand more

_precisely

how viral struc-tures can induce IFNa

_synthesis

in NIP

cells,

future studies will have to

concen-trate on the identification of a

_specialized

putative receptor

on these cells and the nature of its

_ligands.

ACKNOWLEDGMENTS

The authors thank C de Vaureix and M Godet

(INRA,

Jouy-en-Josas,

France) for

providing

anti-IFNa antiserum and for the

_preparation

of Sf9 cells,

respectively.

REFERENCES

Capobianchi

MR, Facchini J, Di Marco P, Anto-nelli G, Dianzani F ₍₁₉₈₅₎ Induction of

alpha

interferon

_by

membrane interaction between viral surface and

_peripheral

blood

mononu-clear cells. Proc Soc

Exp

Biol Med 178,

551-556

Capobianchi

MR, Ankel H,

Ameglio

F,

Paganelli

R, Pizzoli P, Dianzani F

(1992)

Recombinant

glycoprotein

120 of Human Immunodeficien-cy Virus is a _potent interferon inducer. AIDS Res Hum Retroviruses 8, 575-579

Cederblad B, Aim GV

₍₁₉₉₁₎

Interferons and the

colony stimulating

factors IL-3 and GM-CSF

enhance the IFNa response in human blood

leukocytes

induced

by herpes simplex

virus. Scand J Immunol 34, 549-556

Cederblad B, Aim GV

_{(1990) Infrequent}

but effi-cient

interferon-a-producing

human

mononu-clear

_leukocytes

induced

_{by Herpes Simplex}

Virus in vitro studied

by immunoplaque

and

limiting

dilution assays. J Interferon Res 10, 65-73

Cederblad B, Gobl AE, Aim GV ₍₁₉₉₁₎ The in-duction of IFN-a

and

(3 mRNA

in human natu-ral interferon

_{producing (NIP)}

blood

leuco-cytes

requires

de novo

_{protein synthesis.}

J Interferon Res 11, 371-377

Cederblad B,

Sandberg

K, Aim GA ₍₁₉₉₃₎ The

leukocyte

function-associated

antigen

(LFA-1) is involved in _{the interferon-a response} in-duced

by herpes simplex

virus in blood

leu-kocytes.

J Interferon Res 13, 203-208

Charley

B, Laude H (1988) Induction of

_alpha

in-terferon

_by

transmissible Gastroenteritis Co-ronavirus: role of transmembrane

glycopro-tein. E1. J Virol62, 8-11 1

Charley

B, Laude H

(1992)

Interactions

virus-lymphocytes pour la

production

d’interferon

a. Ann Rech Vet 23, 318-322

Chomczyncki

P, Sacchi N ₍₁₉₈₇₎

Single-step

method of RNA isolation

_by

acid

guani-dinium

_{thiocyanate-phenol-chloroform}

extrac-tion. Anal Biochem 162, 156-159

Ito Y, Aoki H, Kimura Y, Takano M, Shimokata K, Maeno K (1981) Natural

interferon-producting

cells in mice. Infect lmmun 31, 519-523

La Bonnardibre C, Laude H (1981)

High

interfer-on titer in newborn

pig

intestine

during

exper-imentally

induced virual enteritis. Infect

Im-mun 32, 28-31 1

Laude H,

Charley

B, Gelfi J _{(1984) Replication} of

enteropathogenic

coronavirus TGEV in swine alveolar

macrophages.

J Gen Virol 65,

327-332

Laude H, Gelfi J, Lavenant L,

Charley

B (1992)

Single

amino-acid

changes

in the viral

glyco-protein

M affect induction of

alpha

interferon

by

coronavirus TGEV. J _Virol66, 743-749 Laude H,

Chapsal

JM, Gelfi J, Labiau S,

Gros-claude J ₍₁₉₈₆₎

_Antigenic

structure of trans-missible

_{gastroenteritis}

virus. I.

_Properties

of monoclonal antibodies directed

against

virion

Lebon P ₍₁₉₈₅₎ Inhibition of

Herpes Simplex

vi-rus _type 1-induced interferon

synthesis by

monoclonal antibodies

_against

viral

glycopro-tein D and

_{by lysosomotropic drugs.}

J Gen

Virol66, 2781-2786

Lebon P,

Commoy-Chevalier

MJ, Robert-Galliot

B,

Chany

C

(1982)

Different mechanisms for

a

_and

_{(3 interferon}

induction.

_Virology

119, 504-507

Lefbvre F, L’Haridon R, Borras-Cuesta F, La Bonnardibre C

(1990)

Production,

purification

and

_{biological properties}

of an Escherichia coli-derived recombinant

porcine alpha

inter-feron. J Gen Virol 71, 1057-1063

Nowacki W,

Charley

B (1993) Enrichment of co-rona virus-induced

interferon-producing

blood

_leukocytes

increases the interferon

yield

per cell: a

_study

with

pig

leukocytes.