Structure-based mutagenesis identifies important
novel determinants of the NS2B cofactor of the
West Nile virus two-component NS2B–NS3
Ilian Radichev,3 Sergey A. Shiryaev,3 Alexander E. Aleshin,
Boris I. Ratnikov, Jeffrey W. Smith, Robert C. Liddington
and Alex Y. Strongin
Alex Y. Strongin
Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla,
CA 92037, USA
Received 7 August 2007
Accepted 20 November 2007
West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in
the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the
productive folding and the functional activity of the two-component NS3 (pro)teinase. Based
on an analysis of the NS2B–NS3pro structure, we hypothesized that the G22residue and the
negatively charged patch D32DD34of NS2B were part of an important configuration required for
NS2B–NS3pro activity. Our experimental data confirmed that G22and D32DD34substitution for S
and AAA, respectively, inactivated NS2B–NS3pro. An additional D42G mutant, which we
designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of
NS2B–NS3pro. Because of the significant level of homology in flaviviral NS2B–NS3pro, our
results will be useful for the development of specific allosteric inhibitors designed to interfere with
the productive interactions of NS2B with NS3pro.
West Nile virus (WNV), a member of the family
Flaviviridae, is an enveloped, positive-strand, 11 kb RNA
virus that is transmitted by mosquitoes (Mukhopadhyay
et al., 2005; van der Meulen et al., 2005). WNV causes
central nervous system damage unless specific treatment is
administered (Madden, 2003; Wang et al., 2004). The
genomic RNA of WNV encodes a polyprotein precursor
which consists of three structural proteins (C, capsid; prM,
membrane and E, envelope) and seven non-structural
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5)
arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3-
NS4A-NS4B-NS5. Polyprotein processing by the host cell
signal peptidase and furin in the endoplasmic reticulum,
and also by the viral two-component NS2B–NS3 proteinase
viral proteins (Beasley, 2005; Cahour et al., 1992;
Mukhopadhyay et al., 2005). Full-length NS3 represents a
multifunctional protein in which the N-terminal 184 aa
residues represent NS3pro and the C-terminal sequence
codes for a helicase. NS3pro is responsible for the cleavage
of the C protein and at the NS2A/NS2B, NS2B/NS3, NS3/
NS4A and NS4B/NS5 boundaries. In addition, the cleavage
of NS4A by NS3pro is required (Lin et al., 1993) for the
subsequent efficient cleavage of the NS4A/NS4B junction
by the signal peptidase (Preugschat & Strauss, 1991).
Inactivating mutations of the NS3pro cleavage sites in the
polyprotein precursor abolished viral infectivity (Chambers
et al., 1993, 2005).
In many flavivirus species including WNV, NS2B functions
as a cofactor and promotes the productive folding and the
activity of NS3pro. The cofactor activity of the 48 aa
central portion of NS2B is roughly equivalent to that of the
entire NS2B sequence (Leung et al., 2001). Structural
studies suggest that NS2B–NS3pro exhibits two alternative,
productive and unproductive, conformations. In the
productive conformation, NS2B wraps around NS3pro,
completing, in a precise and well-defined fashion, the
structure of the active site (Aleshin et al., 2007; Erbel et al.,
2006). In agreement, the NS2B-free NS3pro enzyme is
inactive (Falgout et al., 1991, 1993). These unique
cofactor–protease domain interactions are common for
the multiple flaviviruses (Bessaud et al., 2005; Droll et al.,
2000; Wu et al., 2003). The requirement of these
interactions for catalysis raises the possibility of designing
allosteric inhibitors that interfere with the NS2B fold rather
than directly targeting the catalytic triad of NS3pro. The
inhibitor design, however, requires the precise knowledge
of the functional determinants of the cofactor that are
3These authors contributed equally to this work.
The sequences of the mutant primers are available with the online
version of this paper.
Journal of General Virology (2008), 89, 636–641
636 0008-3359G2008 SGM Printed in Great Britain
essential for the catalytically productive NS2B–NS3pro
interactions. Extensive efforts were expended to determine
the identity of the several residue positions of mutagenesis
that had a deleterious effect on the proteinase activity
(Chambers et al., 1993, 2005; Chappell et al., 2006, 2007;
Droll et al., 2000; Lin et al., 2007; Nall et al., 2004;
Pastorino et al., 2006). These earlier studies were
performed when the catalytically productive structure of
the two-component NS2B–NS3pro was unknown. We
extended these studies by using mutagenesis to identify the
additional and novel functional determinants in the NS2B
sequence. In contrast with earlier work, the knowledge of
the crystal structures of NS2B–NS3pro provided structural
guidance for our studies.
The hydrophilic central sequence has been identified as an
essential region of the of NS2B cofactor, which is required
for the catalytic activity of the two-component NS2B–
NS3pro (Fig. 1a). Deletion and substitution mutagenesis
have already led to a substantial, albeit incomplete,
understanding of the importance of the individual residues
of the cofactor (Chambers et al., 1993, 2005; Chappell et al.,
2006,2007; Droll et al.,
Niyomrattanakit et al., 2004; Pastorino et al., 2006). These
data in combination with the knowledge of the crystal
structure of both the WNV and the dengue 2 virus NS2B–
NS3pro, which has recently become available (Aleshin et al.,
2007; Erbel et al., 2006), suggest that the functional
determinants are localized in the a-helical (a1) and the b-
strand regions (b1–3) of the flaviviral NS2B sequence.
Additional functionally important residues, which are
localized at the C-terminal region of the cofactor, are
proximal to the NS2B–NS3pro junction region.
2000; Linet al., 2007;
The structures of both the catalytically potent and inert
WNV NS2B–NS3pro strain NY99 (PDB 2IJO, 2FP7 and
2GGV) and dengue 2 virus NS2B–NS3pro (PDB 2FOM)
were used to model the structure of the NS2B–NS3pro
mutants. The mutant residues were built using PyMOL.
Energy minimization was done with PyMOL and CNS.
Based on the in-depth analysis of the WNV NS2B–NS3pro
atomic resolution structure, we hypothesized that because of
the tight interactions existing between NS2B and NS3pro,
the presence of the small size G22residue is essential for
maintenance of the productive NS2B–NS3 fold. Conversely,
the insertion of a bulky side chain amino acid, instead of
G22, into the NS2B–NS3pro tight interface would destabilize
the productive association of the cofactor with the NS3pro
domain and render the two-component NS2B–NS3pro
inactive. A close analysis of the NS2B–NS3pro productive
conformation also suggested that the D32DD34sequence
region of NS2B was exposed on the cofactor molecule
surface and that the D32DD34negatively charged patch does
not directly contact the cleavage substrate. The presence of
D32DD34is required, however, for maintaining the negative
charge at the S2 subsite that accommodates the positively
charged Arg/Lys at the P2 substrate position (Fig. 1b, c). In
contrast, according to our in silico modelling, D42which is
localized in the C-terminal sequence region of NS2B and,
Fig. 1. Sequence alignment and structure of the flaviviral NS2B
cofactor. (a) Sequence alignment. DV1–4, dengue virus types 1–
4, YFV, Yellow fever virus; JEV, Japanese encephalitis virus; Kunjin,
Kunjin virus; ALKV, Alkhurma flavivirus. Lower panel: the previously
mutated residues are marked with stars, if they did not, and in a
one letter code, if they did, affect the proteolytic activity of the
constructs. Upper panel: the stars above the alignment show the
mutant positions engineered into WNV NS2B–NS3pro in our
current work. Positions of the a1 helix and b1”b3-strands are
shown above the panel. (b) The structure of NS2B–NS3pro. Both
alternative conformations of NS2B–NS3pro determined in our
previous work are shown (Aleshin et al., 2007). NS2B and NS3pro
are pink and yellow, respectively. DDD/AAA (32–34) and G22S
substitutions are shown as sticks. (c) Electrostatic charge on the
surface of WNV NS2B–NS3pro (left) and DDD/AAA mutant
(right). A portion of the substrate-mimetic inhibitor aprotinin (PDB
2IJO) is shown as green sticks. The position of D42is not shown
because this residue is a part of the unfolded region unavailable in
the NS2B–NS3pro crystal structure.
Structure-based mutagenesis of the NS2B cofactor
therefore, is close to the scissile bond in the NS2B–NS3pro
junction region rather than to the catalytic groove is likely to
have no effect on either self-proteolysis in cis or on the
catalytic activity of NS2B–NS3pro or both.
To acquire experimental evidence that our in silico
predictions were correct, we used site-directed mutagenesis
on the cofactor sequence. The 48 aa residue central portion
of NS2B (residues 1393–1440 of the WNV polyprotein
precursor) and NS3pro (residues 1476–1687 of the
WNV precursor) sequences were linked by a flexible
GGGGSGGGG linker. This wild-type NS2B–NS3pro con-
struct (NS2B–NS3pro-WT) was used as a template for the
PCR mutagenesis to obtain the D42G mutant. The
autolytic cleavage site K48GQGGGSGGGG in the NS2B–
NS3pro junction region was inactivated by the K48A
mutation (Shiryaev et al., 2007). The resulting self-
proteolysis-resistant NS2B–NS3pro K48A construct was
used as a template for constructing the D32DD/A32AA and
G22S mutants, which we have named DDD/AAA and G22S
in the text below. To destroy the tight interactions
involving G22, we mutated this residue to S (G22S). To
eliminate the negative charge at the S2 subsite, we
converted the D32DD34sequence into AAA (DDD/AAA).
The primers we used for the mutagenesis are listed in
Supplementary Table S1 (available in JGV Online). The
mutant PCR products were further amplified using the 59-
TTCGAATCCGGC-39 oligonucleotides as the forward and
reverse primers, respectively, to obtain the final 741 bp
mutant constructs, including the His-tag sequence (the
sequence that encodes a His66 tag is underlined).
The NS2B–NS3pro constructs were then recloned into the
pET101/D-TOPO cloning vector (Invitrogen) to encode
the recombinant sequence, which was C-terminally tagged
with the His66 tag. The NS2B–NS3pro constructs were
expressed in Escherichia coli BL21 (DE3) Codon Plus cells
(Stratagene) and purified using Co2+-chelating Sepharose
Fast Flow chromatography (Shiryaev et al., 2006, 2007).
Collected fractions were analysed using 15% SDS-PAGE.
The presence of the NS2B–NS3pro cleavage activity in the
fractions was confirmed using either butyloxycarbonyl-
AMC) or pyroglutamic acid-Arg-Thr-Lys-Arg-7-amino-4-
methylcoumarin (Pyr-RTKR-AMC) as a substrate. The
peak fractions were pooled, concentrated with a 30 kDa
cut-off concentrator (Millipore) and dialysed against
10 mM Tris/HCl buffer, pH 8.0, containing 100 mM
rechromatography of the pooled fractions was sufficient
to isolate the highly purified NS2B–NS3pro mutant
constructs (Fig. 2a). The presence of the intact C-terminal
sequence in the NS2B–NS3pro constructs was confirmed
by Western blotting using a monoclonal His-tag murine
antibody, followed by the horseradish peroxidase-con-
jugated donkey anti-mouse IgG (Jackson Laboratories)
and a TMB/M substrate (Chemicon).
According to the results of SDS-gel electrophoresis the
original NS2B–NS3pro K48A construct and the G22S and
DDD/AAA mutants were resistant to autoproteolysis at the
flexible linker region (Fig. 2a, upper panel). In agreement,
Western blotting demonstrated that these three constructs
preserved the His66 sequence tagged at the C-terminal
end of NS3pro (Fig. 2a, lower panel). As we expected,
the D42G mutantwas
K48G QGGGSGGGG linker region and it generated the
non-covalently associated NS2B and NS3pro in a manner
that was similar to the NS2B–NS3pro-WT construct
(Shiryaev et al., 2006).
The trans-proteolytic activity of the constructs was
measured using the Boc-RVRR-AMC and Pyr-RTKR-
AMC peptides (both from American Peptide Company)
and the myelin basic protein (MBP; Biodesign) as
substrates. MBP, a protein that is highly sensitive to
NS3pro proteolysis (Shiryaev et al., 2006), was used as a
substrate to demonstrate that the activity loss of the
mutant NS2B–NS3pro construct also includes the endo-
proteolytic activity. The assay for NS2B–NS3pro peptide
cleavage activity was performed in 0.1 ml 10 mM Tris/HCl
buffer, pH 8.0, containing 20% glycerol (v/v) and 0.005%
Brij 35. The cleavage peptide (either Boc-RVRR-AMC or
Pyr-RTKR-AMC) and enzyme concentrations, unless
indicated otherwise, were 24 mM and 10 nM, respectively.
Initial reaction velocities were monitored continuously at
lex5360 nm and lem5460 nm on a Spectramax Gemini
(Molecular Devices). All assays were performed in triplicate
in wells of a 96-well plate. The concentration of active
proteinase was measured using a fluorescence assay by
titration against a standard aprotinin solution of a known
concentration (Shiryaev et al., 2006).
We determined that the specific activity of the D42G
mutant was approximately 50% when compared with the
NS2B–NS3pro-WT or the original NS2B–NS3pro K48A
constructs against both Pyr-RTKR-AMC and Boc-RVRR-
AMC (Fig. 2b). In turn, the G22S mutant retained a low,
3%, residual activity, while the DDD/AAA mutant lost
99% of its activity when compared with the WT and K48A
constructs. Interestingly, the Kmvalue of the G22S mutant
decreased approximately sevenfold, while the kcat value
went down #200-fold resulting in a 30-fold loss of the kcat/
Km parameter when compared with the WT construct
(Km530 and 4 mM of WT and G22S, respectively; kcat52
and 0.008 s21of WT and G22S, respectively). Aprotinin, a
(Shiryaev et al., 2006), efficiently inhibited the proteolytic
activity of both the K48A and D42G constructs, thus
suggesting that the D42G mutation did not affect the active
site conformation of NS3pro (data not shown).
Consistent with our previous results (Shiryaev et al., 2006),
MBP was highly sensitive to NS2B–NS3pro proteolysis.
MBP (4 mg; 11 mM) was co-incubated for 60 min at 37 uC
with the purified NS2B–NS3pro constructs (0.7 mg or
I. Radichev and others
638Journal of General Virology 89
1 mM; an enzyme:substrate ratio #1:10) in 20 ml 10 mM
Tris/HCl buffer, pH 8.0, containing 20% glycerol (v/v).
The reactions were stopped by adding 26 SDS sample
buffer [125 mM Tris/HCl, pH 6.8, containing 4% SDS,
20 mM DTT, 0.005% bromophenol blue and 20% glycerol
(v/v)]. The digested samples were resolved in 4–20%
gradient SDS-gel electrophoresis to determine the conver-
sion of 18 kDa MBP into the 6 and 14 kDa digested
fragments. Fig. 2(c) shows that 18 kDa MBP was almost
totally proteolysed by the NS2B–NS3pro K48A construct
generating, as a result of the proteolysis, the ~6 and
~14 kDa fragments. In contrast, both the G22S and the
DDD/AAA mutants did not cleave MBP.
Circular dichroism (CD) studies confirmed the structural
integrity of the DDD/AAA mutant construct, the spectrum
of which did not differ significantly from that of the
NS2B–NS3pro K48A construct (Fig. 2d). CD spectra (190–
250 nm) of the NS2B–NS3pro purified samples (1.5–
2 mg ml21
in 15 mM potassium
pH 7.8) were collected in a 1 mm path length quartz
cuvette at ambient temperature using a 62ADC spectro-
meter (AVIV Instruments). The results are expressed as the
[H]MRW (mean residue weight) ellipticity. On the other
hand, the CD spectrum of the G22S mutant was
significantly different. Consistent with a potential trans-
ition of this mutant to the unproductive fold (Fig. 1b), the
Fig. 2. The properties of the mutant WNV
NS2B–NS3pro constructs. (a) The K48A,
K48A DDD/AAA and K48A G22S constructs
are resistant to self-proteolysis at the flexible
GGGGSGGGG linker sequence that links the
NS2B cofactor with NS3pro, while both the
wild-type construct (WT) and the D42G
mutant are self-proteolysed. Upper and lower
panels: Coomassie staining and Western
blotting with a His?6 tag antibody, respect-
ively. (b) Specific enzymic activity of the
NS2B–NS3pro constructs against the fluor-
escence Pyr-RTKR-AMC peptide substrate.
(c) The digested reactions were analysed by
SDS-gel electrophoresis in 4–20% gradient
gels. The K48A DDD/AAA and K48A G22S
mutants did not cleave the MBP substrate. (d)
CD spectra of NS2B–NS3pro and the DDD/
AAA and G22S mutants. The spectra suggest
that the structure of NS2B–NS3pro and the
DDD/AAA mutant is similar and that the G22S
Structure-based mutagenesis of the NS2B cofactor
spectrum suggested the presence of additional a- and b-
structural regions in the NS2B–NS3pro DDD/AAA sam-
ples. In agreement with our results, a similar G22A
substitution resulted in a noticeable, albeit insignificant
when compared to the G22S mutation, loss of activity of
NS2B–NS3pro from tick-borne Alkhurma virus (Pastorino
et al., 2006). Our results suggest that the activity loss was
incomplete because of an insufficient increase of the
residue side chain size in the Alkhurma model.
In conclusion, the in-depth analysis of the cofactor–
NS3pro interactions in flaviviruses guided by the crystal
structure parameters led us to identify two novel NS2B
functional determinants critical for NS3pro activation. We
believe this knowledge will be highly valuable for the
precise understanding of the NS2B–NS3pro molecular
complex formation and for the optimization of the
selective allosteric small-molecule inhibitors designed to
target the NS2B–NS3pro interface. We suspect that these
allosteric inhibitors designed to inactivate the two-
component flaviviral proteinase by interfering with the
NS2B–NS3pro interactions, will not cross-react with host
cell serine proteinases and, accordingly, they will exhibit
fewer side effects when compared with the active site-
targeting antagonists of the flaviviral NS2B–NS3pro (Knox
et al., 2006; Lohr et al., 2007; Yin et al., 2006).
The work reported here was supported by NIH Grants RR020843 (to
J.W.S. and A.Y.S.) and XO1MH077601 (to A.Y.S.), and AI055789
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Structure-based mutagenesis of the NS2B cofactor