Vol.

176,

No.

May

15,

1991

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

FULL-LENGTH X PROTEIN

985-992

AND TRUNCATED VERSIONS OF THE HEPATITIS B VIRUS (HBV) (pX) TRANSACTIVATE THE cMYC PROTOONCOGENE AT THE TRANSCRIPTIONAL LEVEL

Clara Balsano*, Maria Laura Avantaggiati* ,Gioacchino Natoli*, Elisabetta De Marzio*, Hans WillA, Michel PerricaudetO,and Massimo Levrero*l * I Clinica Policlinico

Medica and Fondazione Umberto I - 00161

^Max D-8033

Planck Institut Martinsried

bei

Andrea Cesalpino Rome - Italy

fur Biochemie Munchen - FRG

o Laboratoire de Genetique des Virus Institut Gustave Roussy, 94800 Villejuif,

Oncogenes France

Received March 1, 1991 The products of the human hepatitis B virus (HBV) and woodchuck hepatitis B virus X genes (pXs) transactivate homologous and heterologous genes including the HBV-x and core promoters, the human immunodeficiency viruses 1 (HIV-l) and 2 (HIV-2) long terminal repeats and the beta interferon regulatory sequences. We report here that pX is also able to influence the expression of both extrachromosomal transfected c-myc regulatory sequences and endogenous c-myc gene. px acts by increasing transcription of the c-myc gene and do not affect c-myc mRNAs stability. The presence of the first AUG of the X-ORFs is indeed necessary for the production of an active px. The very carboxyterminus of the pX protein is dispensable for this transactivating activity and at least one domain important for its action is located between aminoacids 103 and 117. 0 1991Academic Press, 1°C.

Several of

epidemiological

primary

studies

hepatocellular

HBV infection

(1,2).

carcinoma Integration

been

frequently

observed

with

chromosomal

deletions

few cases identified 1

of

human

have

in

PHC the

as a gene

involved

correlated (PHC)

of human

the

to

the

development

persistence

HBV-DNA into

cellular

PHC and

been

and translocations target

of

in

the

has

(3,4).

HBV-DNA

insertion

control

of

cell

of

DNA has associated Although

in

has

been

growth

and

To whom correspondence should be addressed at Iaboratorio di Espressione Umberto I, Genica, Fondazione A. Cesalpino, I Clinica Medica, Policlinico Viale de1 Policlinico, 00161 Roma, Italy. 0006-291X/91

985

$1.50

Vol.

176,

No.

3, 1991

BIOCHEMICAL

differentiation common

(5,6),

sites

proteins,

of

viral

usually

core

antigen,

any,

these

have known.

contain,

in

addition

protein,

the

envelope

open

reading

(7,8,9,10). under

pX is

the RSV,

During

productive

acting

upon

required the

viral

infected activity during

of

autologous

HIV1

and

in

the

reported

transcriptional

that

saturation

the

cellular

(18,

levels

21).

expression

in In of

from

increase

X protein

of

X gene its

integration,

genes

19,

culture this the

and

are

in

in

the

product expression

HBV genome

NIH-3T3

and

HBV mRNAs

of

(18,

viral

sequences.

of

if

pX

genes

other

this

verify

px expressing

density

of

supplied

spectrum

after

capsid another

regulatory

effect

to

genomes

termed

as

Although

major

broad

or

important

mice

the

well

the

(11).

suggested

replication,

activate

augments

replication

hepatocytes

nude

X protein

HBV genome

viral

other

the

the

expression

as

(12-17)

of

major

a protein

the

HIV2)

if

polymerase,

encodes

(11)1

role,

B virus the

viral

stimulating

the

conversion

encoding

that

no

Viral

which

hepatitis

genes

infection

the

has been

pXs

of

is

an increased

the

capable

transcription

viral

tumors

(ORF)

observed.

but

malignant

and the

frame

on transcription

influence it

the

cases

and infrequently

cells,

mammalian

COMMUNICATIONS

analyzed

been

products

the

proteins

HTLV-1,

for

All

the

have

tumor

in

to

control

(SV40,

in

RESEARCH

of

gene

expressed

not

majority

envelope

proteins is

the

BIOPHYSICAL

DNA integration

the are

hepatocyte

long

in

AND

could

20).

Recently,

cells

displays

able

paper

we demonstrate

c-myc

protooncogene

to

form that at the

level. MATERIALS

AND METHODS

Plasmids The pX expression vectors, pMLP-X and pSV-X, contain the entire X-ORF preceded by the adenovirus type 2 major late promoter (MLP) (12) and the SV40 enhancer/early promoter element (22). The introduction of a translational frameshift mutation in position 1402 generated pMLP-X(FS) and pSV-X(FS) plasmids. To obtain the plasmids pMLP-X(de1) and pSV-X(del), plasmids pMLP-X and pSV-X were digested by NcoI and subsequently treated by Mung Bean

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

nuclease. The elimination of the first potential initiation codon of the X-ORF was confirmed by sequencing. pMLP-WHX contains the entire WHV X-ORF (coordinates 1503 to 2190, according to Galibert et al. (1982)) under the control of the adenoviral MLP. Its derivative pMLP-WHX(de1) lacks the first ATG of the WHV X-ORF (coordinates 1742 to 2190). The pMLP-X(FI), pMLP-X(D1) and pMLPX(HI1) plasmids contain the adenovirus major late promoter (MLP) and the X-ORF deleted of 36 bp, 111 bp, and 153 bp respectively at their 3' end, followed by a fragment containing 75 nucleotides of the CAT gene, the SV40 small t intron and the SV40 early region polyadenilation site (12). In pSV-CAT the CAT gene is under the control of the Sv40 enhancer/early promoter element pRSV-CAT contains the Rous sarcoma virus (RSV) LTR (23). sequences from PvuII to Hind111 upstream of the CAT gene (22). In pE4-CAT and pHBV-CAT the CAT gene is preceded by the early adenovirus E4 enhancer/promoter sequences (positions -329 to +32) or the HBV enhancer linked to the X gene promoter (coordinates 991 to 1402, according to Galibert et al. 1979 (7). In pMyc-CAT a 2.9 kb HindIII-PstI fragment containing the 5' flanking sequences, the promoter and a nearly complete 1st exon of the human c-myc gene, has been inserted into the CAT vector pHP34CAT. Tissue Culture, DNA mediated transfer and transcription assays. Murine cells, human NIH-3T3 fibroblasts, human HeLa hepatoblastoma HepG2 cells and' the adenovirus trasformed human 293 cells were grown in Dulbecco's modified Eagle‘s medium supplemented with 10% fetal bovine serum. Cells at 70% confluence technique were transfected by calcium phosphate coprecipitation and exposed to the precipitate for 12-16 hr. When less than 20 ug of specific DNA was used per lo-cm culture disk, pUC19 plasmid DNA was added to give 20 ug of total DNA. After DNA transfection the cells were harvested and CAT assay nuclear run-on assay (24) and RNAs stability assay (25) (23), were performed as described.

RESULTS The

results

1)

and

of

cotransfection

Hela

cells

transactivation viral kd the

X

(data

potential

regulatory X protein

of

since

a complete

a full

pX

(data

properties initiation

(Table codon

of

shown),

protein The

the

and

and was the

not

the

abolished

elimination in 987

the similar

of the

cells

(table

the

broad

the

plasmids

heterologous due

to

the

aminoterminus

(FS),

but

displays

X-ORFs

in

pSV-X

messanger

not

I).

autologous

mutation

(FS)

of

WHV-X

on

HepG2 confirm

shown)

pX

a frameshift pMLP-X

The

in

The transactivation

transcription

phenomenon.

not

sequences.

(plasmids

length

experiments

allowing translation

17 of the of

transactivation transactivating first

potential

pMLP-X(del)

and

Vol.

176,

No.

Table

I.

HepGX

BIOCHEMICAL

3, 1991

Transactivation

of by

AND

viral tiBV-X

pMLP-X

pMLP-X(FS)

5.1t0.6 7.5+0.y 8.5t0.7 8.8t1.2

0.Yt0.2 1.1to.3 0.8tO.2 1.1to.4

8.Y2O.Y

U.8kO.3

BIOPHYSICAL

and

RESEARCH

cell111ar

COMMUNICATIONS

regulatory

sequences

pMLP-WHV

pMLP-X(del)

protein psv-x

cells

pSV2-CAT pRSV-CA1 p&-CAT pHBV-CA1 pMyc-CA'I

6.5?0.7

7.3to.5 6.3t0.3 7.5to.7 8.3t0.5 8.7t0.3

6.1+0.x 7.1to.3 8.0t1.1 6.8tl.o

1.0+0.1

0.8fO.3 1.0+0.1 1.1to.3

1.220.4

HepGX cells were cotransfected with the different CAT vectors and pMLP-S(FSl, p>b-x ( pMLP-WHX and pMLPeither pMLPl1 (12) or pMLl'-X, phosphate method. Cells were Xldel) vectors usinn the calcium harvested 48 hours post-transfection and CAT activity in cell extracts measured as described in the Materials and Methods. The was Figures experiments were performed in the linear range of the assay. ratio between the percentage of conversion in represent the percentage of with the pX expression vectors to the cotransfection Fleures are the conversion obtained with the control pMLPI1 plasmid. separate experiments mean t st.andard deviation (SD) from at least 3 Ilerformed Ilsinq at least 2 separate plasmid preparations.

pMLP-WHX(de1) proteins

abolished (Table

To

evaluate

the

pSV-X

and

293

and

Hela, CAT.

the

not

effects

px on the

of

pMLP-X

NIH-3T3

cells

sequences

endogenous

c-myc

the

product,

X gene

Vectors

in

increase

in

results

were

find

out

on

analysis

whether of

the

to

be

the

expression

by

mechanisms

of

action

(Table

as

the 988

influenced

by

pX expression

(Figure

la).

not

shown).

NIH-3T3

were

and

(20)(Fig.

induction

cannot

exclude

anti-termination

293

lb).

cells the These

rate of

to run

using

transcriptional

the

order

nuclear

performed

al

strong Similar

In

transcription,

et

we

However,

The

mRNAs

in

in

the

c-myc

I).

a very

increased

event

be

pMyc-

of

induced

c-myc

an

such

increase

of

HepG2,

plasmids

can

(data

Schibler

in

cells

plasmid

pMLP-X

primary

strong

transcripts

that

HBV-X.

cells

increases

by

reveal

recombinant

transfection

c-myc

c-myc

pX

sequences,

cotransfected

expression

NIH-3T3

293

the

both

regulatory

expression

the

in

described

experiments

CAT

state

HBV-X

by

conditions

a

mouse

obtained

transfected

induced

proto-oncogene

steady

were the

driven

quiescent

c-myc

with

since

from

shown).

plasmids

expression

regulatory

transactivation

I and data

the

X gene

likely

the

c-myc

is gene

alternative of

pre-

Vol.

176,

No.

3, 1991

a1

BIOCHEMICAL

2

AND

3

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

b A

B

+

+

pBR322

“_

C-MYC ACT -24

c

A .

MYC

ACT C.MYC

40

0

0

40

Figure 1 a. The HBV-X protein induces an increase in steady state c-myc mRNA levels. NIH-3T3 cells were transfected with either pUC18 (lane l), pMLP-X (lane 2) or pMLP-X(FS) (lane 3) plasmids. The Northern blot was first hybridized with a probe specific for the second and the third exon of murine c-myc gene (a gift of P. Briand, Hop. Necker, Paris) and then stripped and rehybridized with a beta-actin probe. b. Nuclear run-on analysis of c-myc gene in NIH-3T3 (A) and 293 (B) cells transfected with eitper pMLP-X (+) or pMLPl1 (-). Nuclei were prepared from 2x10 cells and the elongation of nascent CAT chdins has been performed using the itions c03~ described bv Schibler et al (24) in oresence of P-UTP. Deproteinated RNA was then hybridized to nylon filters carrying dot spots of 5 ug alkaly denaturated DNA from pBR322, murine Cmyc (A) or human c-myc (B) (a gift from M.A. Buendia, Institut Pasteur, Paris) and beta-actin. c. Actinomycin D chase of c-myc and beta-actin mRNAsfrom NIH-3T3 cells transfected with either pMLPl1 (left) or pMLP-X (right). 36 hours after transfection cells were incubated further with 5 ug of actinomycin D per ml for the indicated times. RNAs were prepared and analyzed by Northern blot as described above.

initiated

transcripts.

regulated

by modulation

performed

RNAs stability

technique

(21).

From these

does not

increase

protein

cause accumulation In

order

to

transactivating constructed. targets

Since

of

of

HBV-X

half

c-myc

mRNAs level life

using

c-myc

it

the

be

systems,

actinomycin is

to

we

D chase

evident

that

the

X

and does not

lc).

deletion

experiments

of

the

mutants

were performed the

989

known

several

localization

carboxytermini

transactivation,

is

mRNAs stability

mRNAs (Fig. to

in the

experiments the

Cotransfection for

its assays

contribute domain,

c-myc

pMyc-CAT

and

pX

the

have been using,

as

pHBV-CAT

Vol.

176,

No.

Table

HepG2

3, 1991

BIOCHEMICAL

11.

of

Effects

HBV-X regulatory

BIOPHYSICAL

RESEARCH

deletion mutants sequences

on

pMI,P-XI

COMMUNICATIONS

c-MYC

and

U1 )

HB\,

pML?-x

pMLP-X(FI)

pMLP-X(HT1)

7.1io.9 8.821.2

7.920.3 7.550.4

7.420.8 8.1+1.0

1.250.2 1.1+0.2

ti.3kl.O 4.521.1

6.820.2 4.921.1

6.1tO.3 3.9f0.7

1.3iO.l 1.5to.4

cells

pMY(‘-C:Al pHBV-CAT HeLa

cells

pMYC-CA'I pHBV-CAT HepG2

and HBV-CAT

Or

deletion Table

HeLa cells were cotransfected and 5 ug of either pMLPl1 plasmids using the calcium

with either 5 ug of or pMLP-X or the various phosphate method. Methods:

pMYC-CAT HBV-X See

r.

plasmids.

As

(pMLP-X(F1)

detailed or

are

to

the

(

deletion

pMLP-X(D1)) are

their

integration

X-ORF in

its

).

12

retain

lost

their

when additional

The

capability

of

transactivational

properties

by the

of

sequencing

process

3'

lacking

still

indeed

as demonstrated the

mutants

pMLP-X(HI1)

retain

HBV integrates, of

(

which

removed

since,

interruption

II,

aminoacids

X proteins

noteworthy

cloned

Table

properties,

14 aminoacids truncated

in

37

transactivating

is

AND

often

several

leads

to

the

end (2).

DISCUSSION We show in

this

paper

that

as a transcriptional and heterologous to

influence

the

transactivator viral the

c-myc

property

is

as demonstrated

in

proteins,

This

study

both

HBV and WHV pX proteins. pX proteins

beginning positioned

at

the

our

characterize

could

but

by the

and other the

or by translation the 990

other

acts

autologous

is

also

able

of

both

and endogenous GSHV pXs

laboratories

(11).

strategy

from

only

WHV and the

of

From a theoretical

within

it

sequences

be made either

X promoter

internally

shared

of

activity

regulatory

this

the

we also

sequences

transcriptional

transfected

not

on a variety

regulatory

extrachromosomal gene.

HBV X gene product

expression point

a minor of

the

known

of

In of

view,

transcript X sequences genomic

or

Vol.

176,

No.

3, 1991

subgenomic the

BIOCHEMICAL

mRNAs.

first

Moreover,

AUG of

Alternatively,

the pX

frameshift

from

consistent

with

required

for

not

obvious

not

and is either

to

or

covalently

factors Alternatively,

mediate

factors.

The

demonstation

responsive

domain

element

transcription the

in

regulatory On the

other

hand,

it

an intrinsic

might

activate

Whatever

the

truncated

pX proteins

integration

involved

in

mechanism, to

expression into

differentiation truncated

HBV

contribute

to the

envelope multistep

or

catalizing

of at

the

represent in

991

the

pX

pX contain

a

directly

first

with specific

a

hypothesis.

reported

that

pX

activity

(27)

and

of both

of

transcription. full

length

transcriptional

control

(28),

process

to

phosphorilation

transactivation proteins

that

an additional the

fused

activate

the

kinase

ability

pX

to

recently

regulation

involved

6)

to

interact

the

activate

could

genes (5,

the

of

targeted

beeen

by

transcription

protein

to

factors.

unidentified,

nuclear

support

pX could

such

so far

domain

transcription

factors

of

suggests

has

pX

transcription

HBV enhancer

when

that

sequence

factors.

able

able

do

suggests

more

serine/threonine

cellular

gene

is

and would

(26),

is

which

regulatory

of

C-EBP

are

AUG codon

of promoters

a hybrid

machinery

sequence

displays

c-myc

the

activating

transcription

or

activation

of

codon.

results

individual

one

that

at

a ribosomal

first

on additional,

pX

begin

X protein.

with

act

the

transcription

modify

that

and

a specific

complexes

pX could

DNA-binding

Our

sequences

multiple

proteins

the

P frames.

a variety

with

influence stable

by

regulatory

directly

form

synthesized

of an active

common

might initiation

model

stimulate

COMMUNICATIONS

a downstream

be

simplest

RESEARCH

initiation

overlapping

HBV-X to

interact

able

even

synthesis

of

does

or at

might

the

The ability

BIOPHYSICAL

translation

X-ORFs

the

the

possess

AND

of hepatic

level way,

of

cell

of

cellular

by

which

and

besides

growth

and

genes HBV

carcinogenesis.

by

could

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS the from: This work was supported by grants C.N.R.S.; Cesalpino, Rome, Italy; the Clara Balsano Forschungsgemeinschaft (Wi 664-2). a short term EMBO fellowship.

Fondazione Andrea Deutsche the was supported by

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R.P.,

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