Unmasking the active helicase conformation of nonstructural protein 3 from hepatitis C virus.

Abstract

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific β-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the β-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3ΔC7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3ΔC7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3ΔC7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3ΔC7 is independent of the substrate 3'-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.

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JOURNAL OF VIROLOGY, May 2011, p. 4343–4353
0022-538X/11/$12.00doi:10.1128/JVI.02130-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 9
Unmasking the Active Helicase Conformation of Nonstructural
Protein 3 from Hepatitis C Virus?†‡
Steve C. Ding,1Andrew S. Kohlway,1and Anna M. Pyle2,3*
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 065201; Department of
Molecular, Cellular, and Developmental Biology and Department of Chemistry, Yale University, New Haven,
Connecticut 065202; and Howard Hughes Medical Institute, Chevy Chase, Maryland 208153
Received 8 October 2010/Accepted 8 February 2011
The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus
(HCV) replication complex. We hypothesized that a specific ?-strand tethers the C terminus of the helicase
domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the
extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the
?-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3?C7).
Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3?C7 adopts an extended
conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation
of NS3?C7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also
show that the unwinding activity of NS3?C7 is independent of the substrate 3?-overhang length, implying that
a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended
conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of
the protein during viral replication.
Nonstructural protein 3 (NS3) is an essential member of the
hepatitis C virus (HCV) replication complex (2, 33). It is a
bifunctional enzyme that contains a serine protease domain
within the N-terminal third of the protein and a nucleic acid-
stimulated NTPase/helicase domain within the C-terminal two-
thirds of the protein (40). Both enzymatic activities are critical
for HCV replication. In the presence of the viral NS4A cofac-
tor protein, the NS3 protease activity cleaves and releases
downstream viral proteins from the precursor polyprotein (13).
Furthermore, the NS3 protease represses the host innate im-
mune response by cleaving cellular proteins such as TRIF and
MAVS, thereby preventing a signaling cascade that leads to an
antiviral cellular response (17, 24, 32, 44, 51). In addition to its
protease activity, NS3 displays robust helicase activity (46).
NS3 helicase activity is essential for replication of the viral
RNA genome (21), potentially functioning together with the
NS5B polymerase during viral replication (16). NS3 also par-
ticipates in the intracellular assembly and packaging of infec-
tious virus particles (30). Given its multiple roles throughout
the viral life cycle, NS3 is an important target for antiviral drug
discovery against HCV (5).
The NS3 helicase (NS3hel) domain belongs to the DExH/D
subgroup of DNA and RNA helicases within helicase super-
family 2 (37, 52). Members of this family contain a core heli-
case structure consisting of two RecA-like folds (domains 1
and 2) arranged in tandem. Together with these two RecA-like
domains, NS3hel has a third domain (domain 3) that forms a
single-stranded DNA/RNA binding groove (Fig. 1A) (20, 29).
The NS3hel construct has been studied extensively and dis-
plays modest helicase activity in isolation. However, a unique
feature of NS3 that distinguishes it from its other family mem-
bers is the covalent attachment of the protease domain. Pre-
vious work has demonstrated a strong functional interdepen-
dence in the enzymatic activities of the protease and helicase
domains (7, 8). The protease domain not only promotes direct
and functional binding of the RNA substrate, but it also im-
proves the translocation stepping efficiency of the helicase (8,
39). Therefore, full-length NS3 is an attractive target for the
development of antiviral strategies.
While previous studies have provided important insights into
the mechanism of RNA unwinding by NS3 (11, 34, 43), we
know very little about the structural rearrangements that occur
when NS3 switches between its protease and helicase activities.
One way NS3 might regulate its functional activities is through
global conformational changes. An increasing body of evidence
suggests that NS3 adopts additional conformations that differ
from the one observed in the crystal structure of the full-length
enzyme, which captured a snapshot of the protein performing
a protease cleavage event in cis (53). One clue to the existence
of alternative conformations came from early kinetic data
showing that NS3 requires an extended incubation time with
RNA substrates prior to reaching its maximal unwinding ac-
tivity (36), suggesting a rate-limiting conformational change.
Second, the structures of NS3 proteins from other Flaviviridae
family members show that the biologically relevant confor-
mation is an extended form where the protease domain sits
beneath the helicase domain (4, 27, 28). These extended
conformations contrast markedly with the compact confor-
mation seen in the crystal structure of full-length HCV NS3
(53). Finally, when HCV NS3 was modeled onto a mem-
* Corresponding author. Mailing address: 266 Whitney Ave., Bass
Bldg., Rm. 334, Yale University, New Haven, CT 06520. Phone: (203)
432-5633. Fax: (203) 432-5316. E-mail: anna.pyle@yale.edu.
† Supplemental material for this article may be found at http://jvi
.asm.org/.
?Published ahead of print on 16 February 2011.
‡ The authors have paid a fee to allow immediate free access to
this article.
4343

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brane bilayer, the helicase domain was positioned in an
impossible configuration within the membrane (9). Based on
this finding, Moradpour and colleagues concluded that a
significant conformational change must occur in order to
orient the helicase domain toward the cytoplasm and prop-
erly position it to interact with other components of the
replication complex. Thus, we sought to understand how
such a conformational change might occur in HCV NS3 and
determine how this change would affect the function of NS3
as a helicase.
Here, we show that the C-terminal ?-strand of HCV NS3
acts as a toggle that alters the structural and functional prop-
erties of the protein. The presence or absence of this ?-strand
changes the global architecture of NS3 and allows the protein
to adopt different conformational states. Moreover, removal of
this ?-strand allows the truncated protein (NS3?C7) to adopt
an extended conformation and form functional complexes with
RNA unwinding substrates faster than WT NS3. In contrast to
NS3?C7, WT NS3 cannot react efficiently under unwinding
conditions that stabilize the resulting ?-sheet substructure.
Finally, we demonstrate that NS3?C7 readily unwinds RNA
with short 3?-overhang strands and behaves like a processive
monomer. Taken together, we propose a model in which
NS3?C7 adopts a conformation that is distinct from the
FIG. 1. Domain orientations of DENV NS3 and HCV NS3 proteins. (A) The NS3 proteins from DENV (Protein Data Bank [PDB] no.
2VBC) and (B) HCV (PDB no. 1CU1) are shown in cartoon form, each with a 90° rotation. The protease and helicase domains are colored
in red and yellow, respectively. Subdomains of the helicase domains are labeled. The linker region connecting the two domains is colored
in blue. Note that the protease domain of DENV NS3 is located “below” the ATP binding pocket, while that of HCV NS3 is located “behind”
the ATP binding pocket. The NS3 protein from MVEV was crystallized in an extended conformation similar to DENV NS3 (data not shown)
(PDB no. 2WV9). (C) The C terminus of HCV NS3 forms a ?-strand and feeds back into the protease active site to form a ?-sheet (circled
in green). The catalytic triad residues of the protease domain are shown as green sticks. Figures were generated by PyMol.
4344DING ET AL.J. VIROL.

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conformation observed in the crystal structure and which
represents the biologically relevant form of NS3 as an active
helicase.
MATERIALS AND METHODS
Materials. DNA oligonucleotides were obtained from Invitrogen. RNA oligo-
nucleotides were synthesized on an automated MerMade 6 synthesizer (Bio-
Automation) by using phosphoramidite chemistry (phosphoramidites purchased
from Glen Research) and deprotected before gel purification (48).
RNA duplex substrates. The sequences of the RNA duplexes used in this study
were described previously (18, 36). RNA duplexes were labeled with [?-32P]ATP
as described previously (6), purified from semidenaturing polyacrylamide gels
(15% acrylamide, 2 M urea, 0.5? Tris-borate-EDTA [TBE]), and stored at
?80°C in ME buffer (10 mM MOPS [morphoplinepropanesulfonic acid], pH 6.5,
1 mM EDTA). Duplex concentrations were determined with a NanoDrop
(Thermo Scientific), and extinction coefficients were calculated from the se-
quences.
Protein purification. Proteins were purified as described previously with mod-
ifications (6). Proteins were expressed in Rosetta II (DE3) cells (Novagen) with
0.25 mM IPTG (isopropyl-?-D-thiogalactopyranoside) for 20 h at 16°C. Proteins
were purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) and heparin
affinity columns (GE Healthcare) before gel filtration over a 16/60 Superdex 200
column (GE Healthcare) equilibrated in storage buffer (10 mM HEPES, 300 mM
NaCl, 10% glycerol, 5 mM ?-mercaptoethanol [?-ME], pH 7.0). Monodisperse
fractions were concentrated, flash frozen in liquid nitrogen, and stored at ?80°C.
Protein concentrations were measured in 6 M guanidinium at ? ? 280 nm using
an extinction coefficient of ε ? 63,580 M?1cm?1(12).
Limited proteolysis assay. Lyophilized carboxypeptidase Y (CPY) was ob-
tained from Worthington Biochemical (Lakewood, NJ) and resuspended in stor-
age buffer (100 mM MOPS, 10% glycerol, 300 mM NaCl, 5 mM ?-ME, pH 6.3)
to a final concentration of 1 mg/ml. WT NS3 and NS3?C7 proteins were diluted
in this buffer to final concentrations of 300 ?g/ml. Proteolysis experiments were
performed at 24°C with 360 ?g protein and either 0, 1, 5, or 10 ?g carboxypep-
tidase Y. Proteolysis was monitored by quenching 15-?g protein aliquots, resolv-
ing 2.5 ?g on 10% SDS-PAGE gels, and staining with Coomassie brilliant blue.
Sedimentation velocity studies. Experiments were performed at 25°C in a
buffer containing 10 mM HEPES, 150 mM NaCl, 5% glycerol, 5 mM ?-ME, pH
7.0, in a Beckman Optima XL-I analytical ultracentrifuge. A four-position AN 60
Ti rotor, together with Epon 12-mm double-sector centerpieces, was used at
42,000 rpm. Radial absorption scans were measured at ? ? 280 nm with a radial
increment of 0.003 cm. Data analyses were performed in Sedfit 8.0 (http://www
.analyticalultracentrifugation.com) (41), using a 95% confidence limit. Sedimen-
tation coefficients at the experimental temperature, buffer density, and viscosity
were corrected to standard conditions (s20, w) using the program SEDNTERP
(http://jphilo.mailway.com).
Functional complex formation assay. Functional complex formation assays
were performed as previously described with modifications (36). A master mix
containing 25 nM protein and 1 nM RNA duplex (final concentrations) was
assembled in helicase reaction buffer (42) for 10 min on ice and then divided into
9-?l reaction aliquots. Each reaction mixture was incubated at 37°C for various
amounts of time, and then 3 ?l of an ATP-trap mixture (16 mM ATP and 4 ?M
protein trap in helicase assay buffer) was added to initiate a single cycle of
unwinding. The trap was a 60-nucleotide (nt), single-stranded DNA oligonu-
cleotide (42). We used a 34-nt, single-stranded RNA oligonucleotide as the trap
when we sought to increase the final unwinding amplitude (6). Reactions
were quenched after 5 min by adding 3 ?l of a 5? quench buffer (42) and
immediately transferred to dry ice. Duplex and single-stranded RNAs were
separated on semidenaturing polyacrylamide gels (described above), visual-
ized with a Storm820 PhosphorImager, and quantified with ImageQuant soft-
ware (GE Healthcare). The fraction of unwound duplex for each time point was
quantitated by the following formula: Aunw? Iss/(Iss? Ids), where Issis the
intensity of the displaced strand and Idsis the intensity of the duplex substrate.
At least three experiments were performed for each kinetic analysis reported
here.
Data analysis. Data were processed with Origin 8.0 (OriginLab Corpora-
tion). Kinetic parameters describing the data for each protein and its set of
unwinding substrates were determined by using global fitting methods. Rate
constants and number of steps were treated as shared global parameters for
each set of substrates. Unwinding amplitudes were treated as unique values
for each substrate.
The data for WT NS3 were modeled to scheme 1:
NS3c- | 0
RNA
NS3c? RNAO
¡
k1
NS3e? RNAO
¡
k2
[NS3e? RNA]*O
¡
fast
unwinding
using equation 1:
Aunw? A0?
A0
k2?k1[k1e?k2t? k2e?k1t](1)
where Aunwis the unwinding amplitude at incubation time t, A0is the apparent
fraction of functional complex formed on the RNA substrate, k1is the rate
constant in which WT NS3 undergoes a conformational change from a compact
(NS3c) to an extended (NS3e) conformation, and k2is the rate constant at which
WT NS3 forms a functional complex with the RNA substrate.
The data for NS3?C7 were modeled to scheme 2:
NS3e- |0
RNA
NS3e?
2 k3
decay
RNAO
¡
k2
[NS3e? RNA]*O
¡
fast
unwinding
using equation 2:
Aunw? A2(1 ? e?k2t) ? A3(1 ? e?k3t)(2)
where Aunwis the unwinding amplitude at incubation time t, A2is the amplitude
of the fast exponential rise in activity with the rate constant k2, and A3is the
amplitude of the decay process with rate constant k3.
Under conditions of increased glycerol and NaCl concentrations, the data
were modeled to the “n-step” mechanism (26) shown in scheme 3:
SO
¡
k
I1O
¡
k
I2O
¡
k
? ? ? O
¡
k
In?1O
¡
k
P
where S is substrate, Inis the nth intermediate state, P is the product, and k is the
kinetic rate constant at each step. Data were fit using the incomplete gamma
function with equation 3:
Aunw? A0(1 ??(n,kt)
?(n))
(3)
where Aunwis the unwinding amplitude at incubation time t, A0is the apparent
fraction of functional complex formed on the RNA substrate, n is the number of
kinetic steps, and k is the observed rate constant.
Linear regression was performed with equation 4:
Aunw? y0? mx(4)
where Aunwis the unwinding amplitude, y0is the offset, m is the slope, and x is
the number of binding sites.
Processivity analysis. The maximum unwinding amplitude for each substrate
duplex was determined by fitting the data to either equation 1 or 2 for WT NS3
or NS3?C7, respectively. Values were plotted as a function of duplex length and
fit to the equation Aunw? xPN,, where Aunwis the unwinding amplitude, x is the
fraction of functional complex, P is processivity, and N is the number of kinetic
steps made on the duplex. For this analysis, we used a kinetic step size of 16 bp,
which had been previously determined for WT NS3 (42). We assume that
NS3?C7 has an identical step size.
RESULTS
Differing domain orientations of Flaviviridae NS3 proteins.
When the crystal structures of NS3 proteins from HCV,
dengue virus (DENV), and Murray Valley encephalitis virus
(MVEV) are compared, it is clear that HCV NS3 differs dra-
matically from the other proteins. Specifically, a ?-strand in the
HCV NS3 structure clamps the protease domain next to the
helicase domain, thereby creating a compact conformational
state that differs from the extended conformations of DENV
and MVEV (Fig. 1A). This ?-strand consists of the C-terminal
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Page 4

seven amino acids of the helicase domain, which feed into the
protease active site at the N terminus of the protein (circled in
Fig. 1B; see Fig. S1 in the supplemental material). The result-
ing ?-sheet interaction between the protease and helicase do-
mains (henceforth called the ?-interaction) serves as a sub-
strate recognition platform for proteolytic cis cleavage during
the maturation of NS3. However, the ?-interaction must be
disrupted in order for the protease to release downstream viral
proteins from the viral polyprotein. We speculated that trun-
cation of the NS3 C terminus would uncouple the protease and
helicase domains, thereby allowing the protease domain to
swing away from the helicase domain and permitting NS3 to
adopt an extended conformation that is similar to those of the
DENV and MVEV NS3 proteins. To test this hypothesis, we
created an NS3 variant (NS3?C7) in which we removed the
C-terminal residues that comprise the ?-strand tether, and we
examined the behavior of this mutant protein.
The C terminus of NS3?C7 is solvent accessible and sensi-
tive to protease. To test whether removal of the ?-interaction
causes the C terminus of NS3?C7 to become solvent accessi-
ble, we subjected both WT NS3 and NS3?C7 to limited pro-
teolysis with carboxypeptidase Y (CPY) (49). In the absence of
CPY, both proteins migrate as single species on an SDS-PAGE
gel (Fig. 2A). In the presence of increasing amounts of CPY,
WT NS3 remained insensitive to the protease (Fig. 2B to D,
left), while CPY digested NS3?C7 with an efficiency propor-
tional to the amount of CPY included in the reaction (Fig. 2B
to D, right). These results show that WT NS3 adopts a con-
formation with a solvent-inaccessible C terminus, whereas the
conformation of NS3?C7 has a solvent-exposed C terminus
that renders it susceptible to CPY. The ability of CPY to digest
NS3?C7 indicates that we have successfully eliminated the
?-interaction between the protease and helicase domains.
Hydrodynamic analysis of the global conformation of
NS3?C7. To directly examine whether WT NS3 and NS3?C7
have different global conformations, we performed sedimenta-
tion velocity experiments to evaluate the shapes and hydrody-
namic properties of the proteins. We observe that NS3?C7
displays a distinct and reproducible 2.3% decrease in the sed-
imentation coefficient (S) relative to WT NS3, together with a
broadening of the sedimentation peak (Fig. 3). For two pro-
teins of similar molecular masses (?1% difference), such a
decrease indicates that NS3?C7 has a higher frictional coeffi-
cient than WT NS3 and therefore adopts a more elongated
conformation. We interpret the broadening of the NS3?C7
sedimentation peak to reflect multiple elongated conforma-
tions as the protease domain moves freely in solution (27, 28).
The presence of a single dominant peak in the data for both
FIG. 2. Limited proteolysis with carboxypeptidase Y. WT NS3 (first four protein lanes) or NS3?C7 (last four protein lanes) was incubated with
increasing amounts of CPY at 24°C. The amounts of CPY and the ratios of protein to CPY (wt/wt) are as follows: (A) 0 ?g CPY; (B) 1 ?g CPY,
360:1; (C) 5 ?g CPY, 72:1; and (D) 10 ?g CPY, 36:1. Asterisks denote stable proteolytic products. Using electrospray mass spectrometry (ES/MS),
the exact masses of these proteolytic products correspond to a region within the NS3 sequence that contains a stretch of consecutive arginine and
glycine residues that partially inhibit CPY activity (48) (see Fig. S1 in the supplemental material).
FIG. 3. Hydrodynamic
Shown is the calculated distribution c(s) versus S of WT NS3 (trian-
gles) and NS3?C7 (squares). Data were acquired at a protein concen-
tration of 5 ?M, a rotor temperature of 25°C, and a rotor speed of
42,000 rpm. The sedimentation coefficients were 4.4S for WT NS3 and
4.3S for NS3?C7.
analysisusing sedimentation velocity.
4346DING ET AL.J. VIROL.

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proteins show that the majority of the population sediments as
monodisperse species in solution. These data are consistent
with gel filtration results indicating that both proteins purify as
stable monomers (data not shown).
NS3?C7 forms functional complexes on RNA duplexes
faster than WT NS3. A fundamental property of helicases is
the ability to bind and engage nucleic acid substrates in a
productive manner that leads to strand separation. Helicases
are often positively charged proteins with a high, nonspecific
affinity for nucleic acids. Sequence composition, chemical iden-
tity (DNA versus RNA), or structure (single stranded versus
double stranded) of the nucleic acid can influence binding
specificity. Thus, direct binding assays for helicase substrate
affinity may not reflect the full functional specificity of a given
helicase. For example, NS3 binds nucleic acid duplexes con-
taining either a 3? or 5? overhang, but it only unwinds a duplex
containing a 3? overhang (46). To monitor the bound states of
NS3 that directly contribute to unwinding, we employed an
activity assay that reports the rate constant at which a helicase
forms functional complexes that are capable of unwinding tar-
get RNA substrates (36).
The functional complex assay begins by combining RNA
duplex substrate with helicase (in excess; see Materials and
Methods) and then dividing this mixture into multiple aliquots
in order to vary the incubation time for each sample at the
desired reaction temperature. Following the incubation period,
unwinding is initiated with the simultaneous addition of ATP
and a helicase “trap,” which is provided in excess to ensure
single-cycle unwinding conditions. (For example, any helicase
that falls off is trapped and cannot rebind.) The trap also
sequesters excess helicase protein in solution from participat-
ing in unwinding after the reaction has already initiated. Each
unwinding reaction is quenched after a constant reaction time,
and the products are separated by PAGE (see Fig. S2 in the
supplemental material). Quantification of the fraction of un-
wound product versus the incubation time yields the rate con-
stant at which the bound helicase forms an activated, func-
tional complex that unwinds RNA.
Previous studies have shown that WT NS3 requires an incu-
bation period of 30 min with RNA duplexes at 37°C prior to
achieving maximal unwinding activity (36). We reasoned that
this long incubation period might reflect the time required to
disrupt the ?-interaction and allow conformational changes to
occur within the protein-RNA complex. NS3?C7, lacking the
?-interaction, would not require such an extended incubation
step since it already adopts an extended conformation that is
poised for immediate, functional binding to RNA substrates.
To test this hypothesis, we measured the rate constants at
which the protein constructs form functional complexes on a
set of RNA unwinding substrates with progressively longer
duplex lengths (36).
Time courses of unwound substrate versus incubation time
reveal that NS3?C7 forms functional complexes more rapidly
than WT NS3 (Fig. 4). However, the amplitude (final extent) of
unwinding is generally higher for WT NS3. The data for WT
NS3 display a prominent lag phase in the evolution of product
for all four duplex substrates. Using global fitting methods, we
fit the WT NS3 data to the mechanism shown in scheme 1 (see
Materials and Methods), which includes two macroscopic rate
constants: k1is the rate constant for a putative conformational
change within the NS3 protein (0.1 ? 0.01 min?1), and k2is the
rate constant at which the helicase-RNA complex adopts an
activated state that proceeds immediately to unwind the duplex
(0.35 ? 0.06 min?1). Such an activated state may reflect partial
invasion at the duplex junction that leads to productive inter-
actions between the helicase and RNA (22).
In contrast, the data for NS3?C7 display a fast exponential
rise in functional complex formation followed by a slow decay
in activity. These data were fit using global fitting methods to
scheme 2 (see Materials and Methods), which lacks the lag
phase (k1) and instead includes two macroscopic rate con-
stants: k2is the rate constant for functional complex formation
(0.36 ? 0.02 min?1), as described above, and k3is a rate
constant for the apparent decay in activity observed late in the
time courses for NS3?C7 (0.03 ? 0.01 min?1). The decay
phase likely reflects an increased instability of the NS3?C7
protein under the experimental conditions.
These results are consistent with a model in which WT NS3
undergoes a slow conformational change prior to forming
activated complexes on RNA substrates. Once this protein
isomerization has occurred (reflected by the NS3 lag phase k1),
WT NS3 forms functional complexes with the same rate con-
stant as NS3?C7 (k2values are within error). Already in the
activated state, NS3?C7 bypasses the need for a slow confor-
mational change and proceeds quickly to form functional com-
plexes that can unwind RNA.
Comparative processivity of WT NS3 and NS3?C7. Proces-
sivity reflects the probability that a helicase will perform a
subsequent unwinding step rather than dissociate from the
substrate (1, 15). Hence, a helicase of infinite processivity will
unwind duplex substrates, independent of duplex length, to
completion and have a processivity value equivalent to unity. A
helicase of finite processivity, such as NS3, will show progres-
sively lower unwinding amplitudes as the length of the duplex
increases and have a processivity value less than unity (Fig. 4)
(26). Under single-cycle conditions, these amplitudes can be
used to calculate and compare the relative processivity values
of the two different NS3 protein constructs. Qualitatively, it
appears that NS3?C7 displays lower unwinding amplitudes
than WT NS3 on all RNA substrates except the 40-bp duplex
(Fig. 4). Based on these uncorrected final amplitudes, proces-
sivity values are P(WT NS3)? 0.50 ? 0.04 and P(NS3?C7)?
0.30 ? 0.02 (see Materials and Methods and Fig. 5A), indicat-
ing that NS3?C7 is a less processive helicase than WT NS3.
However, the final unwinding amplitudes for NS3?C7 are the
sum of two independent amplitudes that describe the expo-
nential rise and the gradual decay in activity (scheme 2). Cor-
rection for protein decay by using only the amplitudes associ-
ated with rate constant k2, the processivity value of NS3?C7
increases to P(NS3?C7; A2)? 0.49 ? 0.05 (Fig. 5B). This finding
leads us to conclude that NS3?C7 and WT NS3 display similar
levels of helicase processivity under these unwinding condi-
tions.
Increased glycerol concentrations inhibit functional com-
plex formation. To test whether the ?-interaction is inhibitory
for helicase activity, we attempted to identify reaction condi-
tions that might strengthen the interaction. We reasoned that
strengthening the ?-interaction would inhibit functional com-
plex formation for WT NS3 (a significant decrease in k1), while
NS3?C7 might be unaffected by comparison. To this end, we
VOL. 85, 2011 STRUCTURAL STATES AND FUNCTIONAL UNWINDING BY HCV NS34347