Journal of Virological Methods 107 (2003) 245 /255

www.elsevier.com/locate/jviromet

A novel high throughput screening assay for HCV NS3 serine

protease inhibitors
Yevgeny Berdichevsky a,1, Romy Zemel b,1, Larisa Bachmatov b, Alex Abramovich b,
Ruth Koren c, Peramachi Sathiyamoorthy d, Avi Golan-Goldhirsh e,
Ran Tur-Kaspa b,f,
,2, Itai Benhar a,

a
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202,
Tel-Aviv University, Ramat Aviv 69978, Israel
b
Molecular Hepatology Research Laboratory, Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University,
Ramat Aviv 69978, Israel
c
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel
d
Gem Research Foundation, Chennai 86, India
e
Ben-Gurion University of the Negev, Sede Boker Campus, Beer-Sheva, Israel
f
Department of Medicine D and Liver Institute, Rabin Medical Center, Beilinson Campus, Petah Tikva, Israel

Received 25 July 2002; received in revised form 28 September 2002; accepted 11 October 2002

Abstract

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver
cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral
proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential
target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed
biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity.
A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease
potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic
activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked
to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of
fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease
inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and
has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: HCV NS3 serine protease; Fluorometric assay; Fluorogenic substrate; High throughput screening assay; Recombinant NS4A-NS3

1. Introduction ley, 1999; Okuda, 2000). Globally, the seroprevalence of

Hepatitis C virus (HCV) is an RNA virus that causes
hepatitis, cirrhosis, liver failure and Hepatocellular
carcinoma (HCC) (Brechot, 1996; Purcell, 1997; Brad-

Corresponding authors. Tel.: /972-3-6407511; fax: /972-3-
6409407
E-mail addresses: rturkaspa@clalit.org.il (R. Tur-Kaspa),
benhar@post.tau.ac.il (I. Benhar).
1
Contributed equally to this work.
2
Tel.: /972-3-9376751; fax: /972-3-9220671.
0166-0934/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 0 9 3 4 ( 0 2 ) 0 0 2 5 5 - 0
HCV is over 100 million (Idilman et al., 1998). Infec-
tions with HCV can manifest in a wide range of clinical
outcomes from an asymptomatic carrier state to acute
or chronic hepatitis. In approximately 20% of chroni-
cally infected patients, the disease progresses to liver
cirrhosis, which is highly associated with the subsequent
development of HCC (Saito et al., 1990; Brechot et al.,
1998). Despite recent advances in the therapy of
hepatitis C, even the most effective treatment with a-
interferon in combination with ribavirin or the more
recent combination of pegylated interferon and ribavirin
246 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
fail to eliminate infection in nearly 50% of those infected
(Bartenschlager, 1997; Pawlotsky, 1999; Manns et al.,
2001; Fried, 2001). This high frequency of treatment
failure points to the need for more specific, less toxic and
more active antiviral therapies for HCV (Pawlotsky,
2000; Clarke, 2000). It is therefore of utmost importance
to identify novel and effective anti-HCV drugs.
HCV is a member of the Flaviviridiae family, it is a
small enveloped virus with a single-stranded, positive-
sense RNA genome packed within a nucleocapsid
(Purcell, 1997). The 9.6 kb RNA genome is organized
to contain a single, large translational open-reading
frame that spans most of its length. This encodes a large
polyprotein precursor of 3010 /3033 amino acids which
is translated in a 5? cap-independent manner under the
control of a highly structured internal ribosome entry
site within the 342-nucleotide long 5? untranslated
region (5?UTR) segment of the genome. Four structural
and at least six nonstructural (NS) proteins are gener-
ated by co-translational cleavage of the polyprotein by
both cellular and virally-encoded proteases. Processing
of the amino-terminal part of the polyprotein by cellular
signalase results in the mature forms of both the core
protein and two envelope proteins plus a small, trans-
membrane protein. Most subsequent proteolytic proces-
sing events are directed by the virally-encoded NS3
serine protease that requires the adjacent NS4A cofactor
for efficient cleavage activity (Failla et al., 1994).
The HCV chymotrypsin-like serine protease activity
resides in the amino-terminal third of the NS3 protein.
The mature form of the NS3 protease, which is a
bifunctional protein with NTPase and helicase activities
located within its carboxy-terminal domain (Kim et al.,
1995), is a noncovalent complex with NS4A. It directs
proteolytic cleavages at the NS3/4A, NS4A/4B, NS4B/
5A, and NS5A/5B junctions (Bartenschlager et al., 1993;
Tomei et al., 1993), and is thus essential for replication
of the virus. The crystal structure of the NS3 serine
protease (complexed with the NS4A cofactor domain)
has been elucidated and much is known about its
structure-function relations (Love et al., 1996; Kim et
al., 1996). The catalytic triads, as well as conserved
cleavage site amino acids, are well-characterized (Koly-
khalov et al., 1994). NS3 appears to be anchored to
intracellular membranes through its association with
NS4A which has a highly hydrophobic, transmembrane
domain, and is suspected to be an essential component
of the viral replicase complex.
In addition to its three associated enzymatic activities,
expression of NS3 has been found to affect the cell
metabolism by histone binding and modulation of
protein phosphorylation mediated by PKA and PKC,
thus interfering with signal transduction pathways
(Borowski et al., 1999a,b). Evidence for the involvement
of NS3 in cell proliferation was provided by Ishido and
Hotta (1998) who reported that the HCV NS3 could
form a complex with wild-type p53, both in the absence
and the presence of the NS4A viral cofactor. Kwun et al.
(2001) demonstrated that NS3 protein could specifically
repress the promoter activity of p21 in a dose-dependent
manner, and the repression is reflected by the stimula-
tion of cell growth. It was also reported that NS3
transforms NIH3T3 cells (Sakamuro et al., 1995) and rat
3T3 cells (Zemel et al., 2001). We have recently reported
that NS3-mediated cell transformation is dependent of
NS3 being catalytically active (Zemel et al., 2001).
The development of new anti HCV protease inhibitors
will be dependent on the expression and purification of
large amounts of the active enzyme for biochemical
characterization and the availability of a rapid reliable
in vitro assay for the screening of potential inhibitors.
The detection of chromogenic or fluorogenic substances
that are generated by proteolytic cleavage of a suitable
substrate is a sensitive and practical approach for
monitoring the reaction of serine proteases. In this
report we describe the expression, purification and
characterization of a recombinant NS4A-NS3 fusion
protein (single-chain NS3; scNS3) and a recombinant
EGFP-NS5A/B-CBD fusion protein serving as a fluor-
escent substrate. We further describe the development of
a fluorometric assay for measurement of NS3 serine
protease activity that is suitable for high throughput
screening (HTS) for potential NS3 inhibitors. Our assay
is the first example of a HTS assay for NS3 catalysis and
inhibition where both enzyme and substrate are both
recombinant proteins.
2. Materials and methods
2.1. Bacterial strains
The following Escherichia coli strains were used: XL-1
Blue (Stratagene) for plasmid propagation and BL-21
(DE-3) (Novagen) for expression of the T7 promoter-
driven NS4A-NS3 single-chain protease and the fluoro-
genic substrate protein.
2.2. Recombinant DNA techniques and vectors
Recombinant DNA techniques were carried out
according to standard protocols or as recommended
by suppliers. Nucleotide sequences were determined
using the ABI373 automated sequencer (Perkin-Elmer
ABI) according to the supplier’s recommendations. The
T7 promoter-based expression vectors pET-11c and
pET-21d were from Novagen (Madison, WI). All
plasmid and DNA fragment purifications were carried
out with a Qiagen Qiaquick PCR purification kit and
Qiaprep Spin Miniprep kit (Qiagen). T4 DNA ligase and
restriction enzymes were purchased from New England
 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255 247

Biolabs (Beverly, MA). DNA ligations were carried out
at 16 8C overnight.
2.3. Construction, expression and purification of NS4A-
NS3 (sc NS3)
The pYB-43 vector (Fig. 1A) used for E. coli
expression of histidine-tagged NS4A-NS3 single-chain
protein have been constructed according to Dimasi et al.
(1998). The NS4A-NS3 gene was obtained by PCR
amplification of the target sequence from plasmid
pcEF321 (kindly provided by Dr C. Brechot, HCV
genotype BK) using primers 5?-TTTTTTCCATGGCG-
CCTATCGGCTCAGTAGTAATCGTAGGCAGAA-
TCATCCTGTCCGGCCGTGGTGGCCCGATCAC-
GGCCTACTCCCAAC-3? that introduces the NS4A
core peptide and a Nco I site and 5?-GCCGGAG-
GATCCTTAGTGATGGTGATGGTGATGGGAGC-
CCCGCATAGTGGTTTCCATAGA-3? that intro-
duces a hexahistidine coding sequence and a Bam HI
site. The PCR conditions were 94 8C, 30 s; 55 8C, 30 s;
72 8C, 60 s for 30 cycles. The PCR product was digested
with Nco I and Bam HI and ligated into pET-21d
linearized with the same enzymes.
The single-chain protease NS4A-NS3 (scNS3) was
expressed from the pYB-43 vector transformed in E. coli
BL-21(DE3). Cultures in shaking flasks were grown in
LB medium supplemented with 4 g/l glucose and 100 mg/
ml ampicillin, at 37 8C. When the culture reached
OD600 nm /0.8, cells were chilled on ice for 10 min
and induced with 1 mM IPTG (isopropyl-b-D-thioga-
lactopyranoside). The culture was then shifted to 16 8C
for 14 h and cells were collected by centrifugation. Cell
pellets from 0.5 l of induced culture were suspended in
40 ml of basic buffer (50 mM Tris(HCl) pH 7.5, 150 mM
NaCl, 0.05% Tween 20, 5 mM 2-mercaptoethanol, 3%
glycerol) and sonicated on ice. The cell-free extract
containing soluble histidine-tagged scNS3 was clarified
by centrifugation at 15 000/g for 30 min. The lysate
was filtered through 0.45 micron filter, adjusted to 5
mM imidazole, and loaded onto 15 ml of Ni-NTA resin
(Qiagen) that was previously equilibrated with the
Binding buffer (Basic buffer/5 mM Imidazole). The
binding to the matrix was performed batchwise at 4 8C
for 1 h with gentle rotation, then the column was washed

Fig. 1. Expression vector pYB-43 (A) and purification of histidine-tagged NS4A-NS3 single-chain protein (sc NS3) (B). (A) Map of plasmid pYB-43
used for cytoplasmic expression of sc NS3. The amino-acid sequence of the NS4A core peptide-NS3 protease-6/His region is shown at the top. (B)
Lane 1, total cell extract from induced BL21(DE-3) cells carrying plasmid pYB-43. Lane 2, soluble fraction of induced cell extract. Lane 3, flow-
through of the Ni-NTA column. Lane 4, Ni-NTA column wash. Lane 5, purified sc NS3. 15 /20 mg of protein were loaded in lanes 1 /4 and 2 mg in
lane 5. MW, Molecular weight marker. Proteins were separated on a 12% SDS-polyacrylamide gel and visualized by staining with GelCode Blue
(Pierce, USA). The arrow marks the position of the sc NS3 protein.
248 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
with 10 column volumes of Binding buffer followed by 5
column volumes of Wash buffer (Basic buffer/20 mM
Imidazole). Finally, the scNS3 was eluted with 2 /3
column volumes of Elution buffer (Basic buffer/250
mM Imidazole) and dialysed overnight at 4 8C against
100 volumes of dialysis buffer (50 mM Tris(HCl) pH
7.5, 150 mM NaCl, 0.05% Tween, 1 mM DTT, 20% (v/v)
Glycerol). The purified protein was stored at /20 8C.
2.4. Vector construction, expression and purification of
the recombinant fluorogenic substrate proteins
To develop a fluorometric assay for NS3 protease
activity we constructed a fluorogenic recombinant sub-
strate protein EGFP-NS5A/B-CBD, where the EGFP
gene coding for enhanced green fluorescent protein
(Clontech) was linked to Clostridium thermocellum
cellulose-binding domain (CBD) gene (Morag et al.,
1995) via the NS5A/B cleavage site (Clarke, 1997;
Rosenberg, 2001). As a specificity control for NS3
mediated cleavage we constructed a fluorogenic protein
where the EGFP gene was fused to the CBD gene via an
enterokinase site.
The plasmid pYB-44 (Fig. 2A) carrying the recombi-
nant fluorogenic substrate EGFP-NS5A/B site*/CBD
for was constructed as follows: the EGFP gene was
obtained by PCR-amplification with 5?-ATTCAGTAC-
CATGGTGAGCAAGGGCGAGGAGCTGTT-3? and
5?-GTCGCCACCTGCGGCCGCCTTGTACAGCT-
CGTCCATGCC-3? primers utilizing plasmid pEGFP-
N1 (Clontech) as template. These primers introduce
Nco I and Not I sites on the N- and C-termini respec-
tively. The CBD gene with the linked N-terminal NS5A/
B site was obtained by PCR-amplification with primers
5?-AAACGGGCGGCCGCAGGTGGCGACTACAA-
GGA CGCTAGCGAGGACGTCGTCTGCTGCTC-
GATGTCCTACACACCGGTATCAGGCAATTTG-
3? and 5?-ACGAATGGATCCTATACTACACTG-3?
using plasmid pCBD (Morag et al., 1995) as template
and PCR conditions as in Section 2.3, above. These
primers introduce Not I and Bam HI sites on the N- and
C-terminus of the CBD gene, respectively. The amplified
EGFP and NS5A/B-CBD fragments were digested with
restriction enzymes Nco I and Not I and Not I and
Bam HI, respectively and were cloned together into
pH6T-FEKCA11c plasmid (Berdichevsky et al., 1999)
previously digested with the Nco I and Bam HI restric-
tion enzymes.
For the construction of plasmid pYB-45 carrying the
negative control protein, i.e. EGFP-Ek site-CBD, the
Nco I /Not I digested EGFP gene was ligated into pH6T-
FEKCA11c previously digested with the same enzymes.
The EGPF-NS5A/B-CBD fusion protein and the
EGFP-Ek-CBD negative control protein were expressed
in E. coli strains BL-21 (DE-3)/pYB-44 and BL-21 (DE-
3)/pYB-45, respectively. The cells were grown in LB
medium supplemented with 100 mg/ml ampicillin at
37 8C, 250 rpm. At OD600 nm $/0.8 /1.0 the cells were
chilled down to 16 8C and induced with 1 mM IPTG
for 16 h at 16 8C, 250 rpm.
The cell pellet was suspended in 50 ml of ice-cold cell
extraction buffer (50 mM Tris(HCl) pH 8, 20 mM EDTA
pH 8, 200 mM NaCl) and disrupted by sonication. The
protein solution was clarified from cell debris and
inclusion bodies by centrifugation at 15 000 /g for 30
min. The clarified cell-free extract was incubated with up
to 1.5 g Sigmacell-50 cellulose for 1 h at 4 8C. The
cellulose matrix was sequentially washed with 50 ml
extraction buffer, 50 mM Tris(HCl) pH 8, 1 M NaCl,
water and 50 mM Tris(HCl) pH 8. The fluorescent fusion
protein was eluted from the cellulose matrix with 5/10 ml
of 0.1 M NaOH for 5 min at room temperature. The eluted
protein was transferred into a new tube and immediately
buffered to neutral pH with 1 M Tris(HCl) pH 7.5.
2.5. NS3 protease activity assay
Digestion of the recombinant substrate protein
(EGFP-NS5A/B site-CBD) with the scNS3 protease
was performed in 100 ml reaction volume at 37 8C for 60
min. The reaction was performed in reaction buffer /1
(50 mM Tris(HCl) pH 7.5, 150 mM NaCl, 0.05% Tween,
20% Glycerol, 1 mM DTT) with 0.25 /1.0 mM ‘GPF-
labeled’ recombinant substrate and 0.01 /0.1 mM of
recombinant protease. Following digestion, the reaction
was transferred into an Eppendorf tube containing 20
mg cellulose pre-equilibrated with reaction buffer. The
cellulose matrix was gently agitated at room tempera-
ture for 5 min followed by centrifugation for 2 min at
maximum speed. The supernatant containing liberated
green fluorescent protein was transferred to a black 96
well plate and data was collected in a fluorometer
utilizing excitation filter 485 nm, and emission filter
538 nm. At each substrate concentration and each time
point the substrate was also incubated in the absence of
the enzyme and the extent of the spontaneous cleavage
reaction was determined. Specific cleavage was the
difference between the fluorescence obtained in the
presence and absence of the enzyme.
For high throughput assay of NS3 catalysis and its
inhibition, the assay described in the previous paragraph
was modified as follows: the reactions were done in 100
ml in individual wells of 96 well-plates containing 40 nM
sc NS3 and 0.25 mM substrate that were incubated for 1
h at 37 8C. Subsequently, 100 ml of a 33% (w/v) cellulose
slurry were added to each well and the plates were
shaken for an additional 15 min. The plates were then
spun and the cellulose-unbound fraction was transferred
to a black walled 96 well plate. Alternatively, the assay
was carried out using MultiScreen 96 well filtration
plates with low-protein binding PVDF membranes
(Millipore, USA). Following the enzymatic reactions,
 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255 249

Fig. 2. Expression vector pYB-44 (A) and purification of the recombinant fluorogenic substrate protein on cellulose (B). (A) The plasmid pYB-44
carrying the fluorogenic substrate EGFP-NS5A/B site-CBD was constructed as described in the text. The amino-acid sequence of the NS5A/B
cleavage site is shown at the top. (B) Expression and purification of EGFP-NS5A/B site-CBD fusion protein: MW, molecular weight marker. Lane 1,
total cell extract from uninduced BL21(DE-3) cells carrying plasmid pYB-44. Lane 2, induced total cell extract. Lane 3, induced soluble cell extract.
Lane 4, flow-through of the cellulose column. Lane 5, purified EGFP-NS5A/B site-CBD protein. 15 /20 mg of protein was loaded in lanes 1 /4 and 3
mg in lane 5. Proteins were separated on a 12% SDS-polyacrylamide gel and visualized by staining with GelCode Blue. The arrow marks the position
of the EGFP-NS5A/B site-CBD fusion protein.

the unbound fractions were recovered by vacuum
aspiration. Fluorescence was recorded using a fluores-
cence plate reader (Bio-Tek FL500, USA). For inhibi-
tion studies, inhibitors (1 ml of a crude herbal extracts)
were mixed with the enzyme in the wells and incubated
for 30 min prior to addition of the substrate. Non
specific effects of the extracts were tested by incubating
the same amount as above with substrate alone and by
measuring the intrinsic fluorescence of extracts in assay
buffer.
days, powdered mechanically and stored at room
temperature for screening. Plant powder was suspended
in distilled deionized water at of 100 mg/ml and
extracted by shaking for 4 h at 40 8C in a water bath.
The extracts were centrifuged at room temperature.
Aliquots of the supernatant were lyophilized and later
dissolved in half of the original volume of the distilled
deionized water. When a precipitate was formed it was
removed by centrifugation and the crude supernatant
was used in the assay.

2.6. Preparation of aqueous extracts from the Indian

Siddha medicinal plant 3. Results and discussion

Plants and plant parts that are used in Indian
traditional medicine were collected in south India based
on ancient literature of ‘Siddha Medicine’. Plants
extracts were prepared according to Sathiyamoorthy et
al. (1999). All the plants were oven-dried at 40 8C for 5
3.1. Expression and purification of the HCV sc NS3
serine protease
The development of new HCV protease inhibitors is
dependent on the availability of large quantities of
250 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
purified enzyme and substrate. For ease of production
we chose the single-chain format of NS3. Previous
studies have shown that the kinetic characteristics of
the single chain construct where the NS4A cofactor was
linked to the N terminus of the NS3 protease domain
suggested that the catalytic efficiency of the single-chain
construct is greater than the NS3 complemented in trans
by the equivalent NS4A peptide (Taremi et al., 1998;
Dimasi et al., 1998). In this study, a His-tagged NS4A /
NS3 single-chain protein (scNS3) was constructed
according to Dimasi et al. (1998) and expressed in E.
coli BL-21(DE3) from plasmid pYB43 (Fig. 1A). Upon
induction at 16 8C, the His-tagged scNS3 accumulated
in the soluble cell fraction from where it was purified by
one-step Ni -affinity chromatography (Fig. 1B). Inter-
estingly, although our expression vector and purification
protocol were slightly different from these reported by
Dimasi et al. (1998) (growth medium and temperature),
our production yield was better, as we could purify
about 8 mg of scNS3 from 1 l of bacterial culture.
3.2. Proteolytic activity of the purified NS3 protease
using a fluorogenic substrate
A variety of NS3 protease in vitro assays have been
described, utilizing various formats of substrates (re-
viewed in Kwong et al., 1998). Initially, cell-free trans-
processing assays for the determination of NS3 protease
activity relied on the use of 35S-radiolabelled substrates
created through an in vitro transcription translation
system. (Bouffard et al., 1995; D’Souza et al., 1995; Lin
and Rice, 1995; Hamatake et al., 1996). As larger
amounts of protease became available for study, a
number of groups applied HPLC cleavage assays of
peptide substrates (Steinkuhler et al., 1996; Kakiuchi et
al., 1998). Substrate proteins and peptides contained
either the NS5A /5B, 4B /5A, or 4A /4B processing
sites.
In our study, an in vitro assay using a fluorogenic
substrate was established. The fluorogenic substrate
consists of three linked modules: a fluorescent protein
EGFP, a peptide (SEDVVCCSMSY), derived from the
boundary between the HCV NS5A and NS5B proteins
where NS3 cleaves more efficiently than at other
cleavage sites in the HCV polyprotein (Kakiuchi et al.,
1999), and a CBD which efficiently binds to cellulose
(Fig. 2A). The EGPF /NS5A/B /CBD fusion protein is
expressed in E. coli , and purified on cellulose matrix
(Fig. 2B). It should be noted that our substrate, as other
CBD fusion proteins (Berdichevsky et al., 1999) is a very
robust protein that binds to cellulose in up to 4 M urea,
50% ethanol and up to 5 M NaCl (not shown). Thus, it
should be suitable for screening of potential inhibitors
under various assay conditions. Our production proto-
col is very simple and efficient. Following IPTG
induction at 16 8C, the recombinant protein was
purified from the soluble cell-free extract with a
production yield of 60 mg from 1 l of bacterial culture.
This quantity is sufficient for /50 000 cleavage reac-
tions. The high production efficiency may be attributed
to the expression of the substrate as a CBD fusion
protein. We have previously shown that such fusion
proteins are expressed at high levels in E. coli and that
their purification is very simple and efficient (Berdi-
chevsky et al., 1999).
The cleavage reaction was performed by adding
purified His-tagged-scNS3 to the purified recombinant
substrate. Following the capture of the released C-
terminal portion of the substrate and the un-cleaved
substrate on cellulose, the released free EGFP is
monitored by fluorometry. The procedure is schemati-
cally illustrated in Fig. 3.
The first objective was to compare substrate cleavage
as quantified by the fluorometric assay and western blot
analysis. We analyzed the time course of the sc NS3
catalyzed cleavage of the purified NS5A-B substrate.
The purified NS3 protein (0.65 mM) was incubated at
37 8C with the fluorogenic substrate (0.125 mM) for 120
min. The site-specific cleavage was assayed by the
release of the fluorescent cleavage product and by
immunoblot where the products of a representative
cleavage reaction were analyzed using anti-GFP anti-
bodies. When the EGFP-Ek-CBD negative control
protein was analyzed under identical conditions, no
cleavage could be detected (not shown). This further
confirms the specificity of cleavage by sc NS3 at the
NS5A/B site that was already demonstrated before
(Dimasi et al., 1998).
The results of a typical experiment are presented in
Fig. 4. It can be easily seen that substrate cleavage, as
Fig. 3. Schematic overview of the fluorometric assay for HCV sc NS3
serine protease activity. The recombinant fluorogenic substrate for
HCV NS3 protease consists of an enhanced green fluorescent protein
(EGFP) linked to a CBD via the NS5A/B site that is cleavable by NS3.
Cleavage of this substrate by sc NS3 followed by capture of the CBD
moiety on cellulose results in emission of fluorescent light that is easily
detected and quantitated by fluorometry.
 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
measured by both methods, increases with time up to 30
min. At this time point the reaction levels off with the
level of cleaved substrate reaching a plateau. This result
is not unexpected in view of the numerous reports of
product inhibition of NS3 cleavage reactions (reviewed
in Kwong et al., 1998). The clear correlation between the
two assay methods validates the use of the fluorometric
assay as a quantitative measure of the substrate
cleavage. To rule out the possibility that the reaction
plateaus due to instability of the enzyme, a stability
assay was carried out. The sc NS3 was pre-incubated at
37 8C for up to 2 h before being added to the reaction.
As shown in Fig. 4C, the enzyme is stable and does not
lose activity during the testing period. The possibility
that the reaction plateaus due to exhaustion of substrate
is also unlikely, as performing the reaction with a large
Fig. 4. Cleavage of the fluorogenic substrate by sc NS3 protease. (A)
fluorometric assay: the fluorogenic substrate was incubated in the
presence (circles) or absence (triangles) of purified sc NS3 at 37 8C for
120 min. Aliquots of the cleavage reaction were taken at the indicated
time points and site-specific cleavage was measured by the release of
fluorescence. (B) Immunoblot analysis: cleavage reaction aliquots were
collected as in A, separated by 12% SDS-PAGE and electroblotted.
Proteins were detected with anti-GFP antibodies. The arrows mark the
position of the intact EGFP-NS5A/B site-CBD fusion protein and the
released EGFP. (C) Stability of sc NS3 assessed by pre-incubation of
the enzyme at 37 8C before initiating the cleavage reaction. Percent
catalysis was calculated from the activity of sc NS3 freshly added to the
reaction. Error bars represent the standard deviation of the data.
251
excess of substrate yields identical reaction curves (not
shown).
Since our substrate is a CBD-fusion protein that is
purified by cellulose-affinity chromatography, it may be
used as an immobilized substrate (by not eluting it from
the cellulose matrix) while the other components of the
reaction are added in the reaction buffer used to suspend
the matrix. However, we found that with immobilized
substrate the reaction is less efficient (about threefold
less fluorescence released vs. a similar reaction in
solution, not shown). This may be attributed to poor
accessibility of the scissile region of the substrate to the
enzyme while immobilized.
We next proceeded to analyze the kinetic parameters
of the cleavage reaction with our model substrate. To
this end we employed only the fluorometric assay that
enabled us to use much lower concentrations of the
enzyme than those employed in the experiment de-
scribed in Fig. 4. Under these conditions the cleavage
reaction was linear up to 60 min. First, we analyzed the
dependence of cleavage rate on enzyme concentration.
Reactions containing increasing concentrations of re-
combinant sc NS3 in the range of 0.01 /0.1 mM were
incubated for 60 min at 37 8C with the fluorogenic
substrate (0.5 mM), after which the initial velocity at
each enzyme concentration was calculated. It is clear
from Fig. 5A that the reaction rate is linear with enzyme
concentration over the entire range tested (R /0.967).
This linearity is a prerequisite for the use of this assay to
determine NS3 activity. A concentration of 40 nM
sc NS3 within the linear range was used in all further
experiments.
Next we examined the dependence of cleavage rate on
substrate concentration. The concentration range we
could use for this propose was limited since at concen-
trations higher than 2.5 mM (125 mg/ml) the background
fluorescence was too high to allow accurate determina-
tion of specific NS3 dependent cleavage. The purified
sc NS3 protein (40 nM) was incubated with the fluoro-
genic substrate in the range of 0.25 /2.5 mM for 60 min,
after which the initial velocity at each substrate con-
centration was calculated (Fig. 5B). As shown, the
cleavage rate is linear with substrate concentration
over the entire range tested. Thus, there is no evidence
for substrate saturation in the experimentally attainable
concentration range. The expected Km of this substrate
is therefore larger than 2.5 mM, and can not be obtained
from this data set. This lower limit is in accordance with
Km values for a wide range of NS3 substrates employed
in a variety of experimental systems (reviewed in Kwong
et al., 1998). However, a measure of the reaction
efficiency can be obtained by the value of kcat/Km, which
in turn can be calculated from the slope of the straight
line of V vs. S in Fig. 5B (when the substrate
concentrations are much lower than the Km, the
Michaelis /Menten equation can be approximated by a
252 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
Fig. 5. Kinetic parameters of sc NS3 protease catalysis. (A) Depen-
dence of catalytic rate on enzyme concentration. The reactions
consisting of 0.5 mM fluorogenic substrate and 0.01 /0.1 mM recombi-
nant sc NS3 were incubated for 60 min at 37 8C. The catalytic rate
was calculated from the slopes of fluorescence/time obtained at each
enzyme concentration. (B) Dependence of catalytic rate on substrate
concentration. The reactions consisting of 0.25 /2.5 mM fluorogenic
substrate and 40 nM recombinant sc NS3 were incubated for 60 min at
37 8C. The catalytic rate was calculated from the slopes of fluores-
cence/time obtained at each substrate concentration and by using a
calibration curve of fluorescence vs. substrate concentration.
straight line the slope of which is kcatE /Km). This value
was found to be 4170 (s1 M1), which is similar to
values obtained for a variety of NS5A/B substrates
(Kwong et al., 1998). This value represents the first
order rate constant of substrate cleavage at concentra-
tions lower than the Km.
3.3. Application of the fluorogenic substrate in a HTS
format for the identification of NS3 protease inhibitors
Before proceeding to HTS, we analyzed the perfor-
mance of our assay by applying a statistical analysis of
data collected from four independent activity assays.
The data was evaluated as described (Macarrón and
Hertzberg, 2002). The signal to background ratio (S/B )
was 3.87, the coefficient of variation of the signal was
7.24% and the Z factor, which is an indication of the
separation of the signal and background populations
was 0.44. Thus we can conclude that our assay satisfies
the criteria for a HTS assay. In addition, the recovery of
cellulose-unbound fractions for measuring fluorescence
that was initially carried out by centrifugation was
changed to a vacuum aspiration system that is adaptable
to robotics automation.
With the aim of performing a HTS of a large number
of compounds as potential NS3 inhibitors we modified
our assay to a 96 well plate HTS format as described in
Section 2. We first examined commercially available
protease inhibitors including serine protease inhibitors
such as, PMSF and TPCK at the concentrations
commonly used (Takeshita et al., 1997). None of these
inhibitors markedly inhibited the enzymatic reaction
(data not shown). However, when we used these
inhibitors at higher concentrations, a dose dependent
inhibition of catalysis could be observed (Fig. 6) which
is in agreement with the report of Kwong et al. (1998).
We next applied our assay to screen for an inhibitory
effect of crude aqueous herbal extracts from the tradi-
tional Indian Siddha medicinal plant on the sc NS3
protease activity. The results of an initial screen of 30
crude samples are shown in Fig. 7A. As shown, various
extracts inhibited the reaction at varying percentages
while other extracts had no inhibitory effect. The HTS
format allowed us to check in parallel the effect of the
extracts on the substrate itself (incubation without
enzyme). This built-in control is important particularly
when crude extracts are tested for inhibitory effect, since
such preparations may have intrinsic fluorescence, may
inadvertently quench the fluorescence of the substrate or
contain a non-specific proteolytic activity that could
flaw the results. The extracts we tested had no intrinsic
fluorescence (not shown). However, extracts that
quenched the fluorescence could be identified (Fig. 7A
extracts 4, 15, 17, 20 and 31), as well as extracts that
probably contain non-specific hydrolytic activity (Fig.
7A extracts 3, 5, 10, 12, 18 and 24). A combination of
Fig. 6. Inhibition of NS3 catalysis by known serine protease inhibi-
tors. The reactions were done in 100 ml in individual wells of 96 well-
plates containing 40 nM sc NS3 and 0.25 mM substrate that were
incubated for 1 h at 37 8C. For inhibition studies, PMSF (A) or
TPCK (B) at the indicated concentrations were mixed with the enzyme
in the wells prior to addition of the substrate. Percent inhibition was
calculated from control reactions done without inhibitors. Error bars
represent the standard deviation of the data.
 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255 253

Fig. 7. High throughput screen for inhibition of NS3 catalysis by crude herbal extracts. The reactions were done in 100 ml in individual wells of 96
well-plates containing 40 nM sc NS3 and 0.25 mM substrate that were incubated for 1 h at 37 8C. (A) For inhibition studies, inhibitors (1 ml of a
crude herbal extracts) were mixed with the enzyme in the wells prior to addition of the substrate (filled bars). For each extract, a control reaction was
carried out without enzyme (diagonally hatched open bars). Percent catalysis was calculated from control reactions done without inhibitors. (B)
Candidate inhibitors that were identified in A were re-evaluated in triplicate using 0.5 ml of extract per reaction. Error bars represent the standard
deviation of the data.

quenching and non-specific hydrolysis could be ob-
served for extracts 19 and 25. Extracts 1, 14, 16 and
23 were suspected to be genuine inhibitors and were
selected for further evaluation. These extracts were
tested in three additional repetitions and in triplicate
in each experiment. As shown in Fig. 7B, they were
confirmed as inhibitors of scNS3 catalysis. We are
currently fractionating these inhibitory extracts to
elucidate the nature of the inhibitory component. It is
noteworthy, that since all our assays are performed at
substrate concentration which are below the Km for
cleavage by NS3, our assay does not allow us to
determine whether the inhibitors act in a competitive
or non competitive manner. In addition, as any high-
throughput screen for enzyme inhibitors, our assay does
not check for specificity of inhibition, which, following
identification of candidate inhibitors, should be done
separately.
While initial NS3 catalytic assays were of a low-
throughput nature, the importance of high throughput
assays was appreciated which resulted in attempts to
adapt the peptide-based assays to an appropriate setting
(reviewed in Kwong et al., 1998). Subsequently, reports
of advances in high throughput assays, still applying
HPLC analysis of cleavage products and based on
peptide substrates have appeared (Sudo et al., 1996).
An ELISA based assay of cleavage activity was later
reported by the same group (Takeshita et al., 1997). This
assay was based on a biotinylated C-terminal 17-mer
peptide corresponding to the NS5A/B cleavage site.
After NS3 catalyzed proteolysis, the P1 cysteine was
acetylated with iodoacetate and reacted with N -hydro-
xysuccinimide-digoxigenin. The labeled product was
captured onto streptavidin coated microtiter plates and
detected with an anti-digoxigenin-alkaline phosphatase
conjugated antibody. Eventually, p-nitrophenyl phos-
254 Y. Berdichevsky et al. / Journal of Virological Methods 107 (2003) 245 /255
phate gave a colorimetric quantitation of the amount of
cleaved peptide. For such an analysis of a large number
of samples, the cost of reagents may become inhibitory.
Taliani et al. (1996) utilized a depsipeptide for the
development of a fluorometric assay based on resonance
energy transfer (FRET). The peptide contained a
fluorescent donor 5-[(2?-aminoethyl)amino]naphthalene
sulfonic acid (EDANS), and an acceptor group 4-[[4?-
(dimethylamino)phenyl]azo]benzoic acid (DABCYL).
When the substrate is intact, the fluorescence of the
donor is intramolecularly quenched by the acceptor
through resonance energy transfer. As the peptide is
cleaved, the quencher is no longer attached, and the
fluorescence of the donor generates an enhanced signal.
A similar approach was taken by Liu et al. (1999) and by
Kakiuchi et al. (1999), using other fluorescence donors
and acceptors. The latter work also demonstrated the
applicability of their high-throughput assay for the
identification of potential NS3 inhibitors. The high
sensitivity of the FRET based assay allows the use of
minimal enzyme concentrations, which would be re-
quired for screening compounds in nanomolar amounts
as found in some combinatorial libraries or natural
product collections. The fluorescent signal contributes
to the high sensitivity of the assay, but caution should be
practiced so that inhibitor quenching of the signal does
not contribute to false positives in the assay. A different
approach was undertaken by Zhang et al. (1999) who
developed a continuous spectrophotometric assay for
NS3 catalysis. In that case the substrate peptide was C-
terminally esterified with chromophoric alcohols so that
intact peptides having a different absorbance spectra
than that of the cleaved products. These authors claimed
that their assay should be suitable for the identification
of NS3 inhibitors. A radiometric in vitro assay for
discovery of NS3 protease inhibitors, suitable for high-
throughput screening was developed by Cerretani et al.
(1999). Recombinant NS3 proteases from different HCV
strains, purified from E. coli were used with a synthetic
radiolabeled peptide substrate that mimics the NS4A/B
junction. Upon incubation with the enzyme the sub-
strate was separated from the radiolabeled cleavage
product by addition of an ion exchange resin. The assay
was performed in a microtiter plate format and offered
the potential for assaying numerous samples using a
laboratory robot. It was claimed that the sensitivity of
the assay makes it suitable for detection and detailed
mechanistic characterization of inhibitors with low-
nanomolar affinities for the NS3 protease.
The above-described high-throughput NS3 catalysis
assays were all based on recombinant enzyme and
chemically synthesized peptide substrates, and in most
cases required chemical modification of the substrate
peptide or of the cleavage product for its detection. In
contrast, our assay is, to the best of our knowledge, the
first report where both enzyme and substrate are
recombinant proteins. Thus, our system may be applied
in any ordinary lab with no need for special instrumen-
tation or costly reagents. With regard to sensitivity, we
use nanomolar concentrations of enzyme and substrate
that are in the same range of the peptide-substrate-based
assays.
Acknowledgements
This research was supported in part by a Grant from
the Israel Health Ministry.
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