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
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
