NS1 protein in patients with type C hepatitis

BIOCHEMICAL

Vol. 189, No. ‘1, 1992 November

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 565-571

30. 1992

DETECTION

OF ANTIBODY

TO HEPATITIS

C EZ/NSl

PROTEIN

IN PATIENTS

WITH TYPE C HEPATITIS Osamu Yokosuka, Yoshimi Masao Ohto, ar.d

First

Received

Chiba University School of Medicine, Inohana, Chuou-ku, Chiba, JAPAN (260)

Department

October

Ito, Fumio Imazeki, Masao Ornatal

23,

of

Medicine,

1992

SUMMARY: Putative E2/NSl sequence of hepatitis C virus was expressed in E. coli as a fusion protein with maltose binding protein. Approximately 80 kDa protein was obtained containing 38 kDa E2/NSl protein. The antibody to this protein was detectable in the same serum from which the sequence was amplified. It was also detectable in none of 7 acute hepatitis, in 2 of 12 in 3 of 25 chronic active hepatitis, and in 2 of chronic persistent hepatitis, 4 cirrhosis. It was detectable in none of 10 normal subjects. In 3 cases who were positive for the antibody before the interferon treatment, it became undetectable after the treatment. Thus, it seems that the antibody is not a 0 1992 neutralizing antibody and is related to active viral replication. Rcademic Press, Inc.

Hepatitis disease

C virus

and hepatocellular

molecularly

cloned

sequences

revealed

polyprotein, 5).

From

proteins (NS)

(HCV) is a major

and

(core

that

to

lies

immediately

(3-5). amplified

The

biological the

putative

were

viruses, near

them.

should lTo whom correspondence Med:clne, Faculty of Medicine, JsE‘an. Fax: 3-3814-0021.

of

supposed

protein

HCV E2/NSl

protein

is

and expressed

present 7-3-l

a large (1,3-

structural

end and 5 non-structural

protein

at

on HCV

encoding

to encode

envelope

be adciressed Tokyo University,

studies

was

and pestiviruses

the

sequence

liver

C virus

The

RNA virus

The glycosylated

of

E2/NSl

11,3-5).

amino-terminal

chronic

hepatitis

flaviviruses

HCV is

its

downstream role

to

of non-B

Recently,

stranded

related

these

agent

reported

HCV was single

and envelope) following

(1,2).

sequences

was distantly

proteins

protein

carcinoma

and the

similarity

etiologic

the

designated in the not

as EZ/NSl polyprotein

revealed

encoded

yet. protein

as a

address:Second Hongo, Funkyn-ku,

Department '?olq~o

:f

Copyright 0 1992 rights of reproduction

by Academic Press, Inc. in any form reservrd.

111,

0006-291X/92 565

All

We

$4.00

Vol.

BIOCHEMICAL

189, No. 1, 1992

fusion

protein

antibody

with

to

this

maltose

protein

AND BIOPHYSICAL

binding

protein

in patients

A

sample

serum

chronic

hepatitis

were

from

acids

were

reverse

virus

amino

acids

includes of

protein

start

from

acids

serum to

amino

acid

95% of

cDNA, then

384-702)(5).

Putative

end

protein

amplification

are

at is

(nucleotides

primer;

5'-ATGGTGGGGAACTGGGCTAA-3'

primer;

5'-TATAGGTATTGCACGTCCA-3'(nucleotides numbers

The amplified Engl

Biolabs,

was

transfected

clone

fusion

E. coli

the

vector

2 hours

in with

The

The strips room

Western

with

various

with

0.5%

liver tween

TBI

to blot

I

of 5 ul

was performed

diseases. 20 (TTBS),

acid

EZ/NSl

acid

the

inner

of

the

E.

then

coli

as described buffer

incubated

with

The filters

containing

incubated 566

so

The primers primer;

5'-

primer;

5'[sense

antisense acid

and

(41. p-mal-C

(New

inserted

(9).

The

DNA

E. coli

DNA was incubated the

EZ/NSl

protein

expressing

to

nitrocellulose

(101.

Briefly

diluted

washed with

(5,8),

with

as a E2/NSl filter

for

sera tris

at

as follows:

2 % gelatine

loo-fold were

729

DNA with

lysate

blotted

to

vector

amplified

to express

supposed

and

by ampicillin the

IPTG

et al.

The vector

selected

is

The amino

expression

(9).

319

primers

1429-1438) 2419-2437)l.

site

13 amino

[sense

and

by Kato

The sequence

sequence.

primers

C

and

protein

amino in

hepatitis

371-3831,

2535-255311

with

then

(amino

nucleic

364-3701,

antisense

12.5% SDS-PAGE,

then

acid

and

inserting

presence

treated

(amino

to

acids

(7).

1287-1306)

inserted

and

MBP. Then

of NCF were temperature,

Stu

correctly the

was applied

(NCF).

at

to

was

its

PCR method

outer

report

Nucleic

encoding

(nucleotides

on the

DNA segment

protein

protein

based

MA, USA) at

with

37OC for

are

with

sequence

included

(nucleotides

CTATCAGCAGCATCATCCA-3'

of

patient

The extracted

around

as follows:

female

(6).

by nested

site

E2/NSl

presence blot.

antibody.

the

1 protein

and

old

anti-C-loo-3

recongition

384

TGGGACATGATGATGAACTG-3'

nucleotide

a 53 years

was amplified

acid

C by western

as described

of envelope

(amino

the

of

peptidase

approximately for

for

364-702

signal

EZ/NSl

positive

transcribed

7 amino

acids

from

100~1

hepatitis

the

and Methods

obtained

who is

extracted

used

was

(MBP) and examined

with

Materials

RESEARCH CObdMUNICRTlONS

from buffered

biotinylated-anti-human

2 hours patients saline IgG,

Vol.

BIOCHEMICAL

189, No. 1, 1992

followed with

by incubation

TTBS between

detect

the

Patients

acute

each

signal

from

10

and

examined

(11).

were

liver

avidin-biotin

diamino

from

complex,

benzidine

samples treated

who are

washed

was performed

were

examined

the

from

positive

25 chronic

hepatitis,

which

for

sequence

3 patients

successfully

BCV RNA was detected kit

disease

subjects

serum

serum

who

by a commercial

with

persistent

normal

The

Serial

antibody

Reaction

with

12 chronic and

examined.

labelled

to

samples

antibody.

EZ/NSl

steps.

48 patients

hepatitis,

4 cirrhosis)

peroxidase

RESEARCH COMMUNICATIONS

(10).

and serum

Sera

with

AND BIOPHYSICAL

as reported

active

the

HCV RNA (7 hepatitis,

presence

of

was amplified who

by

for

were

(6).

was

positive

interferon

also

for

(IFN)

Anti-C-loo-3

anti-

were

the also

was detected

(2). Results

Expression By

of

HCV EZ/NSl

amplifying

approximately

the 1.0

ligating

the

sequence

as a fusion

EZ/NSl

protein

protein extracted

kilobase

sequence

to protein

nucleic

putative expression

from

EZ/NSl

sequence

vector

p-mal-c,

, approximately

and 42 KDa maltose

acid

binding

a was and

80 KDa protein protein

patient

serum,

obtained.

After

expressiny including

the 38 KDa

was obtained.

Fig.l. Detection of antibody against the EZ/Nsl protein expressed as a fusion protein with maltose binding protein. A: The serum from which this EZ/NSl protein was made. B: HCV RNA negative serum. Arrow indicates the Position of 80 KDa protein. 567

Vol.

189, No. 1, 1992 Table

BIOCHEMICAL

1. Prevalence

of anti-E2/NSl AH

positive

2

3

2

0

number

7

12

25

4

10

(%)

0

17

12

50

0

hepatitis, LC;

antibody

to

of

2 of

positive

positive

for

sequence

was

(Fig.1).

12 patients

for

anti-E2/NSl

the

after

detected

CAH$

(Table

antibody

before treatment

in

was also

patients.

None

chronic

hepatitis,

antibody

the

hepatitis,

It

with

active

the

normal

chronic

active

liver.

protein

chronic

anti-EZ/NSl

undetectable

persistent

HCV infection

antibody

this

the

the

HCV RNA positive

with

were

of

anti-EZ/NSl

with

patients

CPH; chronic

cirrhosis

of

amplification

the

anti-E2

LC

with

0

for

hepatitis,

in patients

CAR

Detection

patients

antibody

RESEARCH COMMUNICATIONS

CPH

AH; acute hepatitis,

The

AND BIOPHYSICAL

and

detected of

2 of None

In three

serum

in

7

used of

7 patients

4 patients of

with

with

were

positive

antibody

for became

2).

N. I(.

ZOY,O Y

CPM

IFN II NCV RNA +

+

*-

-

EZ--lb

+.

+z

+

+

--

-

\ 3.1

Fig.2a. with line

9b.r

Sequential analysis chronic persistent

indicates

of hepatitis

the movement

WV-RNA and who was

of ALT (IU/L). 568

0l.l

anti-E2/NSl treated

with

25

cirrhosis

subjects

who were the

acute 3 of

10 normal

treatment,

for

48 (15%)

hepatitis,

patients

interferon (Fig.

same

persistent

antibody. 1).

the

antibody in a patient IFN alpha-The solid

Vol.

189,

No.

BIOCHEMICAL

1, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNIC~ITIONS

12345676

Fig.Zb. Detection of anti-EZ/NSl antibody in serial serum samples from the patient. Lane 1; 12 months before the IFN treatment, lane 2; 6 months before the treatment, lane 3: just before the treatment, lane 4; at the end of the treatment (4 weeks after the beginning of the treatment), lane 5; 4 months after the end of treatment, lane 6: 10 months after the treatment, lane 7; 13 months after the treatment, lane 8; 18 months after the treatment. The 80 KDa EZ/NSI band indicated by an arrow gradually decreased after the IFN treatment. The fourth band which increased after the IFN treatment was considered to be a non-specific band as discussed in the text.

Discussion The

cooing

sequence

determined.

Because

acid

390 is

around

a ending

site

putative

structural

revealed

amino

includes

majority

expressed

of

the

in

relation

viral

the

protein the

(14).

to viral

membrane

component not

analogy

of

a component As

the

the

of

other

This

indicates

the

the

virion,

HCV EZ/NSl

HCV

that

is

probably of

(13). is

is

not

is 569

heavily

protein.

is

protein to elicit

glycosylated

of

this

during

formation. fully

the is

of

from

produced

produced

the to

we

in sera

protein

pestiviruses

believed

protein

EZ/NSl

in viral

related

The NSl

we expressed so the

was

the

proteins

disappearance

role

protein

E2/NS1

of

translated

EZ/NSl

729 is

study

to

protein

the

amino

around

antibody

The this

acid

antibody EZ/NSl

partially

peptidase,

sequence,

of

is

The protein

and pestiviruses,

it protein

(8).

of

that

The E2 protein membrane

of the

indicating

replication

viral

sequence

replication.

flaviviruses

(12).

of

presence

probably role

translation

detection

suggests

replication

Recent

site

the

signal

and amino

HCV E2/NSl

the

viral

treatment

during

viral

for

active

direct

reported

patients.

The biological From the

the

(5).

C virus

for

site

starting

revealed

by IFN

active

the

be applicable

to

antibody

and

hepatitis

sequences

protein

384 is

(95%) of

infected

of

of

to be a starting

sequence acid

protein

presence

HCV E2/NSl

Our examination chronically

EZ/NSl

supposed

for

must

of

understood.

production

of

organization supposed

the neutral

the of

to

flaviviruses

be a is

antibodies compared

to

Vol.

BIOCHEMICAL

189, No. 1, 1992

flaviviruses than

and

the

HCV E2/NSl

of

viral

The evidence with

chronic

antibody the

and

the

in

exsist

epitope

and

exists

However,

made

the

the

the

membrane

the virus

antibody. after

The evidence

(data

not

the as

However,

and clone

as used shown),

of

for so this

without lane

8 in

fourth

also

when

on

were

in

this

figure

clone

the

the

from

surface

viral

which

of

the

to

as

inside

in the

disappearance

not

a neutralizing increased

presence

plasmid side

virus

antibody.

but after

we

antibody

membrane

is

the

a neutralizing

of

p-mal-c

by side

same 42 KDa band

was considered

occur

presence

E2/NSl

the

examined

was might

So the

2b (42 KDa)

with

of

by changing

surface

reflecting

were 2b,

surface

could

not

increase

that

E. coli

figure

patients

protein

host. is

the

(13).

a neutralizing

same serum

the

not

fabor

plasmid

band

of

band

it

in

as a neutralizing

did

fourth if

situation

on the

a component

would

vaccine

in

regions

the

HCV and

virus

a role

on the

EZ/NSl

the

protein

antibody

not

the

antibody in

exist

the

exist

by the

of

the

play

antibody

functioning

not

the

treatment

antibody.

sequence

same serum

as

case,

this

E2/NSl not

may

that

intensity

IFN

neutralizing EZ/NSl

is

by IFN treatment The

the

antibody

epitope

that

of

hypervariable of

detected

of

exists

the

of

coexistence

of epitope

protein

pestiviruses, membrane.

The

is

not

attack

mean

for

neutralizing

immunological

pestiviruses

was detectable

does

epitope

of

to

might

antibody

is the

was also

protein.

the

the this

necessarily

absence

EZ/NS~

If

the

presence

presence

the

antibody

and that

the

(15).

HCV E2/NSl

antiqen

and

in the

not

EZ/NSl

indicates

If

regions

does

the the

sequence,

by escaping

of antibody

of

protein

be a candidate

that

However,

E2jrI.91

virus

to

C fabors

epitope(s)

in these the

antibody

similar

be a component

HCV E2/NSl

and could

RESEARCH COMMUNICATIONS

more

might

This

the

membrane.

reported

One.

that

is

protein

membrane

hepatitis

viral

that

profile

as in pestiviruses(l3).

organization

of

hydrophobicity

flaviviruses,

membrane

AND BIOPHYSICAL

with

a

with the

was detected

be a non-specific

one.

References Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. (1989) Science 244, 359-362. 2. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Honino F, Colonbo M, Lee WS, 1.

570

Vol.

i89,

No. 1, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M. (1989) Science 244, 362-264. 3. Houghton M, Choo QL, Kuo G.(1990) European patent application. Application number 90302866.0. 4. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. (1990) Proc Nat1 Acad Sci USA 81, 9524-9528. 5. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Ohnishi E, Andoh T, Yoshida I, Okayama H. (1991) J Virol 65, 1105-1113. K, Ohto M.(1990) J Clin Invest 86, 6. Kato N, Yokosuka 0, Omata M, Hosoda 1764-1767. 7. Hosoda K, Omata M, Yokosuka 0, Kato N, Ohto M. (1992) Gastroenterology 102, 1039-1043, 1992. 8. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Shimotohno K. (1991) Proc Nat1 Acad Sci USA 88, 5547-5551. 9. Guan C, Li P, Riggs PD, Inouye H. (1988) Gene 67, 21-30. lO.Yokosuka 0, Omata M, Ito Y. (1988) Virology 167, 82-86. 11. Omata M, Ito Y, Yokosuka 0, Imazeki F, Uchiumi K, Takano S, Hosoda K, Ohto M.(1989) Dig Dis Sci 34, 330-337. 12. Spaete RR, Alexander D. Rugroden ME, Choo QL, Berger K, Crawford K, Kuo C, Leng S, Lee C, Ralston R, Thudium K, Tung JW, Kuo G, Houghton M.(1992) Virology 188, 819-830. 13.Collett MS, Larson R, Belzer SK, REtzel E. (1988) Virology 165, 200-208. 14Schlesinger JJ, Brandriss MW. Walsh EE. (1985) J Immunol 135, 2805-2809. 15. Weiner AJ, Brauer MJ, Rosenblatt J, Richman KH, Tung J, Crawford K, Bonino F, Saracco G, Choo QL, Houghton M, Han JH. (1991) Virology 180, 842-848.

571