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J Virol, January 1998, p. 868-872, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Hepatitis G Virus Encodes Protease Activities Which Can Effect
Processing of the Virus Putative Nonstructural Proteins
Alexander S.
Belyaev,1,*
Susan
Chong,1
Alexander
Novikov,1
Ana
Kongpachith,1
Frank R.
Masiarz,2,3
Moon
Lim,1 and
Jungsuh P.
Kim1
Genelabs Technologies, Inc., Redwood City,
California 940631;
Protein Structure
Analysis Group, Chiron Corporation, Emeryville, California
94608-29162; and
Department of
Pharmaceutical Chemistry, University of California, San Francisco,
San Francisco, California 941433
Received 23 May 1997/Accepted 20 September 1997
 |
ABSTRACT |
The genome of a recently identified virus, hepatitis G virus (HGV),
shows considerable homology to hepatitis C virus (HCV). Two HGV
proteases similar to nonstructural proteins NS2 and NS3 of HCV were
identified, and their cleavage site specificity was investigated. Amino
acids essential for the protease activities were determined by mutation
analysis. NS4A of HGV was demonstrated to be a cofactor for
NS3-mediated proteolysis, with a region critical for activity residing
between Leu1561 and Ala1598.
| |
TEXT |
Hepatitis G virus (HGV) is a new
transfusion-transmissible agent identified in the sera of hepatitis
patients (18). Recently, two nonhuman viruses, GBV-A and
GBV-B, and a human virus, GBV-C, were detected (26, 28).
Comparison of the polyprotein amino acid sequence from the HGV
isolates with that from GBV-C showed that these viruses are nearly
identical (33); therefore, the HGV nomenclature will be used
throughout this paper.
The HGV genome is a 9.4-kb positive-stranded RNA molecule which
contains an open reading frame (ORF) encoding a 2,900-amino-acid (aa)
precursor polyprotein. The polyprotein sequence resembles those of
viruses in the Flaviviridae family, especially hepatitis C
virus (HCV), with which it shares 25.5% overall identity at the amino
acid level. Regions of conservation cover the whole nonstructural
region of the polyprotein, reaching 40 to 60% identity in parts of the
putative NS3 and NS5B regions (18). Therefore, the putative
HGV nonstructural region is expected to be organized similarly to
the NS2-NS3-NS4A-NS4B-NS5A-NS5B structure of HCV (22). The HGV polyprotein contains consensus sequences
typical of the Flaviviridae family, such as a putative
serine protease and helicase in the NS3 region as well as an
RNA-dependent RNA polymerase in the NS5B region. It also contains an
NS2 protease motif, characteristic of HCV (18). In HCV, NS2,
NS3, and NS4A activities are responsible for the proteolytic processing
of the nonstructural region of the polyprotein. NS3 of HCV is a
chymotrypsin-like serine protease responsible for cleavages at
NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B sites (1, 7, 9, 16,
20, 24). Processing at these sites is abolished when
Ser1165 in the catalytic site of the NS3 is mutated to
alanine (7, 30). NS4A of HCV is a cofactor required for the
NS3-mediated cleavages at the NS3/NS4A, NS4A/NS4B, and NS4B/NS5A sites,
but it is not obligatory for the NS5A/NS5B cleavage (2, 3, 13, 16, 19, 21, 27, 29). Proteolytic activity located in the NS2 region
of HCV is required for the NS2/NS3 cleavage (8, 9). His952 and Cys993 (numbered as in HCV-H
sequence) were shown to be essential for the NS2 activity
(8).
In these studies, we used the recombinant baculovirus expression system
to study the proteolytic processing of the HGV polyprotein. Using
site-directed mutagenesis of the putative HGV protease motifs and
deletion mapping, we demonstrate that HGV polyprotein encodes protease
activities which are functionally similar to the NS2, NS3, and NS4A
activities of HCV.
Construction of recombinant baculoviruses.
Plasmid p3ZHGV-6,
encoding the entire HGV (PNF2161) polyprotein (18a), was
used as a cloning source of the HGV genes, either as DNA restriction
fragments or as a template for PCRs. Portions of the HGV genome were
cloned into baculovirus plasmid transfer vectors pAcG1 and pAcG3X
(5). These vectors contain the glutathione S-transferase (GST) gene, allowing the expression of HGV
genes as amino-terminal fusion proteins under the control of the very late baculovirus polyhedrin promoter. The
BglII-StuI fragment coding for the
Ile806-Glu1658 region of the HGV polyprotein
was cloned into the BamHI-SmaI-cut pAcG3X vector,
which enabled expression of the part of the HGV polyprotein containing
the NS2 and NS3 protease motifs and a putative NS4A region. Amino acids
are numbered from the first methionine encoded by AUG461 in
the polyprotein ORF of HGV (PNF2161). This construct was designated P.
Mutations at the NS2 and NS3 protease motifs were made by
oligonucleotide-directed mutagenesis (15).
Oligonucleotides for the His849-to-Tyr and
Cys890-to-Leu mutations in the NS2 protease motif were
5'-CGAATAAACAAGCTAATATACCCGAG and
5'-CCTGAAACAGGAATCCCGTCACGCAG, respectively.
Nucleotides which differ from those of the HGV (PNF2161) strain are
underlined. Amino acids are numbered from the first methionine of the
long ORF encoded by AUG461 in the HGV (PNF2161) genome
(18). Oligonucleotide
5'-GCACCGAGCCGACCGAGTGG was used to mutate
Ser1062 to Ala in the NS3 serine protease putative active site. In addition, some of the constructs were provided with a carboxy-terminal tag, derived from herpes simplex virus (HSV) glycoprotein D. This allowed the carboxy-terminal part of the HGV
polyprotein encoded in these constructs to be monitored during the
polyprotein processing studies. A synthetic EcoRI DNA
fragment encoding the 11-aa Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu tag was cloned into the unique EcoRI site of the
NS2-NS3-NS4A-expressing constructs in frame with the carboxy terminus
of the HGV polyprotein fragment. Constructs P23 and P234 were derived
by making carboxy-terminal truncations of the NS4 region in the P
construct. Construct P23 was largely devoid of the putative NS4
sequence and encoded the Ile806-Arg1550 segment
of the HGV polyprotein containing the NS2 and NS3 sequences. Construct
P234 spanned the beginning of the NS4 region and encoded the
Ile806-Ala1598 segment of the HGV polyprotein. Constructs 4, 41, and 42 encoded the
Arg1550-Lys1655,
Leu1561-Lys1655, and
Leu1576-Lys1655 portions of the HGV
polyprotein, respectively. They were derived by cloning PCR fragments
from the putative NS4 region into the pAcG1 vector.
Construct 45 encodes an Asp
1806-Lys
2235 segment
of the HGV polyprotein, which includes the putative NS4B/NS5A junction.
It
was obtained by ligating a
HindIII-
BglII
fragment of the p3ZHGV-6
plasmid into the pAcG1 vector. Construct 55 encodes a part of
the HGV polyprotein containing the putative NS5A/NS5B
junction.
Plasmid p3ZHGV-6 was digested with the
Eco47.III
and
EcoRI restriction
endonucleases. The
Eco47.III-
EcoRI fragment encoding the
Arg
2078-Gly
2873 segment of the HGV polyprotein
was ligated into the
EcoRI- and
SmaI-digested
pAcG1 vector.
The plasmid transfer vectors containing the HGV genes were
cotransfected with the BaculoGold linearized baculovirus DNA
(PharMingen,
San Diego, Calif.) according to the manufacturer's
instructions.
The resultant white plaques were selected and purified by
two
sequential plaque assays (
11,
12).
NS2 mediates cleavage at the NS2/NS3 junction.
In order to
elucidate the role of the putative NS2 protease active site in the HGV
polyprotein cleavage, Sf21 insect (Spodoptera frugiperda)
cells were infected either with the NS2-NS3-NS4A construct containing
the wild-type NS2 putative protease (P) or with constructs carrying
mutations of His849 or Cys890 in the NS2 active
site (PH or PC) or with a construct carrying a Ser1062
mutation in the NS3 serine protease active site (PS). Cells were
harvested at 60 h postinfection, and cell proteins were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with rabbit polyclonal antibodies raised against the NS2 (Ser872-Ser889) or
NS3 (Gln1488-Pro1504) peptides (Fig.
1). GST-NS2 was completely
cleaved from NS3 in the P construct, because GST-NS2 was the only
product reacting with anti-NS2 antibodies, and there was no
protein-associated reactivity at a higher molecular mass (Fig. 1C).
Analysis of the same lysate with anti-NS3
(Gln1488-Pro1504) antibodies indicated a major
product with an apparent molecular mass of 70 kDa, which is consistent
with the predicted molecular mass of NS3 (Fig. 1D). Mutation of the NS3
serine protease motif in the PS construct did not significantly affect
NS2/NS3 cleavage, because the release of the GST-NS2 product was
comparable in both P- and PS-infected cell lysates (Fig. 1C and D).
However, mutations of the NS2 putative active site in the PH and the PC
constructs were found to have a profound effect on this cleavage. Free
GST-NS2B and -NS3 products were not observed on immunoblots of PH- and
the PC-infected cell lysates, indicating that NS2 was the major factor
responsible for the NS2/NS3 cleavage (Fig. 1C and D).
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FIG. 1.
Western blot analysis of proteolytic processing of the
NS2B-NS4A polyprotein expressed by recombinant baculoviruses. S. frugiperda SF21 cells were infected at a multiplicity of 5 PFU per
cell with recombinant baculoviruses or wild-type (wt) Autographa
californica nuclear polyhedrosis virus. Cells were harvested at
60 h postinfection. Proteins were separated by SDS-PAGE,
electroblotted onto nitrocellulose membranes, and probed with either
rabbit antisera raised to HGV synthetic peptides or with the monoclonal
anti-HSV tag antibodies. (A) Schematic representation of constructs.
(B) Mutations of HGV protease motifs. Phe154 of the HCV
substrate specificity pocket is indicated by an asterisk and numbered
from the N-terminal residue of NS3. (C) Western blot analysis of
NS2B/NS4A polyprotein cleavage with an anti-NS2
antibody. Samples were separated on an SDS-PAGE gel (12%
polyacrylamide). (D) Western blot analysis of NS2B/NS4A polyprotein
cleavage with anti-NS3 polyclonal antibody. Samples were separated on
an SDS-PAGE gel (12% polyacrylamide). (E) Western blot analysis of
NS2B/NS4A polyprotein cleavage with anti-HSV tag antibody. Samples were
separated by SDS-PAGE (18% polyacrylamide) and probed with anti-HSV
tag monoclonal antibody. MW, molecular mass markers (kilodaltons).
|
|
NS3 mediates cleavages at the NS3/NS4, NS4/NS5A, and NS5A/NS5B
junctions.
Cleavage at the NS3/NS4 junction was studied with the
PT and PST constructs. These constructs expressed the NS2-NS3-NS4
portion of HGV polyprotein containing an easily detectable HSV tag at the carboxy terminus of NS4. Consequently, release of NS4 from the
polyprotein could be studied with commercially available HSV tag
monoclonal antibodies (Novagen, Madison, Wis.). The PT construct expressed wild-type NS3, while the PST construct expressed NS3 with a
Ser1062 mutation in the putative serine protease active site. Cleaved NS4 product with a molecular mass of 15 kDa was observed
when the cells were infected with the PT construct, whereas no cleaved
NS4 product was detected on immunoblots of the cell lysates infected
with the PST construct containing a mutated NS3 serine protease
motif (Fig. 1E). This finding indicates that the NS3 serine protease
was essential for the NS3/NS4 cleavage. In the HCV polyprotein,
NS4A/NS4B cleavage occurs 54 aa downstream of the NS3/NS4
cleavage. This cleavage event was not observed in our experiments with
HGV. The size of the cleavage product containing the HSV tag
corresponded to the size of the intact NS4 portion expressed in cells
infected with the construct PT (Fig. 1E). However, the PT construct
contains an incomplete putative NS4B; therefore the possibility that
the intact NS4B region is required for this cleavage has not been ruled
out.
NS3-mediated cleavage at the NS4B/NS5A and NS5A/NS5B junctions was
studied in
trans and not in
cis, because
cytotoxicity associated
with the middle part of the NS4 region
precluded efficient expression
of the NS3-NS4-NS5 constructs. For this,
constructs 45 and 55
containing NS4B/NS5A and NS5A/NS5B junctions,
respectively, were
coinfected with constructs expressing wild-type or
mutated NS3
and NS2B, with or without NS4A. As in HCV, these cleavages
were
found to be dependent on the NS3 serine protease activity, but
not
on the NS2 protease activity. Mutations in the NS2 protease
active site
(constructs PC and PH) or deletion of the NS4A region
(construct P23)
did not impair NS5A/NS5B cleavage (Fig.
2B). Therefore,
the NS3 protease was
sufficient in mediating NS5A/NS5B cleavage.
However, as in HCV,
NS4B/NS5A cleavage required expression of
both NS3 and NS4A. Deletion
of the NS4A region (construct P23)
abolished the cleavage, but this was
restored when construct P23
was coinfected with construct P4, which
expresses the NS4A region
(Fig.
2C).
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FIG. 2.
Analysis of cleavage at the NS4B/NS5A and NS5A/NS5B
sites. (A) Schematic representation of the recombinant baculovirus
constructs. NS4A sequences are shown and compared with those of HCV-H
NS4A. Borders of the putative NS4A region are indicated by arrows.
Identical amino acids are underlined. HCV-H NS4A (17) and
HGV activity domains are indicated inside shaded rectangles. (B)
Western blot analysis of NS5A/NS5B cleavage products. S. frugiperda SF21 cells were infected, at a multiplicity of 5 PFU
per cell, with each type of recombinant baculovirus or wild-type
Autographa californica nuclear polyhedrosis virus (wtAcNPV).
Cells were harvested at 60 h postinfection. Proteins containing
the GST tag were purified by affinity chromatography on
glutathione-Sepharose, separated by SDS-PAGE (12% polyacrylamide), and
electroblotted onto nitrocellulose membranes, which were reacted with
rabbit anti-GST tag antibodies (Sierra Biosource). (C) Western blot
analysis of NS4B/NS5A cleavage. Cells were infected with recombinant
baculoviruses, and proteins containing the GST tag were purified and
reacted with NS4B(Ala1884-Tyr1899) antibodies.
MW, molecular mass markers (kilodaltons).
|
|
Identification of the NS4A active region.
In the HCV
polyprotein, the NS3 protease is followed by NS4A, which serves as
a cofactor for the NS3-mediated cleavages (22). Identification of the NS4A active region was performed by making truncations of the NS4 region in constructs P and 4. Construct P
encoded the NS2, NS3, and NS4 portions of the HGV polyprotein, and construct 4 encoded only the NS4 portion. The activity of the
truncated constructs P234, 41, and 42 was investigated by determining
the ability of these constructs to complement the NS3-mediated
cleavage at the NS4B/NS5A junction following coinfection with construct
45 (Fig. 2A and C). Cells were harvested at 60 h postinfection,
and the GST-tagged proteins were purified by affinity chromatography on
glutathione-Sepharose (7). The purified proteins were
separated by SDS-PAGE, and the GST-NS4B cleavage product was identified
by Western blot analysis with polyclonal antibody raised against the
NS4B (Ala1884-Tyr1899) peptide (Fig. 2C).
Construct 4, covering all of the putative NS4A region; construct 41, with an amino-terminal truncation; and construct P234, with a
carboxy-terminal truncation in the region, retained full NS4A activity.
Construct 42 exhibited very little residual NS4A activity, indicating
that the NS4A active domain was impaired in this construct. Therefore,
the NS4A active domain was located within the
Leu1561-Ala1598 HGV polyprotein sequence. Amino
acid sequence comparison between the HCV NS4A and HGV NS4A active
domains indicated limited homology between these sequences; however,
there was a considerable conservation on the secondary structure level,
as predicted by the Chou-Fasman algorithm (4) (data not
shown).
Analysis of the HGV polyprotein cleavage sites.
The sequence
at the NS2 protease-mediated NS2/NS3 cleavage site was established by
amino-terminal sequence analysis of the NS3 cleavage product, which was
identified in cell lysates of the insect cells infected with the P
construct (Fig. 1D). Amino-terminal sequence analysis of the NS3
product yielded the Ala-Pro-Val-Val-Ile sequence, which indicates that
the NS2/NS3 cleavage occurred within the
Gly-Phe-Val-Pro-Thr/Ala926-Pro-Val-Val-Ile
sequence. Alignment of the HCV, HGV, GBV-A, and GBV-B sequences at the
NS2/NS3 junction shows strict conservation of amino acids at positions
P5, P1', and P2' (Fig. 3). The reason for
such strict conservation remains unclear and could reflect other
functions distinct from proteolytic processing. As previously
demonstrated for HCV, NS2B/NS3 cleavage was not strictly dependent on
the Gly, Ala, and Pro in these positions; conservative, as well as some
nonconservative P5, P1', and P2' amino acid substitutions were
tolerated (24).
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FIG. 3.
Alignment of the HCV, HGV, GBV-A, and GBV-B sequences at
the NS2B/NS3 and NS4B/NS5A junctions. The cleavages have been
experimentally established for HGV (this study) and HCV only (7,
8). The amino acids demonstrated to be conserved in cleavage
sites found in HCV are shaded.
|
|
The sequence at the NS3-mediated NS4B/NS5A cleavage site was
established by amino-terminal sequence analysis of the NS5A cleavage
product present in the extracts of the cells coinfected with construct
45, which contained the putative NS4B/NS5A junction, and construct
P,
which had HGV protease activities. The NS5A cleavage product
was
identified in the cell extracts by Western blot analysis with
anti-NS5A
(Ser
2196-Gly
2211) peptide antibodies (data not
shown).
Amino-terminal sequence analysis of the NS5A product yielded
the
sequence Tyr-Val-Trp-Asp-Leu-Trp-Glu-Trp-Ile-Met, indicating that
the NS4B/NS5A cleavage occurs within the
Glu-Val-Gln-Val-Gly/Tyr
1899-Val-Trp-Asp-Leu
sequence.
Unlike NS2/NS3 cleavage, HCV and HGV NS4B/NS5A cleavage
sites have
little in common apart from the conserved acidic amino
acid residue at
position P6 and the presence of small residues
at positions P1 and P1'
(Fig.
3). The acidic amino acid at P6
was conserved in all NS3-mediated
HCV cleavage sites; however,
it was not essential for efficient
cleavage (
14). The P1 position
was shown to be more
important for this cleavage (
14,
31,
32). P1 Cys is
conserved in all
trans-cleaved HCV sites, and
a recent study
demonstrated that it was critical for the
trans cleavage at
the NS5A/NS5B site (
32). P1 Cys is likely to interact
with
the S1 substrate pocket of HCV NS3 protease, with a cysteine
sulfhydryl
group probably playing a critical role in substrate
binding (
10,
19,
23,
32). However, Gly instead of Cys
was found in the P1
position of the
trans-cleaved NS4B/NS5A site
of HGV (Fig.
3). Moreover, Phe
154, which determines the preference
of
the HCV NS3 for cysteine in the P1 substrate position (
6,
32), is substituted for Leu
154 in HGV NS3, probably
determining
the different substrate specificity of HGV NS3 (Fig.
1B).
Interestingly,
GBV-B NS3 protease, which shares substrate specificity
with HCV
NS3, has both Phe
154 in its substrate binding
pocket (
25) and
Cys in the P1 position of the putative
NS4B/NS5A cleavage site
(Fig.
3).
In summary, the mode of the HGV polyprotein cleavage in the
nonstructural protein region appears to be very similar to that
of HCV,
except for the substrate specificity of the NS3 protease.
HCV NS3
serine protease activity is required for all of the cleavages
downstream of NS3, while the NS4A activity is absolutely required
for
all of the cleavages except for NS5A/NS5B (
2,
16).
Similarly,
HGV NS3, but not NS2B, was found to be required for the
NS4B/NS5A
and NS5A/NS5B cleavages, and NS4A was found to be absolutely
required
for the NS4B/NS5A cleavage but not for the NS5A/NS5B cleavage.
As in the case of HCV, the HGV NS4B/NS5A and NS5A/NS5B cleavages
could
occur in
trans. Similarity between HGV and HCV is not
surprising,
because these viruses share considerable amino acid
sequence similarity
in the nonstructural regions of their polyproteins
(
18). In
both viruses, processing of the nonstructural part
of the polyprotein
is mediated by the NS2 and NS3 proteases and the
NS4A cofactor,
which are encoded at corresponding parts of the genomes
of these
viruses, have similar active domains, and can effect cleavages
at analogous sites of the polyprotein in a similar fashion.
| |
ACKNOWLEDGMENTS |
We express our gratitude to Kirk Fry, Jeffrey Linnen, and Patrice
Yarbough for helpful discussions during the course of this study and to
Nancy Alexi and Jane Bardwell for comments during the preparation of
the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Genelabs
Technologies, Inc., 505 Penobscot Dr., Redwood City, CA 94063. Phone:
(415) 369-9500, ext. 566. Fax: (415) 368-0709. E-mail:
alexb{at}genelabs.com.
| |
REFERENCES |
| 1.
|
Bartenschlager, R.,
L. Ahlborn-Laake,
J. Mous, and H. Jacobsen.
1993.
Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.
J. Virol.
67:3835-3844[Abstract/Free Full Text].
|
| 2.
|
Bartenschlager, R.,
L. Ahlborn-Laake,
J. Mous, and H. Jacobsen.
1994.
Kinetic and structural analyses of hepatitis C virus polyprotein processing.
J. Virol.
68:5045-5055[Abstract/Free Full Text].
|
| 3.
|
Bartenschlager, R.,
V. Lohmann,
T. Wilkinson, and J. O. Koch.
1995.
Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation.
J. Virol.
69:7519-7528[Abstract].
|
| 4.
|
Chou, P. Y., and G. D. Fasman.
1978.
Prediction of the secondary structure of proteins from their amino acid sequence.
Adv. Enzymol. Relat. Areas Mol. Biol.
47:45-148[Medline].
|
| 5.
|
Davies, A. H.,
J. B. M. Jowett, and I. M. Jones.
1993.
Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.
Bio/Technology
11:933-936[Medline].
|
| 6.
|
Failla, C. M.,
E. Pizzi,
R. De Francesco, and A. Tramontano.
1996.
Redesigning the substrate specificity of the hepatitis C virus NS3 protease.
Folding Design
1:35-42.
[Medline] |
| 7.
|
Grakoui, A.,
D. W. McCourt,
C. Wychowski,
S. M. Feinstone, and C. M. Rice.
1993.
Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.
J. Virol.
67:2832-2843[Abstract/Free Full Text].
|
| 8.
|
Grakoui, A.,
D. W. McCourt,
C. Wychowski,
S. M. Feinstone, and C. M. Rice.
1993.
A second hepatitis C virus-encoded proteinase.
Proc. Natl. Acad. Sci. USA
90:10583-10587[Abstract/Free Full Text].
|
| 9.
|
Hijikata, M.,
H. Mizushima,
T. Akagi,
S. Mori,
N. Kakiuchi,
N. Kato,
T. Tanaka,
K. Kimura, and K. Shimotohno.
1993.
Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus.
J. Virol.
67:4655-4675.
|
| 10.
|
Kim, J. L.,
K. A. Morgenstern,
C. Lin,
T. Fox,
M. D. Dwyer,
J. A. Landro,
S. P. Chambers,
W. Markland,
C. A. Lepre,
E. T. O'Malley,
S. L. Sharbeson,
C. M. Rice,
M. A. Murcko,
P. R. Caron, and J. A. Thomson.
1996.
Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A peptide.
Cell
87:343-355[Medline].
|
| 11.
|
King, L. A., and R. D. Possee.
1992.
.
The baculovirus expression system: a laboratory guide.
Chapman & Hall, London, United Kingdom.
|
| 12.
|
Kitts, P. A., and R. D. Possee.
1993.
A method for producing recombinant baculovirus expression vectors at high frequency.
BioTechniques
14:810-817.
[Medline] |
| 13.
|
Koch, J. O.,
V. Lohmann,
U. Herian, and R. Bartenschlager.
1996.
In vitro studies on the activation of the hepatitis C virus NS3 proteinase by the NS4A cofactor.
Virology
221:54-66[Medline].
|
| 14.
|
Kolykhalov, A. A.,
E. V. Agapov, and C. M. Rice.
1994.
Specificity of the hepatitis C virus serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.
J. Virol.
68:7525-7533[Abstract/Free Full Text].
|
| 15.
|
Kunkel, T. A.
1985.
Rapid and efficient site-directed mutagenesis without phenotypic selection.
Proc. Natl. Acad. Sci. USA
82:488-492[Abstract/Free Full Text].
|
| 16.
|
Lin, C.,
B. M. Prágai,
A. Grakoui,
J. Xu, and C. M. Rice.
1994.
The hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.
J. Virol.
68:8147-8157[Abstract/Free Full Text].
|
| 17.
|
Lin, C.,
J. A. Thomson, and C. M. Rice.
1995.
A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine protease complex in vivo and in vitro.
J. Virol.
69:4373-4380[Abstract].
|
| 18.
|
Linnen, J.,
J. Wages,
Z.-Y. Zhang-Keck,
K. Fry,
K. Z. Krawczynski,
H. Alter,
E. Koonin,
M. Gallagher,
M. Alter,
S. Hadziyannis,
P. Karayiannis,
K. Fung,
Y. Nakatsuji,
J. Shih,
L. Young,
M. Piatak,
C. Hoover,
J. Fernandez,
S. Chen,
J. C. Zou,
T. Morris,
K. C. Hyams,
S. Ismay,
J. D. Lifson,
G. Hess,
S. K. H. Foung,
H. Thomas,
D. Bradley,
H. Margolis, and J. P. Kim.
1996.
Molecular cloning and disease association of hepatitis G virus: a new transfusion transmissible agent.
Science
271:505-508[Abstract].
|
| 18a.
| Linnen, J., et al. Personal communication.
|
| 19.
|
Love, R. A.,
H. E. Parge,
J. A. Wickersham,
Z. Hostomsky,
N. Habuka,
E. W. Moomaw,
T. Adachi, and Z. Hostomska.
1996.
The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.
Cell
87:331-342[Medline].
|
| 20.
|
Manabe, S.,
I. Fuke,
O. Tanishita,
C. Kaji,
Y. Gomi,
S. Yoshida,
C. Mori,
A. Takamizawa,
I. Yoshida, and H. Okayama.
1994.
Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3.
Virology
198:636-644[Medline].
|
| 21.
|
Mori, A.,
K. Yamada,
J. Kimura,
T. Koide,
S. Yausa,
E. Yamada, and T. Miyamura.
1996.
Enzymatic characterization of purified NS3 serine proteinase of hepatitis C virus expressed in Escherichia coli.
FEBS Lett.
378:37-42[Medline].
|
| 22.
|
Neddermann, P.,
L. Tomei,
C. Steinkuhler,
P. Gallinari,
A. Tramontano, and R. De Francesco.
1997.
The nonstructural proteins of the hepatitis C virus: structure and functions.
Biol. Chem.
378:469-476.
[Medline] |
| 23.
|
Pizzi, E.,
A. Tramontano,
L. Tomei,
N. La Monica,
C. Failla,
M. Sardana,
T. Wood, and R. de Francesco.
1994.
Molecular model of the specificity pocket of the hepatitis C virus protease: implication for substrate recognition.
Proc. Natl. Acad. Sci. USA
91:888-892[Abstract/Free Full Text].
|
| 24.
|
Reed, K. E.,
A. Grakoui, and C. M. Rice.
1995.
Hepatitis C virus-encoded NS2-3 protease: cleavage site mutagenesis and requirements for bimolecular cleavage.
J. Gen. Virol.
69:4127-4136.
|
| 25.
|
Scarselli, E.,
A. Urbani,
A. Sbardellati,
L. Tomei,
R. De Francesco, and C. Traboni.
1997.
GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.
J. Virol.
71:4985-4989[Abstract].
|
| 26.
|
Schlauder, G. G.,
G. Dawson,
J. N. Simons,
T. J. Pilot-Matias,
R. A. Gutierrez,
C. A. Heynen,
M. F. Knigge,
G. S. Kurpiewski,
S. L. Buijk,
T. P. Leary,
A. S. Muerhoff,
S. M. Desai, and I. K. Mushahwar.
1995.
Molecular and serologic analysis in the transmission of the GB hepatitis agents.
J. Med. Virol.
46:81-90[Medline].
|
| 27.
|
Shimizu, Y.,
K. Yamaji,
Y. Masuho,
T. Yokota,
H. Inoue,
K. Sudo,
S. Satoh, and K. Shimotohno.
1996.
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.
J. Virol.
70:127-132[Abstract].
|
| 28.
|
Simons, J. N.,
T. P. Leary,
G. J. Dawson,
T. J. Pilot-Matias,
A. S. Muerhoff,
G. Schlauder,
S. M. Desai, and I. K. Mushahwar.
1995.
Isolation of novel virus-like sequences associated with human hepatitis.
Nat. Med.
1:564-569[Medline].
|
| 29.
|
Tanji, Y.,
M. Hijikata,
S. Satoh,
T. Kaneko, and K. Shimotohno.
1995.
Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.
J. Virol.
69:1575-1581[Abstract].
|
| 30.
|
Tomei, L.,
C. Failla,
E. Santolini,
R. De Francesco, and N. La Monica.
1993.
NS3 is a serine protease required for processing of hepatitis C virus polyprotein.
J. Virol.
67:4017-4026[Abstract/Free Full Text].
|
| 31.
|
Urbani, A.,
E. Bianchi,
F. Narjes,
A. Tramontano,
R. De Francesco,
C. Steinkuhler, and A. Pessi.
1997.
Substrate specificity of the hepatitis C virus serine protease NS3.
J. Biol. Chem.
272:9204-9209[Abstract/Free Full Text].
|
| 32.
|
Zhang, R.,
J. Durkin,
W. T. Windsor,
C. McNemar,
L. Ramanathan, and H. V. Le.
1997.
Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.
J. Virol.
71:6208-6213[Abstract].
|
| 33.
|
Zuckerman, A. J.
1996.
Alphabet of hepatitis viruses.
Lancet
347:558-559[Medline].
|
J Virol, January 1998, p. 868-872, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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