(Received for publication, August 7, 1995; and in revised form, November 18, 1995)
From the
A recombinant Baculovirus expression system was used for the
production of a 20-kDa protein encompassing the hepatitis C virus NS3
protease domain. The protein was purified to apparent homogeneity after
detergent extraction of cell homogenates. It was shown to be a monomer
in solution and to cleave the in vitro translated precursor
proteins NS4A-NS4B and NS5A-NS5B, but not the NS4B-NS5A or the NS3-NS4A
precursors. The enzyme also cleaved a 20-mer peptide corresponding to
the NS4A-NS4B junction with k/K
= 174 M
s
. Peptides harboring
NS4A sequences comprising amino acids 21-54
(Pep4A
) and 21-34
(Pep4A
) were found to induce an up to 2.8-fold
acceleration of cleavage. Kinetic analysis revealed that this
acceleration was due to an increase in k
,
whereas no significant effect on K
could
be detected. Pep4A
was also an absolute
requirement for cleavage of in vitro translated NS4B-NS5A by
the purified protease. From these data we conclude that: (i) the
purified protease domain shows substrate specificity and cleavage
requirements similar to those previously reported on the basis of
transfection experiments, (ii) activation of the purified protease by
the NS4A co-factor can be mimicked by synthetic peptide analogs, and
(iii) a central hydrophobic region of NS4A with a minimum core of 14
amino acids is responsible for the interaction with NS3.
The hepatitis C virus (HCV) ()is the causative agent
of parenterally transmitted non-A non-B
hepatitis(1, 2) . The virus contains a positive
stranded RNA genome of 9.5 kilobases with a single open reading frame
encoding for a polyprotein of 3010-3033 amino
acids(3, 4, 5, 6) . Upon translation
this polyprotein is proteolytically processed into nine different
polypeptides, which are encoded as follows on the viral RNA:
5`-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3`.
The mature structural proteins, C (the nucleocapsid protein), E1, and E2 (the two envelope proteins) have been shown to arise via proteolytic processing by host signal peptidases(7, 8) .
Conversely, generation of the mature nonstructural proteins NS2 through NS5B relies on the activities of virally encoded proteases. Thus, cleavage at the NS2-NS3 junction is accomplished by an as yet poorly characterized protease encoded between NS2 and NS3(9, 10) . We (11) and others (12, 13, 14, 15, 16) have shown all subsequent cleavages downstream of NS3, i.e. at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions, to be catalyzed by a serine protease contained within the N-terminal region of NS3.
Characterization of cleavage events has shown that there are kinetic differences in processing of the single junctions. Thus, while processing between NS3 and NS4A is an intramolecular event, cleavage at all other sites has been demonstrated to occur in trans.
Homology modeling of the active site of the NS3 protease has permitted us to predict the preference for a cysteine residue at the P1 (according to the nomenclature introduced by Schechter and Berger(17) ) positions of the substrates(18) . Subsequent sequencing of the single cleavage sites has partially confirmed our predictions and yielded the consensus sequence D/E-X-X-X-X-C-A/S for all trans cleavage sites, with X being any amino acid and the scissile bond being located between C and A or S(12, 18) . Notably, the intramolecular cleavage site between NS3 and NS4A differs from this consensus having a threonine residue in the P1 position.
NS3 is necessary, but not sufficient, for cleavage events within the polyprotein between NS3 and NS5B. As a matter of fact we (19) and others (20) have shown the NS3 protease to be a heterodimeric protein in vivo consisting of both NS3 and NS4A. Truncation experiments have mapped the N terminus of NS3 as the domain responsible for interaction with NS4A(21) . The same region has been recently shown to be sufficient for NS4A binding when fused to a heterologous protein (22) . In transfection experiments the interaction between NS3 and NS4A accelerates basal cleavage rates at the NS4A-NS4B and NS5A-NS5B junctions, whereas no cleavage occurs at the NS4B-NS5A site in the absence of NS4A, making the correct processing of the NS4B-NS5A precursor completely dependent upon the presence of NS4A(19, 20) .
NS4A is a 54-residue protein. Deletion mutagenesis experiments have demonstrated that a central region of NS4A spanning from residue 22 through 34 is sufficient for co-factor activity(23) .
Neither the mechanism of protease activation by NS4A nor the reasons for the different cleavage kinetics are known. Mechanistic investigations are hampered by difficulties in obtaining sufficient amounts of pure, active protease. As a matter of fact, the NS3 protein is a multidomain protein of 70 kDa, which, in addition to the protease domain at the N terminus, contains a putative RNA-helicase at its C terminus. C- and N-terminal truncation experiments (21) have demonstrated that a 20-kDa N-terminal fragment of NS3 is capable of performing all cleavages in in vitro translation and in transfection experiments with an efficiency indistinguishable from that of the wild type enzyme, retaining its ability to interact with NS4A (21) .
In this paper, we report the expression of this NS3 protease domain in a Baculovirus expression system, the purification and the characterization of the in vitro activity of this protein. We further show that peptides encompassing sequences of NS4A are capable of activating the purified protease and demonstrate that a minimum central core region of 14 amino acids in fact mediates the interaction of NS4A with NS3.
The pBacNS5AB plasmid was obtained inserting into the BamHI site of the pBlueBacIII vector a DNA fragment encoding HVC polyprotein from amino acids 1973 to 3011. Linearized AcNPV DNA (Invitrogen) was co-transfected with each plasmid into the insect cell line Sf9 to obtain recombinant Baculovirus vBacPro and vBacNS5AB expressing the NS3 protease domain or the NS5AB polyprotein, respectively. Viral plaques were isolated and amplified according to the protocol recommended by the manufacturer.
The plasmids pCiteNS3-4A, pCiteNS4AB, pCiteNS4B5A, and
pCiteNS5AB expressing, respectively, the HCV proteins NS3-4A from
residue 992 to residue 1711, NS4AB from residue 1649 to residue 1964,
NS4B5A from residue 1775 to residue 2380, and NS5AB from residue 1965
to residue 3010 were described previously(19) .
NS3-NS4Apro was obtained from pCiteNS3-4A by digestion with MscI and SalI, resulting in a transcript expressing
residues 1462-1711. NS4BNS5A
C216 was obtained from
pCiteNS4B5A by digestion with NheI, resulting in a transcript
encompassing residues 1712-2203, while NS5A-NS5B
C51 was
obtained by digestion of pCiteNS5AB with BstEI. In vitro transcription was done with T7 RNA polymerase. The transcripts
were translated for 1 h at 30 °C in the presence of
[
S]methionine using an RNA-dependent rabbit
reticulocyte lysate (Promega). Aliquots of purified NS3 protease were
added to the translated proteins, and the mixture was incubated for up
to 2 h at 22 °C. Cleavage of labeled precursors was assessed by
SDS-PAGE on 12.5% gels. Control experiments were included to verify the
identity of precursors and cleavage products by immunoprecipitation
using specific antisera as described previously(11) . Data were
analyzed on a PhosphorImager and quantified by volumetric integration
using ImageQuant software.
The following peptides were used: Pep4AB,
Fmoc-Y-Q-E-F-D-E-M-E-E-C-A-S-H-L-P-Y-I-E-Q-G;
Pep4A,
G-S-V-V-I-V-G-R-I-I-L-S-G-R-P-A-I-V-P-D-R-E-L-L-Y-Q-E-F-D-E-M-E-E-Abu;
Pep4A
,
G-R-P-A-I-V-P-D-R-E-L-L-Y-Q-E-F-D-E-M-E-E-Abu;
Pep4A
, G-S-V-V-I-V-G-R-I-I-L-S-G-R with Abu
= amino butyric acid substituting the cysteine residue present
in the original sequence.
Cleavage assays were performed using 300
nM to 1.6 µM enzyme in 30 µl of 50 mM Tris, pH 7.5, 50% glycerol, 2% CHAPS, 30 mM DTT, and
appropriate amounts of substrate and/or NS4A-peptide, such that the
final concentration of MeSO did not exceed 10%. This
Me
SO concentration was shown not to affect enzyme activity.
After incubation for variable time intervals at 22 °C, the reaction
was stopped by addition of 70 µl of H
O containing 0.1%
trifluoroacetic acid. pH dependence experiments were carried out using
the following buffers: pH 6.0-7.5, sodium phosphate; pH
7.5-9.0, Tris; pH 9.0-10.5, sodium borate. At overlapping
pH values activity was determined with two different buffer systems and
shown not to be affected by buffer composition. Ionic strenghth was
kept constant at 20 mM.
Cleavage of peptide substrates was
assessed by HPLC using a Merck-Hitachi chromatograph equipped with an
autosampler. 90-µl samples were injected on a reversed phase HPLC
column (C18 Lichrospher, 5 µm, 0.4 12.5 cm, Merck) and
fragments were separated using a 30-100% acetonitrile gradient at
2%/min. Peak detection was done by monitoring both absorbance at 220 nm
and the fluorescence of the N-terminal Fmoc group (excitation, 260 nm;
emission, 305 nm). Peptide fragments eluting from the HPLC column were
collected and identified by mass spectrometry.
Cleavage products
were quantified by integration of chromatograms with respect to the
standard peptide Fmoc-Y-Q-E-F-D-E-M-E-E-C. Initial rates of cleavage
were determined at <20% substrate conversion. The kinetic parameters
of the proteolysis reaction were calculated from least squares fit of
initial rates as a function of substrate concentration assuming
Michaelis-Menten kinetics with the help of a Grafit or a Kaleidagraph
software. k/K
values were
calculated from initial rates determined at substrate concentrations
< K
.
Binding of
Pep4Ato the Purified
Protease-To estimate binding parameters of
Pep4A
to the isolated protease rate enhancements
relative to samples containing the protease alone were determined as a
function of Pep4A
concentration. 200 nM to 3.2 µM Pep4A
were added to
solutions of 600 nM protease in 50 mM Tris, pH 7.5,
50% glycerol, 2% CHAPS, 30 mM DTT and incubated for 10 min,
after which the reaction was started by addition of 60 µM (2 K
) substrate Pep4AB. The reaction was
stopped at <20% substrate conversion.
V was defined as
the enhancement of velocity of substrate conversion at a given
concentration of Pep4A
, and
V
as the velocity enhancement at
Pep4A
-saturation. The enzyme-bound
Pep4A
at a given
V can then be
calculated from , assuming a 1:1 stoichiometry of the
NS3-Pep4A
complex (see
``Results''),
with [E] being the total
concentration of protease in the assay. Free and bound species were
calculated from total Pep4A
concentrations, and
binding parameters were obtained from fitting data to a Scatchard plot.
Figure 1:
Expression of enzymatically active,
membrane bound NS3 protease domain in Sf9 cells. A, Sf9 cells
were infected with recombinant Baculovirus as described under
``Materials and Methods.'' 1.3 10
cells
were collected by centrifugation at the indicated time points, lysed in
SDS sample buffer, and loaded on a 15% polyacrylamide gel. The protease
was visualized by immunoblotting. The migration positions of molecular
mass markers are indicated on the left. B,
homogenates of Sf9 cells expressing the NS3 protease were prepared in
20 mM HEPES, pH 8.0, 1.5 mM MgCl
, 0.5
mM EDTA, 3 mM DTT and centrifuged for 1 h at 100,000
g. Pellet (lane 1) and supernatant (lane
2) were loaded on a 15% polyacrylamide gel followed by an
immunoblot using anti-NS3 antibodies. The pellet was subsequently
loaded on a 38-65% sucrose gradient in 20 mM Tris, pH
7.5, 150 mM KCl, 2 mM MgCl
and
centrifuged for 16 h at 100,000
g. The presence of the
protease in the single fractions was monitored by immunoblotting. The triangle indicates increasing sucrose concentration. C, Sf9 cells were co-infected with recombinant Baculoviruses
encoding the NS3 protease and the NS5A-NS5B substrate as described
under ``Materials and Methods.'' After labeling with
S, proteins were immunoprecipitated using anti-NS3
(
3), anti-NS5A (
5A), or anti-NS5B (
5B) antibodies. The
immunoprecipitates were loaded on 12% polyacrylamide gels and revealed
by autoradiography. The molecular masses were estimated from the
migration positions of
C-labeled molecular mass
markers.
Upon
centrifugation at 100,000 g, the protein was almost
quantitatively detectable in the pellet (Fig. 1B, lanes S and P). To discriminate whether this was attributable to
formation of protein aggregates or to a partitioning of the protein
into cell membranes the pellet was loaded on sucrose gradients (Fig. 1B). During these centrifugation experiments the
immunoreactive NS3 protein migrated at low density, demonstrating that
the protease was not present under the form of an aggregate and was
most likely membrane-bound in Sf9 cells.
To determine whether the
protein was also expressed in an enzymatically active form Sf9 cells
were co-infected with recombinant Baculovirus encoding the NS5A-NS5B
precursor protein. Cells were labeled with
[S]methionine, and proteins were
immunoprecipitated with anti NS3, anti-NS5A, or anti-NS5B, respectively (Fig. 1C). The appearance of immunoprecipitable
polypeptides, migrating at the expected molecular mass positions of
mature NS5A and NS5B, indicated the expression of enzymatically active
NS3 protease in Sf9 cells (Fig. 1C). In control cells
not expressing NS3, on the other hand, only the uncleaved NS5A-NS5B
precursor was detectable. These findings prompted us to attempt the
purification of the enzyme.
Figure 2: Purification of the NS3 protease domain. Samples deriving from single steps of the purification procedure were loaded on an SDS-12.5% polyacrylamide gel, and bands were visualized by Coomassie staining. Lane 1, molecular mass markers, lane 2, homogenate; lane 3, S-Sepharose pool; lane 4, Superdex-75 pool; lane 5, Mono S pool.
Sf9
cells expressing the NS3 protease were collected 72 h post-infection.
After homogenization and centrifugation of the homogenate, the
resulting 120,000 g supernantant was chromatographed
on a cation exchange column at pH 6.5, followed by gel filtration
chromatography, which simultaneously shifted the pH of the sample to
7.5. A final ion exchange step performed at pH 7.5 removed residual
contaminants and yielded a protein that was homogeneous as judged by
SDS-PAGE (Fig. 2).
Since molecular mass determination under native and under denaturing conditions yielded the same results (molecular mass of 22.8 kDa as judged from SDS-PAGE, 23.6 kDa as determined by gel filtration, versus 20.1 kDa calculated from the primary sequence), we conclude that the protein was present as a monomeric species in solution.
Figure 3:
Activity of the purified protease on in vitro translated precursor substrates. NS3-NS4APro,
NS4A-NS4B, NS4B-NS5A
C216, and NS5A-NS5B
C51 precursor proteins
were produced by in vitro translation of the appropriate RNAs
in the presence of
S-labeled methionine as described under
``Materials and Methods.'' A stock solution containing 11
µM protease was diluted in 50 mM Tris, pH 7.5,
50% glycerol, 2% CHAPS, 30 mM DTT to yield a series of diluted
solutions. 5 µl of these dilutions or 5 µl of buffer alone were
added to 5 µl of in vitro translated precursor and
incubated for 1 h at 30 °C. Final protease concentrations were 0,
203 nM, 610 nM, 1.8 µM, and 5.5
µM. Triangles indicate increasing protease
concentration. Each set of experiments was repeated in the presence of
100 µM final concentration of Pep4A
(+Pep4A) added from a 2 mM stock solution
in Me
SO. Reactions were stopped by addition of 15 µl of
SDS sample buffer. Samples were run on 12.5% polyacrylamide gels and
proteins detected by autoradiography (A) or quantified by
densitometry (B: squares, +Pep4A; circles, control). One experiment, representative of five, in
which the same trend was consistently observed, is
shown.
We wanted to address
the question of the role of NS4A in the cleavage efficiency of the
protease at different sites and synthesized a 34-mer peptide
corresponding to the C terminus of NS4A by solid phase synthesis
(Pep4A, see ``Materials and Methods'').
We compared the effects of this peptide on the activity of the protease
on all precursors. As shown in Fig. 3, Pep4A
caused a modest increase of processing at the NS4A-NS4B and
NS5A-NS5B sites. The former precursor already contains the NS4A
sequences present in Pep4A
, which should render
the processing at this site less NS4A-dependent. Still no cleavage was
detectable at the cis cleavage site, NS3-NS4A, after addition of
Pep4A
. However, processing was now also
detectable at the NS4B-NS5A site, although at higher enzyme
concentrations as those required for efficient cleavage of the other
two trans sites.
Next, the cleavage of in vitro translated
NS5A-NS5BC51 was used as an assay for testing the inhibitory
potential of a series of protease inhibitors on NS3 (Fig. 4).
Classical serine protease inhibitors such as PMSF and diisopropyl
fluorophosphate, the chymotrypsin inhibitors TPCK as well as aprotinin
were effective in inhibiting the NS3 protease. On the other hand, other
trypsin, chymotrypsin, cysteine- and metalloproteinase inhibitors were
unable to yield significant inhibition of the activity of NS3. Together
with previous mutagenesis studies(11) , these findings about
its reactivity confirm the identity of the NS3 protease as serine
protease.
Figure 4:
Effect of protease inhibitors on the
processing of the NS5A-NS5BC51 precursor by the purified protease.
Pretranslated radiolabeled NS5A-NS5B
C51 precursor was incubated in
the presence of purified NS3 protease and different canonical protease
inhibitors as described under ``Materials and Methods.''
Uncleaved precursor and cleavage products were separated on an SDS-12%
polyacrylamide gel and visualized by autoradiography. The percentage of
cleavage in the presence of added inhibitors was determined by
densitometric analysis and data were expressed as percent residual
activity with respect to appropriate control samples. Inhibitors and
their final concentrations were: DFP, diisopropyl
fluorophosphate (1 mM or 10 mM); PMSF,
phenylmethylsulfonyl fluoride (1 mM); TPCK,
tosylphenylalanyl chloromethyl ketone (1 mM); TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone (0.5 mM); aprotinin (0.5 mg/ml);
chymostatin (0.5 mg/ml); phenanthroline (2 mM); EDTA (2
mM); ZnCl
(2 mM); leupeptin (0.5 mg/ml).
Data are from one experiment representative of
three.
The cleavage efficiency was highly dependent on the
detergent concentration in the assay mix, drastically declining at
CHAPS concentrations below the critical micelle concentration value
(not shown). The cleavage reaction of Pep4AB had a pH optimum around pH
8.5 and activity titration yielded an apparent pK = 7.0, which are common values for most serine proteases.
We next addressed the question of whether synthetic NS4A analogs
were able to increase cleavage efficiency of the purified protease also
using Pep4AB as substrate. Furthermore, we wanted to verify that the
minimum core region of NS4A is still capable of eliciting full
activation of the isolated NS3 protease domain. To this purpose we
compared the effects of Pep4A and the truncated
peptides Pep4A
and Pep4A
.
As shown in Table 1Pep4A
and
Pep4A
, but not equivalent amounts of
Pep4A
, were able to stimulate the activity,
expressed as k
/K
, of the
purified NS3 protease. These data directly confirm the region between
amino acids 21 and 34 of NS4A as being responsible for the interaction
with NS3 and show that the effect of NS4A is to enhance the efficiency
of enzymatic catalysis. We wanted to further dissect the effect of NS4A
by determining whether the peptide was increasing the affinity of the
enzyme for its substrate or enhancing the catalytic rate. To this
purpose substrate titration curves were fitted to the Michaelis-Menten
equation and the kinetic parameters were calculated (Table 1).
These experiments demonstrate that Pep4A
did not
significantly affect K
values but acted on the
rate of catalysis by increasing k
values.
To
evaluate the affinity of Pep4A for the NS3
protease a Pep4A
titration experiment was done
monitoring the relative rate enhancement (Fig. 5). Since we
found the protease to be still monomeric after complex formation with
NS4A (not shown), a 1:1 stoichiometry was assumed for the
NS3-Pep4A
complex. Based on this assumption an
apparent K
of 0.22 µM was calculated
from a Scatchard plot (Fig. 5).
Figure 5:
Binding parameters of
Pep4A to the NS3 protease calculated from kinetic
data. To 600 nM protease different amounts of
Pep4A
in Me
SO were added and the
reaction was started by addition of 60 µM substrate
peptide, Pep4AB. After 90-min incubation at 23 °C, the reaction was
stopped by addition of 0.1% trifluoroacetic acid. The activity
increase, relative to samples incubated with Me
SO alone,
produced by a given amount of Pep4A
4 was plotted
against the concentration of Pep4A
(A).
From these data the amount of bound peptide was calculated (see
``Materials and Methods''), and binding parameters were
determined from a Scatchard plot (B). The data obtained were
apparent K
= 0.22 µM with 0.84 binding sites/enzyme
molecule.
We here describe the purification of the hepatitis C virus NS3 protease domain from recombinant Baculovirus and the characterization of its enzymatic activity in vitro.
The enzyme was found to be presumably membrane-associated in Sf9 cells, and detergent extraction was necessary to recover appreciable amounts of soluble protein.
The purified enzyme showed a very low specific
activity, which is reminiscent of what has been reported for other
viral proteases. As a matter of fact, both human cytomegalovirus
protease (k/K
=
12-400 M
s
,(27) ) and herpes simplex virus
protease (k
/K
=
17-37 M
s
(28, 29) ) display cleavage
kinetics that are comparable with the parameters we have determined for
the HCV protease (k
/K
= 174 M
s
).
While this manuscript was in preparation, two reports, describing the
purification of fusion proteins encompassing both the protease and the
helicase domains of the NS3 protein, were
published(30, 31) . One report (31) describes
the activity on a peptide substrate corresponding to the NS5A-NS5B
junction. The kinetic parameters that can be calculated from the
published data (k
/K
= 100 M
s
)
are in good agreement with our own findings.
From transfection
experiments the temporal hierarchy of cleavage events of NS3-dependent
junctions within the nonstructural region has been determined as being:
NS3-NS4A > NS5A-NS5B > NS4A-NS4B NS4B-NS5A, the latter
cleavage being completely dependent upon the presence of NS4A. It is
likely that this hierarchy reflects physiological requirements of the
viral life cycle that are still elusive. Nor do we understand what
factors govern the different cleavage kinetics at the single sites.
Many open questions probably will have to be addressed by means of
kinetic studies on purified proteins. As a first approach in this
direction we investigated the activity of the purified NS3 protease
domain on precursor proteins bearing all cleavage sites and on a
synthetic peptide substrate.
The purified enzyme showed the highest activity on in vitro translated NS5A-NS5B, followed by NS4A-NS4B, while NS4B-NS5A was not detectably cleaved. The latter precursor was cleaved only in the presence of a peptide corresponding to the 34 C-terminal amino acids of NS4A. The same peptide had only very modest effects on the cleavage efficiency at the NS5A-NS5B and NS4A-NS4B sites. Furthermore, no processing was observed in either experimental condition at the NS3-NS4A site. This site, however, is known to be cleaved in cis only, suggesting that steric factors may hinder the cleavage of the NS3-NS4A precursor by the added purified protease.
The activation of NS3 by Pep4A deserves some further comment. It has been shown recently that
NS3 and NS4A form an immunoprecipitable complex in transfected
cells(21, 22) . On the basis of these data, the
activated form of the NS3 protease therefore appears to be a
heterodimeric protein consisting of both polypeptides. Several
different mechanisms have been suggested to explain the activation of
the NS3 protease by the co-factor
NS4A(19, 20, 21, 22, 23, 32) ,
including stabilization, membrane anchoring, alteration of cleavage
site specificity, direct contribution to substrate recognition, or
induction of structural changes in the substrate binding pocket(s).
Although we have some indication of stabilization of the protease
toward thermal inactivation upon binding to
Pep4A
, our kinetic data favor the latter
hypothesis. In fact, we have shown the major effect of NS4A to be on
the catalytic rate constant k
. This rate
constant could be increased due to structural rearrangements altering
the nucleophilicity of the active site serine residue or affecting
transition state binding. It has to be pointed out, however, that these
kinetic differences were observed using a peptide derived from an
NS4A-independent cleavage site. Comparison of the very small effects of
Pep4A
on the processing efficiency of the in
vitro translated NS4A-NS4B precursor with the absolute
Pep4A
requirement for NS4B-NS5A precursor
processing (Fig. 3) suggests that different mechanisms might
account for the effects of NS4A on the processing at the single
cleavage sites.
The interaction domain with NS4A has been mapped to
the N terminus of NS3(21, 22) . In this work we have
addressed the question of what region of NS4A interacts with NS3. A
recent report, using a recombinant vaccinia/transfection system comes
to the conclusion that a 13-amino acid region spanning residues
22-34 of NS4A is crucial for the interaction with
NS3(23) . These findings are confirmed by our observation that
deletion of 12 amino acids at the N terminus of Pep4A abolishes its ability to activate NS3, strongly arguing for an
involvement of these residues in the interaction with NS3. A further
proof of this assumption is given by the fact that the 14-mer peptide
Pep4A
binds with high affinity to the purified
protease and has a potential of activating NS3, which is
undistinguishable from the effect observed upon addition of
Pep4A
. It is interesting to notice that structure
predictions of NS4A predict two
-helices with a highly hydrophobic
region in their middle. This prediction has been partially confirmed by
CD spectra of Pep4A
(
)Notably, the
residues which are apparently crucial for the interaction with NS3 fall
exactly in this region, indicating that a hydrophobic extended
structure of NS4A contacts the N-terminal domain of NS3. Proteolytic
events mediated by the NS3 protease are likely to be absolute
requirements for the generation of an active viral replication
apparatus. Sequence alignments point to NS5B as harboring the HCV
RNA-dependent RNA-polymerase. As a matter of fact, this protein is
generated by an NS3-dependent cleavage. Thus, HCV represents an
intriguing example of regulation of both gene expression and
replication by crucial proteolysis steps. The elucidation of the role
of NS4A in these processes awaits further detailed studies in vitro as well as in vivo. Furthermore, based on these
considerations, the enzyme appears as being an attractive candidate
target for the development of anti-HCV therapeutics. A deeper
understanding of the regulation and the substrate requirements of the
protease will help to develop first generation inhibitors.