JOURNAL OF VIROLOGY, June 2010, p.

56785686
0022-538X/10/$12.00 doi:10.1128/JVI.02451-09
Copyright 2010, American Society for Microbiology. All Rights Reserved.

Vol. 84, No. 11

Inhibition of Dengue Virus Polymerase by Blocking

of the RNA Tunnel
Pornwaratt Niyomrattanakit,1 Yen-Liang Chen,1 Hongping Dong,1 Zheng Yin,1 Min Qing,1
J. Frasier Glickman,2 Kai Lin,3 Dieter Mueller,2 Hans Voshol,2 Joanne Y. H. Lim,1
Shahul Nilar,1 Thomas H. Keller,1 and Pei-Yong Shi1*
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore1; Novartis Institutes for
BioMedical Research, Basel CH4002, Switzerland2; and Novartis Institutes for
BioMedical Research, Cambridge, Massachusetts 021393
Received 20 November 2009/Accepted 9 March 2010

single open reading frame encodes a long polyprotein that is

processed by viral and host proteases into 10 mature viral
proteins. Three structural proteins (Capsid [C], premembrane
[prM], and envelope [E]) are components of virus particles.
Seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A,
NS4B, and NS5) are responsible for viral replication (40),
virion assembly (19, 21, 24, 33), and innate immunity antagonism (4, 16, 23, 29, 30). Two viral proteins encode enzymatic
activities that have been targeted for antiviral development.
NS3 functions as a protease (with NS2B as a cofactor), helicase, 5-RNA triphosphatase, and nucleoside triphosphatase
(7, 14, 42). The N-terminal part of NS5 is a methyltransferase
that methylates the N7 and 2-O positions of the viral RNA cap
structure (13, 18, 37); the C-terminal part of NS5 has an RNAdependent RNA polymerase (RdRp) activity (1, 39). The
RdRp activity is unique to RNA viruses and therefore represents an attractive antiviral target.
Two types of inhibitors could be developed to suppress viral
polymerases. Type 1 inhibitors are nucleoside/nucleotide analogs that function as RNA or DNA chain terminators; about
half of the current antiviral drugs are nucleotide analogs (10).
For flaviviruses, a nucleoside analog (7-deaza-2-C-methyladenosine), originally developed for hepatitis C virus (HCV)
RdRp, showed anti-DENV activity (32, 38). We recently reported a similar adenosine analog (7-deaza-2-C-acetyleneadenosine) that potently inhibited DENV both in cell culture
and in mice; unfortunately, this compound showed side effects
during a 2-week in vivo toxicity study (44). Nevertheless, these

The family Flaviviridae consists of three genera: Flavivirus,

Pestivirus, and Hepacivirus. The genus Flavivirus contains about
73 viruses, many of which are arthropod-borne and pose major
public health threats worldwide (15). The four serotypes of
dengue virus infect 50 to 100 million people each year, with
approximately 500,000 cases developing into life-threatening
dengue hemorrhage fever (DHF) and dengue shock syndrome
(DSS), leading to about 20,000 deaths. In addition to DENV,
West Nile virus (WNV), Japanese encephalitis virus (JEV),
yellow fever virus (YFV), and tick-borne encephalitis virus
(TBEV) also cause significant human diseases. No antiviral
therapy is currently available for treatment of flavivirus infections. Human vaccines are only available for YFV, JEV, and
TBEV (15). Development of antiviral therapy and new vaccines is urgently needed for flaviviruses.
The flavivirus genome is a single-stranded RNA of plussense polarity. The genomic RNA contains a 5 untranslated
region (UTR), a single open reading frame, and a 3 UTR. The
* Corresponding author. Mailing address: 10 Biopolis Road,
#05-01 Chromos, Singapore 138670. Phone: (65) 67222909. Fax:
(65) 67222916. E-mail: pei_yong.shi@novartis.com.
P.N. and Y.-L.C. contributed equally to this study.
Present address: College of Pharmacy and The State Key Laboratory of Elemento-Organic chemistry, Nankai University, Tianjin
300071, China.
Present address: High Throughput Screening Resource Center,
The Rockefeller University, 1230 York Avenue, Box 203, New York,
NY 10065.

Published ahead of print on 17 March 2010.

5678

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor
antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives
are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified
through high-throughput screening of one million compounds using a primer extension-based RdRp assay
[substrate poly(C)/oligo(G)20]. Chemical modification of the initial hit improved the compound potency to
an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 M. In addition to suppressing the
primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV
subgenomic RNA, but at a lower potency (IC50 of 5 M). Remarkably, the observed anti-polymerase activity
is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 M against
hepatitis C virus RdRp and human DNA polymerase and . UV cross-linking and mass spectrometric
analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of
DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template
tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the
assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound
inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the
hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

VOL. 84, 2010

INHIBITION OF DENGUE VIRUS RdRp

MATERIALS AND METHODS

Compound synthesis. Compound synthesis and structure-activity relationship
(SAR) studies were recently published (45). Compound NITD-1 was an initial
hit identified from a DENV RdRp HTS. Over one million compounds from
the Novartis library collection were screened. Compound NITD-2 was a lead
obtained after an SAR study. NITD-29 contained a photoalkylating group benzophenone (2) and was synthesized for a UV cross-linking experiment to map the
compound-binding site on the DENV NS5 protein. The synthesis of NITD-29
was described elsewhere (45).
Primer extension-based DENV RdRp assays. Three types of primer extensionbased RdRp assays were performed. All three assays were in a scintillation
proximity assay (SPA) format using poly(C) (Sigma-Aldrich) as a template and
a 5-biotinylated oligo(G)20 (Sigma) as a primer. The RdRp activity was quantified by the incorporation of [3H]GTP using SPA beads coated with streptavidin
(28, 43). All 50% inhibitory concentrations (IC50s) reported in the present study
were calculated using Prism (Graphpad) or ActivityBase software (ID Business
Solutions). Full-length NS5 of DENV-2 and DENV-4, each with an N-terminal
His6 tag, were generated using a standard baculovirus expression system and
Escherichia coli expression system, respectively; the recombinant proteins were
purified using a HiTrap chelating column according to the manufacturers protocol (GE Healthcare).
The first assay type was used for HTS in 384-well white plates. Compounds
from the library were spotted onto the plates followed by assembly of reactions
(30 l) containing 50 mM Tris-HCl (pH 7.0), 10 mM KCl, 2 mM MgCl2, 2 mM
MnCl2, 0.05% CHAPS, 0.05 U of RNasin/l, 50 nM protein, 0.8 M GTP, 0.5
Ci of [3H]GTP, 4 g of poly(C)/ml, and 0.2 g of oligo(G)20/ml. After incubation at 23C for 20 min, the reactions were terminated by adding 150 g of
streptavidin-coated SPA beads to stop solution (75 mM Tris-HCl [pH 7.0], 37.5
mM EDTA, 225 mM NaCl). The plates were further incubated at 23C for 30
min and subjected to quantification on LEADSeeker (GE Healthcare).
The second assay type was used to allow a single cycle of RNA synthesis. This
assay used heparin to trap unbound DENV RdRp and was performed in 96-well
plates. Briefly, RNA-NS5 complex was preformed in the HTS assay buffer (described above) at 23C for 1 h, after which various concentrations of heparin (0
to 96 ng/ml) were added. The reaction mixtures were incubated for another 15
min. Compound NITD-2 and [3H]GTP were then added to the reactions (final
volume of 50 l). After incubation at 23C for 1 h, the reactions were terminated
by adding an equal volume of 2 stop buffer containing 3-mg/ml SPA beads in
50 mM Tris-HCl (pH 7.5), 40 mM EDTA, and 150 mM NaCl. The incorporation
of [3H]GTP was measured by a MicroBeta counter (Perkin-Elmer).
The third assay type was used to examine the effect of order of addition of
RNA template and inhibitor (during reaction assembly) on the compound potency. Different schemes of RNA template/inhibitor addition are described in
Results. For each experimental scheme, various concentrations of NITD-2 were

tested to derive the IC50s. The assay buffer and the RNA template concentration
were as described for the type 1 assay.
De novo RdRp assay. For DENV, the de novo RdRp reaction (25 l) contained 50 mM HEPES (pH 8.0), 10 mM KCl, 5 mM MgCl2, 2 mM MnCl2, 10 mM
dithiothreitol (DTT), 0.5 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 2.5 Ci of
[-33P]GTP (10 Ci/l, 3,000 Ci/mmol; Perkin-Elmer), 0.25 g of NS5, 0.5 g of
RNA template, and indicated concentrations of compound NITD-2. The RNA
template was in vitro transcribed from a PCR product using a T7 MEGAscript kit
(Applied Biosystems). The PCR template contained a T7 promoter, followed by
a cDNA fragment representing a DENV-2 subgenome with a deletion from
nucleotides 169 to 10263 (GenBank accession number AF038403.1). The de novo
RdRp reactions were incubated at 23C for 30 min; the mixtures were passed
through a MicroSpin G-25 column (GE Healthcare) to remove unincorporated
nucleoside triphosphates (NTPs); and the RNA elute was extracted with phenolchloroform and precipitated with ethanol. The RNA pellet was dissolved in 10 l
of RNase-free water, mixed with an equal volume of denaturing Gel Loading
Buffer II (Applied Biosystems), and loaded onto a 10% denaturing polyacrylamide gel with 7 M urea. A PhosphorImager was used to quantify the 33P-labeled
RNA products.
For WNV, the RdRp assay (20 l) was performed in 50 mM Tris-HCl (pH
8.0), 5 mM MgCl2, 20 mM NaCl, 2 mM DTT, 0.5 mM ATP, 0.5 mM UTP, 0.5
mM CTP, 5 M GTP, 15 Ci of [-33P]GTP (10 Ci/l, 3,000 Ci/mmol), 1 g
of WNV subgenomic RNA, and 2 g of WNV full-length NS5. The reactions
were incubated at 33C for 2 h and analyzed on a denaturing PAGE as described
for the DENV RdRp assay (above). The expression and purification of the WNV
NS5, and the preparation of the WNV subgenomic RNA (containing the 5terminal 269 nucleotides directly connected to the 3-terminal 622 nucleotides of
the WNV genome) were reported previously (46).
Spectrum of polymerase inhibition. Activity profiling against HCV NS5B,
DNA polymerase and were similarly examined using SPA-based assays in
96-well half-area white plates. The HCV NS5B (from genotype 1B strain Con1)
contained a deletion of the C-terminal 21 amino acids. The protein (with a
C-terminal His tag) was generated in an E. coli expression system and purified
through a Ni2 column (47). The HCV RdRp reaction (40 l) contained 50 nM
NS5B, 50 mM HEPES (pH 7.3), 10 mM KCl, 5 mM MgCl2, 0.5 U of RNasin/l,
5 mM DTT, 3.6 g of poly(C)/ml, 0.36 g of 5-biotinylated oligo(G)13/ml, 1.33
M GTP, and 0.16 Ci of [3H]GTP. The reaction was incubated at 30C for 2 h
and stopped by the addition of an equal volume of 2 stop solution (containing
61.2 mM potassium oxalate [pH 1.68] and 50 mM EDTA) and 5-mg/ml streptavidin SPA beads. The plate was quantified by using a MicroBeta counter.
Human polymerase (Chimerx) and (Trevigen) were commercially purchased. The polymerase assay was performed in 56.25 mM Tris-HCl (pH 8.0),
2.5 mM NaCl, 10 mM MgCl2, 0.05% Tween 20, 2.5% glycerol, 5 mM DTT, 3.7
g of poly(dA)/ml, 0.47 g of 5-biotinylated oligo(dT)18/ml, 2 M total dTTP
(with a 1:10 ratio of [3H]dTTP [0.9 Ci/reaction] to cold dTTP), and 0.2 U of
polymerase . The reaction was incubated at 37C for 60 min. The polymerase
DNA assay was performed in 56.25 mM Tris-HCl (pH 8.0), 150 mM KCl, 2.5
mM NaCl, 10 mM MgCl2, 0.05% Tween 20, 2.5% glycerol, 10 mM DTT, 3.7 g
of poly(dA)/ml, 0.47 g of 5-biotinylated oligo(dT)18/ml, 2 M total dTTP (with
a 1:10 ratio of [3H]dTTP [0.9 Ci/reaction] to cold dTTP), and 0.2 U of polymerase . The reaction was incubated at 37C for 60 min. Both reactions were
stopped and quantified as described for the HCV RdRp assay.
Virus titer reduction assays. Virus titer reduction assays were performed on
Vero cells. Cells were seeded in a 96-well plate (2 104 cells per well). At 24 h
post-seeding, the cells were infected with DENV-2 (NGC strain), YFV (17D
vaccine strain), chikungunya virus at a multiplicity of infection (MOI) of 0.1. The
infected cells were immediately treated with compound at the indicated concentration. Culture fluids were collected at 44 h postinfection (p.i.) for DENV-2 and
YFV and at 22 h p.i. for chikungunya virus. Virus titers were quantified by
standard plaque assays on BHK cells (36).
Photoaffinity cross-linking of compound-protein complex. Labeling of the
photoreactive derivative (NITD-29) was performed by incubating 20 M fulllength DENV-2 NS5 protein with 200 M NITD-29 in a total volume of 30 l.
The sample was exposed to a table top UV light source (UVitec) at a wavelength
of 365 nm. The reaction assembly and UV cross-linking were performed in a 4C
room. Samples were withdrawn at 15, 30, and 60 min, and a small aliquot (5 l)
was diluted and used to assess the RdRp activity using the SPA assay. The SPA
assay was performed to ensure that the inhibitor had irreversibly inactivated the
RdRp by forming a covalent bond with the protein; the non-cross-linked compound was sufficiently diluted such that it would not affect the enzyme activity.
The remaining sample was subjected to size exclusion spin column to remove the
unincorporated inhibitor and subjected to mass spectrometric (MS) analysis (41).

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

studies have proved the concept that nucleoside analogs could

potentially be developed for flavivirus therapy. Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the
mechanism of action of NNI includes structural alteration of
polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or
impeding the processivity of polymerase elongation (11). A
broad range of chemical classes have been identified as NNI,
including inhibitors of HIV (9, 35) and HCV (3, 5, 11, 25).
In the present study, we performed high-throughput screening (HTS) to search for NNI of DENV RdRp. The HTS and
chemistry synthesis led to the identification of N-sulfonylanthranilic acid derivatives as inhibitors of DENV RdRp. The
compounds specifically inhibit DENV RdRp. UV cross-linking
experiments mapped the compound binding site to the RdRp
domain of DENV NS5. Amino acid Met343, located at the
entrance of RNA template tunnel of the DENV RdRp, was
cross-linked to the compound. These results, together with
biochemistry experiments, suggest that the compound blocks
the RdRp activity through binding to the RNA template tunnel
of the polymerase.

5679

5680

NIYOMRATTANAKIT ET AL.

J. VIROL.

RESULTS
Identification of NITD-1. We identified NITD-1 (Fig. 1A) as
a DENV-2 RdRp inhibitor after screening a library of over one
million compounds in a SPA-based RdRp HTS. The HTS
assay measured the DENV-2 RdRp-mediated primer extension activity using poly(C)/oligo(G)20 as a RNA template. The
HTS results showed a Z factor of 0.73 and a hit rate of 0.7%.
We focused on NITD-1 after analyzing the hit list for druglike properties such as potency, aqueous solubility, and metabolic stability (data not shown). NITD-1 contained N-sulfonylanthranilic acid and suppressed DENV-2 RdRp activity in a
dose-responsive manner, with an IC50 of 7.2 M (Fig. 1B).
Chemistry synthesis was performed to study the SAR of the
hit. The details of the SAR results were recently published
(45). The SAR study generated NITD-2 which had an improved IC50 of 0.7 M (Fig. 1B). Compared to NITD-1,
NITD-2 has a replacement of dihydropyrazolone with N-benzyl-pyrazole. In addition to improving the compound potency,
the SAR results allowed us to synthesize a photoreactive derivative (NITD-29) that could be used to map the compoundbinding site of NS5 through UV-cross-linking (see below).
Selectivity of NITD-2 for DENV RdRp. To examine the
spectrum of the anti-polymerase activity, we tested NITD-2
against three other polymerases, including HCV NS5B RdRp,
and human DNA polymerases and (Fig. 1C). At 100 M
concentration, NITD-2 reduced 32, 50, and 23% of the HCV
NS5B, polymerase , and polymerase activities, respectively.
Therefore, the IC50s of the compound in these polymerases
were 100 M. In contrast, 33 M NITD-2 suppressed ca.
86% of the DENV-2 RdRp activity. As controls (Fig. 1D), compounds with known activities against HCV RdRp (LCY967) (8),
polymerases (aphidicolin) (17), and polymerases (lithocholic)
(31) showed expected anti-polymerase results, with IC50s of 50
nM, 40 M, and 40 M, respectively. These results indicate that
NITD-2 selectively inhibits DENV-2 polymerase.

FIG. 1. Specific inhibition of DENV-2 NS5 RdRp activity. ()

Structure of NITD-1 and NITD-2. NITD-1 was identified from the
RdRp HTS. NITD-2 was obtained after the SAR study. (B) Inhibition
of DENV-2 NS5 RdRp. Various concentrations of NITD-1 and
NITD-2 were incubated with the RdRp reaction containing poly(C)/
oligo(G)20 as a template. A SPA-based detection method was used to
quantify the incorporation of [3H]GTP. The relative RdRp activities
are presented; signals from the mock-treated reaction are set as 100%.
(C) Anti-polymerase activity of NITD-2 against HCV RdRp and human DNA polymerase and . The inhibition curve of DENV-2 NS5
from panel B is included in this plot to show the relative sensitivity of
the different polymerases to the compounds. (D) Inhibition of different
polymerases using control inhibitors. Control inhibitors with known
activities against HCV RdRp (LCY967), polymerases (aphidicolin),
and polymerases (lithocholic) were included. See the experimental
details in Materials and Methods. The results were derived from 3
independent experiments. Error bars represent the standard deviations.

Inhibition of elongation in a single round RNA synthesis.

The HTS assay used poly(C)/oligo(G)20 as a RNA template to
measure DENV-2 RdRp-mediated elongation activity; no initiation event was involved. Thus, NITD-2 was assumed to
block RNA elongation. However, because multiple rounds of
RNA synthesis could occur in the HTS RdRp assay, it was
possible that the compound inhibits the transition between
multiple rounds of RNA synthesis rather than through a direct
inhibition of RNA elongation. To address this question, we

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

MS analysis of the compound-NS5 complex. Samples of untreated and compound-cross-linked NS5 were subjected to SDS-PAGE on 4 to 20% Tris-glycine
gels and stained with Coomassie blue. Protein bands at 100 kDa were excised
and in-gel digested with trypsin as described previously (41). After in-gel digestion, tryptic hydrolysates were eluted from the bands with 5% formic acid and
collected in a microtiter plate. For matrix-assisted laser desorption ionization
(MALDI)-MS and tandem MS (MS/MS) analyses, the tryptic peptides were
purified on ZipTips (Millipore Corp., Bedford, MA) by using a Tecan Genesis
ProTeam 150 system (Tecan, Maennedorf, Switzerland). After two washes with
5 l of 80% acetonitrile (ACN)0.1% trifluoroacetic acid (TFA), the tips were
equilibrated twice with 5 l of 0.1% TFA, and the hydrolysate was applied; after
four washes with 5 l of 0.1% TFA, the peptides were directly eluted or spotted
onto ABI 4700 MALDI targets (100-well plates) with 2 l of a solution of
-cyano-4-hydroxycinnamic acid (5 mg/ml in 50% ACN and 0.1% TFA containing 2 mM NH4H2PO4) applied to the backend of the ZipTips. MALDI spots
were analyzed on the Applied Biosystems 4700 Proteomics Analyzer (ABI,
Framingham, MA) in MS and MS/MS mode. Both MS and MS/MS data were
acquired with a Nd:YAG laser with 200 Hz repetition rate; 2,000 shots were
accumulated for each spectrum in MS mode and 4,000 shots were accumulated
for each precursor ion in MS/MS mode. MS/MS mode was operated with 1 keV,
and the products of metastable decomposition at elevated laser power were
detected. MS data were acquired with proximal external calibration and MS/MS
data using default instrument calibration.
Peptides with potential cross-links were identified by comparing MALDI-MS
spectra of control and compound-cross-linked NS5. MS/MS spectra of parent
ions with crosslink-specific mass shifts were analyzed manually to confirm and
localize the cross-linked residue.

VOL. 84, 2010

examined the activity of NITD-2 in a single round of RNA

synthesis by incubating the RdRp reaction with heparin, a
highly sulfated glycosaminoglycan polymer that can trap the
unbound RdRp. First, we determined the optimal heparin
concentration required for the single round RNA synthesis.
This was accomplished by incubating various concentrations of
heparin (0 to 96 ng/ml) with the binary NS5-RNA complex,
which was preformed by incubating NS5 (50 nM) with poly(C)/
oligo(G)20 RNA (0.25 g/ml) for 60 min. After the mixtures
were incubated for 15 min, [3H]GTP was added to initiate the
RNA synthesis. As shown in Fig. 2A, the addition of heparin to
the RdRp reactions reduced the [3H]GTP incorporation in a
dose-responsive manner. The reduction of RdRp activity was
presumably due to the heparin-mediated trapping of the unbound RdRp after its completion of the first round of RNA
synthesis. A saturated concentration of heparin was reached at
50 ng/ml; at this concentration, heparin decreased the total
GTP incorporation by two-thirds. Based on this result, we

5681

FIG. 3. Inhibition of de novo RNA synthesis of DENV RdRp.

(A) Recombinant proteins of full-length NS5. Full-length NS5 from
DENV-2 (1 g), DENV-4 (2 g), and WNV (2 g) were analyzed by
SDS-PAGE and stained with Coomassie blue. Molecular masses of
protein markers are labeled. (B) DENV de novo RNA synthesis. Subgenomic RNA of DENV-2 was incubated with recombinant DENV
NS5 in the presence or absence of NITD-2. The RdRp products
were analyzed on a denaturing PAGE and quantified by using a
PhosphorImager. The relative amount of RNA product was indicated
below the autoradiograph of the PAGE gel. The concentrations of
NITD-2 are labeled above the autoradiograph. As a maker of the input
RNA template, the subgenomic RNA was 5-end 33P-labeled (12) and
analyzed on the PAGE. (C) WNV de novo RNA synthesis. A subgenomic RNA of WNV was incubated with WNV NS5 in the presence
indicated concentrations of NITD-2. The RdRp products were similarly analyzed as described in panel B. See experimental details in
Materials and Methods.

decided to use 50 ng of heparin/ml in our single-round RNA

synthesis experiments.
Next, we determined the IC50s of NITD-2 in the presence or
absence of heparin (Fig. 2B). The compound clearly inhibited
RdRp activity in the single-round RNA synthesis. However,
the presence of heparin reduced the IC50 from 0.7 M
(without heparin) to 0.32 M (with heparin). As a control,
3ddGTP, a known RNA chain terminator, also decreased
the IC50 from 2 nM (without heparin) to 0.4 nM (with
heparin) (Fig. 2C). Overall, the results clearly demonstrate
that NITD-2 inhibits the DENV RdRp-mediated RNA
elongation.
Specific inhibition of de novo RNA synthesis of DENV. Since
the above assays used an artificial RNA template [poly(C)/
oligo(G)20] and DENV-2 NS5, it is important to validate the
compound activity in an authentic de novo RdRp assay. In
addition, we sought to determine whether the compound inhibits NS5 from other serotype of DENV. To address these
questions, we prepared recombinant full-length NS5 from both
DENV-2 and DENV-4 (Fig. 3A). The NS5 from both serotypes generated an RNA product using the DENV-2 sub-

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

FIG. 2. Inhibition of RNA elongation in a single cycle RNA synthesis assay. (A) Heparin titration (0 to 96 ng/ml) in the standard SPA
reaction. Heparin was added to 60-min preincubated NS5-RNA complex. The mixture was incubated for another 15 min before addition of
[3H]GTP to initiate the reaction. The reaction was allowed to proceed
for 60 min. (B) Analysis of IC50s for NITD-2. (C) Analysis of IC50s for
3ddGTP. For each compound, the IC50s were determined in the
presence or absence of heparin (50 ng/ml). The experimental procedures were described in Materials and Methods. Average results from
three experiments are presented.

INHIBITION OF DENGUE VIRUS RdRp

5682

NIYOMRATTANAKIT ET AL.

FIG. 4. Photoreactive inhibitor of DENV-2 NS5 RdRp. ()

Structure of the photoreactive inhibitor NITD-29. (B) Inhibition of
DENV-2 NS5 RdRp. An RdRp SPA assay was performed using
poly(C)/oligo(G)20 as a RNA template. The experiment procedure was
identical to that described in the legend to Fig. 1B. Average results
from three independent experiments are shown. Error bars represent
standard deviations. (C) Cytotoxicity of NITD-29 in Vero cells. Cytotoxicity was examined by incubation of Vero cells with the indicated
concentrations of NITD-29. Cell viability at 48 h posttreatment was
measured by an MTT assay kit (American Type Culture Collection)
and presented as a percentage of colorimetric absorbance derived
from the compound-treated cells compared to that from the mocktreated cells (with 1% DMSO). (D) Virus titer reduction assays. Vero
cells were infected with indicated viruses (MOI of 0.1) and treated
immediately with compound at 6 and 17 M. For DENV-2 and YFV,
culture medium were collected at 44 h p.i.; for chikungunya virus,
culture medium was collected at 22 h postinfection. Virus titers of all
samples were determined by plaque assays on BHK cells.

N-terminal b5 ion, all of which match the sequence QTGSAS

SMVNGVVR, as shown in the bottom panel of Fig. 5B. These
results indicate that the modification is located in the sequence
stretch SSM of amino acids 341 to 343.
If the Met343 residue is modified, two isomers could provide
a possible explanation of the additional fragments (1,344.75,
1,376.73, and 1,392.76, labeled in green in Fig. 5B) observed in
the MS/MS spectrum of m/z 2048.87; these isomers were depicted in the upper inset of Fig. 5B. However, a proportion of
the C-terminal y5 and y6 ions was shifted by one mass unit [Fig.
5B, unshifted peaks labeled as y5(a) and y6(a), shifted peaks as
y5(b) and y6(b)], indicating partial hydrolysis of Asn345 to
Asp345; this reaction is frequently observed for the sequence
element NG and is further supported by the pronounced formation of y4 promoted by Asp345 (Fig. 5B). Collectively, the
results strongly indicate that NITD-29 was cross-linked to
Met343 within the RdRp domain of NS5. Sequence alignment
showed that Met343 is absolutely conserved among the four
serotypes of DENV as well as YFV, but it is not conserved in
the WNV and JEV NS5.

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

genomic RNA (containing the first 168 nucleotides and the last
461 nucleotides of the viral genome) (Fig. 3B). The length of
the RNA product was similar to that of a 5-end 33P-labeled
input RNA template, indicating that the RNA product was
derived from the de novo RdRp activity. The DENV-4 NS5
exhibited more robust RdRp activity than the DENV-2 NS5;
the difference between the two polymerase activities could be
due to a difference in protein folding during the NS5 preparation. Nevertheless, NITD-2 reduced the amount of RNA products in a dose-responsive manner for both DENV-2 and
DENV-4 NS5, with IC50s of 4.6 and 5.2 M, respectively (Fig.
3B). These results demonstrate that NITD-2 inhibits de novo
RNA synthesis (Fig. 3B), although the potency is reduced
compared to the potency derived from the poly(C)/oligo(G)20
elongation RdRp assay (Fig. 1B). The discrepancy in IC50s
between the two assays is most likely due to the difference in
experimental conditions.
To test whether NITD-2 could inhibit other flavivirus RdRp,
we expressed full-length NS5 of WNV as described before
(46). SDS-PAGE analysis of the purified protein showed a
major band of 100 kDa, representing WNV NS5 (Fig. 3A). A
de novo RdRp assay, using a subgenomic RNA template containing the 5 terminal 269 nucleotides directly connected to
the 3-terminal 622 nucleotides of the WNV genome, was
performed. As shown in Fig. 3C, addition of NITD-2 to the
WNV RdRp reactions did not affect the yield of de novo RNA
product. Overall, these results suggest that the compound specifically inhibits DENV RdRp.
Antiviral activity of a photoreactive inhibitor. To map the
compound-binding site on the protein, we synthesized a photoreactive derivative of the inhibitor, NITD-29. Compared to
NITD-2, NITD-29 contained an extra photoalkylating group
benzophenone (Fig. 4A). NITD-29 inhibited the RdRp activity
of DENV-2 NS5 [using the poly(C)/oligo(G)20 primer extension HTS assay], with an IC50 of 1.5 M (Fig. 4B). Cytotoxicity
assay indicated that NITD-29 is not toxic up to 50 M (Fig.
4C). Importantly, virus titer reduction experiments showed
that NITD-29 at 6 and 17 M suppressed DENV-2 yields by
3.4- and 4.5-fold, respectively (Fig. 4D). In contrast, the compound only slightly inhibited YFV at 17 M; almost no inhibition was observed when chikungunya virus (a plus-strand
alphavirus)-infected cells were treated with the compound
(Fig. 4D). These results indicate that NITD-29 selectively inhibits DENV through suppression of the viral RdRp activity.
MS analysis of UV cross-linked compound/NS5 complex.
NITD-29 was UV cross-linked to DENV-2 NS5. The crosslinked complex was subjected to trypsin digestion, followed by
MALDI MS analysis. The NITD-29/NS5 complex without UV
cross-linking was similarly analyzed as a negative control. A
search of the peptide mass fingerprints of the cross-linked
sample versus the non-cross-linked sample for a mass difference of 656.17 (NITD-29) revealed a peptide ion at m/z
2,048.87, which was specific for the cross-linked sample (Fig.
5A, left panel); this ion was not observed in the non-crosslinked analog (right panel). The peptide mass matched the
sequence of amino acids 336 to 349 of DENV-2 NS5 (QTGS
ASSMVNGVVR) modified by 656.17 atomic mass units
(amu). The MS/MS spectrum of m/z 2,048.87 from the crosslinked sample showed 6 unmodified C-terminal fragment ions
(Fig. 5B, labeled as y1 to y6 in red) and one unmodified

J. VIROL.

VOL. 84, 2010

INHIBITION OF DENGUE VIRUS RdRp

5683

Inhibition of RdRp activity through binding to the RNA

template tunnel. Based on the crystal structure, Met343 is
located next to the thumb subdomain of the DENV RdRp
(Fig. 6A). Docking and molecular simulation suggest that the
compound binds to the RNA template formed between the
fingers and thumb subdomains; the detailed methods for modeling was recently reported (45). Two RdRp residues, Arg737
and Thr413, form distinct hydrogen bond interactions with the
inhibitor; the observed interactions were maintained during
the course of the dynamics simulations, indicating their roles in
stabilizing the compound in the allosteric binding pocket (45).
Sequence alignment showed that residue Arg737 is conserved

among mosquito-borne flavivirus NS5, whereas residue Thr413

is divergent among various members of flavivirus.
These results suggest that the compound inhibits RdRp activity through competing with RNA template in the RNA template tunnel. If this is the case, the order of addition of inhibitor and RNA template during the assembly of RdRp assay
should affect the compound potency. We performed two sets of
experiments to test this hypothesis (Fig. 6B). For the first set of
experiments, we preincubated NS5 with RNA template for 1 h
before adding the inhibitor (NITD-2) and [3H]GTP to initiate
the reaction; under this assay scheme, the compound displayed
an IC50 of 1.80 M. Reversing the order of addition between

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

FIG. 5. MS analysis of UV-cross-linked DENV-2 NS5 and NITD-29 complex. (A) Partial MALDI-MS traces of compound-cross-linked (left)
and control (right) NS5 tryptic digests showing the differential peak at m/z 2,048.87. (B) MSMS spectrum of m/z 2,048.87 with insets to illustrate
compound cross-linking (top) and peak assignment of the observed fragment ions (bottom).

5684

NIYOMRATTANAKIT ET AL.

J. VIROL.

the RNA template and inhibitor significantly improved the

IC50 to 0.48 M (p value of 0.05 [Student t test]). For the
second set of experiments, we incubated NS5 with RNA template for 1 h, followed by the compound addition. After incubating the mixture for another 1 h, [3H]GTP was added to start
the reaction. This experimental scheme yielded an IC50 of 4.63
M. Reversing the order of addition between the RNA template and inhibitor significantly improved the IC50 to 0.66 M
(P value of 0.05). These results clearly indicate that the
compound and RNA template compete against each other
during the RdRp reactions.
DISCUSSION
To develop flavivirus antivirals, we have taken both nucleoside analog and NNI approaches to target DENV RdRp. As
the first step toward the NNI approach, we identified an Nsulfonylanthranilic acid derivative (NITD-1) as an inhibitor of
DENV-2 RdRp through HTS. SAR study improved the
compound potency. We used two distinct assays to demonstrate the anti-DENV RdRp activity: assay 1 measured the
activity against RNA elongation of poly(C)/oligo(G)20, and
assay 2 measured the activity against de novo RNA synthesis
using a DENV-2 subgenomic RNA template. The compound
inhibited RNA production in both assays, indicating that the
anti-RdRp activity is not template specific. In contrast, the
compound (NITD-2) selectively inhibited DENV RdRp; it did
not inhibit a closely related WNV RdRp (Fig. 3C), despite the
structural similarity of the two polymerases (26, 43). Furthermore, we showed that the compound did not inhibit HCV
RdRp or human DNA polymerase and at higher concen-

trations (Fig. 1C). The observed DENV-specific activity is desirable for antiviral development because it avoids potential
side effects from nonspecific inhibition of host polymerases.
Our biochemical and structural experiments strongly indicate that the compound inhibits RdRp through binding to the
RNA template tunnel of the polymerase. This conclusion was
supported by three lines of evidence. First, UV cross-linking
and MS analyses showed that NITD-29 could be cross-linked
to residue M343 within the RdRp domain of NS5 (Fig. 5). This
residue is located at the entrance of the RNA template tunnel.
Computational docking and simulation suggest that the compound binds to the RNA template tunnel (Fig. 6A) (45). Second, crystal structure analysis of the DENV-3 RdRp in complex with the compound revealed an extra electron density in
the RNA template tunnel (J. Lescar et al., unpublished results). Unfortunately, the current cocrystal form did not allow
us to trace the complete compound structure in the RdRp
structure. Lastly, biochemical RdRp assay showed that the
order of addition of RNA template and inhibitor affected
the IC50 of the compound. Addition of the compound before
the RNA template during assay assembly displayed a better
potency; reversing the order of addition reduced the compound potency (Fig. 6B).
How does the identified compound inhibit RdRp activity
through binding to the RNA template tunnel? Crystal structures show that RdRp from DENV-3 and WNV are in a
closed conformation (26, 43). The proteins are expected to
change to an open conformation during RNA initiation and
elongation. Such conformational switches are required to allow
(i) the single-stranded RNA template to enter the template
tunnel and (ii) the newly synthesized RNA (together with the

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

FIG. 6. Binding site of NITD-29 in the DENV RdRp domain. (A) Three-dimensional model of NITD-29 in complex with DENV RdRp. The
model was built on the results of UV cross-linking, MS analysis, and the crystal structure of DENV-3 RdRp (43). Three RdRp subdomains (thumb,
fingers, and palm) are indicated. The RNA tunnel and NTP entrance are circled with dotted lines. NITD-29 is shown in green. Three critical
residues for interaction with the compound are labeled in red: M343 was cross-linked to NITD-29; R737 and T413 are within a hydrogen bond
distance from the compound. (Left panel) Surface view; (right panel) ribbon view; (middle panel) magnified surface view of the compound binding
site (the RdRp protein was set to partial transparency to view the complete molecule of NITD-29). The images were produced by using PyMOL
software (6). (B) IC50s from RdRp assays with different orders of reagent assembly. Different orders of reagent addition (RNA template and
NITD-2 inhibitor) during reaction assembly are depicted. The IC50s were determined for each experimental scheme. The experiments were
performed using poly(C)/oligo(G)20 as a template in the SPA format. The abbreviations are indicated. See Materials and Methods for reagent
concentrations.

VOL. 84, 2010

INHIBITION OF DENGUE VIRUS RdRp

ACKNOWLEDGMENTS
We thank Subhash Vasudevan, Julien Lescar, Thai Leong Yap, and
Wan Yen Lee for helpful discussions and technical support. We also
thank Ka Yan Chung and Siew Pheng Lim for providing the plasmid
template used for DENV-2 subgenomic RNA synthesis.

REFERENCES
1. Ackermann, M., and R. Padmanabhan. 2001. De novo synthesis of RNA by
the dengue virus RNA-dependent RNA polymerase exhibits temperature
dependence at the initiation but not elongation phase. J. Biol. Chem. 276:
3992639937.
2. Al-Mawsawi, L. Q., V. Fikkert, R. Dayam, M. Witvrouw, T. R. Burke, Jr.,
C. H. Borchers, and N. Neamati. 2006. Discovery of a small-molecule HIV-1
integrase inhibitor-binding site. Proc. Natl. Acad. Sci. U. S. A. 103:10080
10085.
3. Beaulieu, P. L. 2007. Non-nucleoside inhibitors of the HCV NS5B polymerase: progress in the discovery and development of novel agents for the
treatment of HCV infections. Curr. Opin. Invest. Drugs. 8:614634.
4. Best, S. M., K. L. Morris, J. G. Shannon, S. J. Robertson, D. N. Mitzel, G. S.
Park, E. Boer, J. B. Wolfinbarger, and M. E. Bloom. 2005. Inhibition of
interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and
identification of NS5 as an interferon antagonist. J. Virol. 79:1282812839.
5. Biswal, B. K., M. Wang, M. M. Cherney, L. Chan, C. G. Yannopoulos, D.
Bilimoria, J. Bedard, and M. N. James. 2006. Non-nucleoside inhibitors
binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of
inhibition. J. Mol. Biol. 361:3345.
6. Brunger, A., P. Adams, G. Clore, W. DeLano, P. Gros, R. Grosse-Kunstleve,
J. Jiang, J. Kuszewski, M. Nilges, N. Pannu, R. Read, L. Rice, T. Simonson,
and G. Warren. 1998. Crystallography & NMR system: a new software suite
for macromolecular structure determination. Acta Crystallogr. D 54:905
921.
7. Chambers, T. J., A. Grakoui, and C. M. Rice. 1991. Processing of the yellow
fever virus nonstructural polyprotein: a catalytically active NS3 proteinase
domain and NS2B are required for cleavages at dibasic sites. J. Virol. 65:
60426050.
8. Chan, L., S. K. Das, T. J. Reddy, C. Poisson, M. Proulx, O. Pereira, M.
Courchesne, C. Roy, W. Wang, A. Siddiqui, C. G. Yannopoulos, N. NguyenBa, D. Labrecque, R. Bethell, M. Hamel, P. Courtemanche-Asselin, L.
LHeureux, M. David, O. Nicolas, S. Brunette, D. Bilimoria, and J. Bedard.
2004. Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV
NS5B polymerase and HCV subgenomic RNA replication. 1. Sulfonamides.
Bioorg. Med. Chem. Lett. 14:793796.
9. De Clercq, E. 2009. Anti-HIV drugs: 25 compounds approved within 25 years
after the discovery of HIV. Int. J. Antimicrob. Agents 33:307320.
10. De Clercq, E., and J. Neyts. 2009. Antiviral agents acting as DNA or RNA
chain terminators. Handb. Exp. Pharmacol. 2009:5384.
11. De Francesco, R., L. Tomei, S. Altamura, V. Summa, and G. Migliaccio.
2003. Approaching a new era for hepatitis C virus therapy: inhibitors of the
NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase.
Antivir. Res. 58:116.
12. Dong, H., B. Zhang, and P. Y. Shi. 2008. Terminal structures of West Nile
virus genomic RNA and their interactions with viral NS5 protein. Virology
381:123135.
13. Egloff, M. P., D. Benarroch, B. Selisko, J. L. Romette, and B. Canard. 2002.
An RNA cap (nucleoside-2-O-)-methyltransferase in the flavivirus RNA
polymerase NS5: crystal structure and functional characterization. EMBO J.
21:27572768.
14. Falgout, B., R. H. Miller, and C. J. Lai. 1993. Deletion analysis of dengue
virus type 4 nonstructural protein NS2B: identification of a domain required
for NS2B-NS3 protease activity. J. Virol. 67:20342042.
15. Gubler, D., G. Kuno, and L. Markoff. 2007. Flaviviruses, p. 11531253. In
D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed., vol. 1. Lippincott/The William & Wilkins Co., Philadelphia, PA.
16. Guo, J., J. Hayashi, and C. Seeger. 2005. West Nile virus inhibits the signal
transduction pathway of alpha interferon. J. Virol. 79:13431350.
17. Huberman, J. 1981. New views of the biochemistry of eucaryotic DNA
replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase
alpha. Cell 23:647648.
18. Koonin, E. V. 1993. Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of
reovirus. J. Gen. Virol. 74:733740.
19. Kummerer, B. M., and C. M. Rice. 2002. Mutations in the yellow fever virus
nonstructural protein NS2A selectively block production of infectious particles. J. Virol. 76:47734784.
20. Le Pogam, S., A. Seshaadri, A. Kosaka, S. Chiu, H. Kang, S. Hu, S. Rajyaguru, J. Symons, N. Cammack, and I. Najera. 2008. Existence of hepatitis
C virus NS5B variants naturally resistant to non-nucleoside, but not to
nucleoside, polymerase inhibitors among untreated patients. J. Antimicrob.
Chemother. 61:12051216.
21. Leung, J. Y., G. P. Pijlman, N. Kondratieva, J. Hyde, J. M. Mackenzie, and
A. A. Khromykh. 2008. Role of nonstructural protein NS2A in flavivirus
assembly. J. Virol. 82:47314741.
22. Lindenbach, B. D., H.-J. Thiel, and C. M. Rice. 2007. Flaviviridae: the virus
and their replication, p. 11011152. In D. M. Knipe and P. M. Howley (ed.),
Fields virology, 5th ed., vol. 1. Lippincott/The William & Wilkins Co., Philadelphia, PA.
23. Liu, W., X. Wang, V. Mokhonov, P.-Y. Shi, R. Randall, and A. Khromykh.

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

template strand) to exit the RNA output channel. The compound identified in the present study may interfere with the
conformational change, leading to the reduction of RNA synthesis. Alternatively, the compound binding to the RNA tunnel
may sterically block the entrance of the RNA template to the
polymerase. Structural studies are needed to further define the
mechanism of inhibition.
Development of NNI is often faced with the challenge of
virus heterogeneity. The amino acids that form an allosteric
binding site for NNI are often under less selective pressure,
resulting in quick emergence of resistance strains. In fact, allosteric inhibitors that were developed against one genotype of
HCV were often not potent against another genotype (34); in
addition, resistant isolates may already exist in the clinical
quasispecies (20), which can quickly dominate viral population
upon monotherapy of NNI. For flaviviruses, NS5 is the most
conserved viral protein (22), among which the NS5 from the
four serotypes of DENV has an amino acid homology of 73%.
Although we showed that NITD-2 inhibited RdRp activities of
both DENV-2 and DENV-4 NS5, the compound did not inhibit WNV RdRp (Fig. 3). The selective inhibition is most
likely due to a subtle conformational difference and amino acid
variation between the DENV and WNV enzymes.
Inhibitors identified from target-based HTS are often inactive in cell culture. Although NITD-29 inhibited DENV-2 in a
virus titer reduction assay (Fig. 4D), neither NITD-1 nor
NITD-2 exhibited any antiviral activity in cell culture (data not
shown). Three common reasons could account for the discrepancy between the enzyme activity and the cell culture activity:
(i) lack of cell permeability, (ii) metabolic instability, and (iii)
inaccessibility of the binding site (due to steric hindrance of
other components in the replication complex). In vitro pharmaco-analysis showed that NITD-2 had a logPe value of
6.0 in the PAMPA (for parallel artificial membrane permeability assay), suggesting that the compound penetrates cell
membrane poorly. Chemistry effort is needed to improve the
cell permeability of the current lead compound NITD-2.
To our knowledge, the present study reports the first allosteric inhibitor of RdRp from members of the genus Flavivirus.
For development of allosteric inhibitors, Malet et al. recently
proposed two allosteric pockets that were conserved in the
crystal structures of DENV-3 and WNV RdRp (27). Both
pockets are located in the thumb subdomain and could be
targeted for rational design of small molecular inhibitors.
However, before launching an antiviral effort, it would be important to validate the biological relevance of these pockets.
This could be accomplished by mutagenesis of recombinant
NS5 and analysis of the RdRp/MTase activities of the mutant
proteins; the role of these allosteric pockets in viral replication
could be examined by mutagenesis of an infectious cDNA
clone of DENV. These studies should provide new opportunities for development NNI of DENV.

5685

5686

24.

25.

26.

27.

28.

30.

31.

32.

33.

34.

35.

2005. Inhibition of interferon signaling by the New York 99 strain and Kunjin
subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins. J. Virol. 79:19341942.
Liu, W. J., H. B. Chen, and A. A. Khromykh. 2003. Molecular and functional
analyses of Kunjin virus infectious cDNA clones demonstrate the essential
roles for NS2A in virus assembly and for a nonconservative residue in NS3
in RNA replication. J. Virol. 77:78047813.
Ma, H., V. Leveque, A. De Witte, W. Li, T. Hendricks, S. M. Clausen, N.
Cammack, and K. Klumpp. 2005. Inhibition of native hepatitis C virus
replicase by nucleotide and non-nucleoside inhibitors. Virology 332:815.
Malet, H., M. P. Egloff, B. Selisko, R. E. Butcher, P. J. Wright, M. Roberts,
A. Gruez, G. Sulzenbacher, C. Vonrhein, G. Bricogne, J. M. Mackenzie, A. A.
Khromykh, A. D. Davidson, and B. Canard. 2007. Crystal structure of the
RNA polymerase domain of the West Nile virus nonstructural protein 5.
J. Biol. Chem. 282:1067810689.
Malet, H., N. Masse, B. Selisko, J. L. Romette, K. Alvarez, J. C. Guillemot,
H. Tolou, T. L. Yap, S. Vasudevan, J. Lescar, and B. Canard. 2008. The
flavivirus polymerase as a target for drug discovery. Antivir. Res. 80:2335.
McKercher, G., P. L. Beaulieu, D. Lamarre, S. LaPlante, S. Lefebvre, C.
Pellerin, L. Thauvette, and G. Kukolj. 2004. Specific inhibitors of HCV
polymerase identified using an NS5B with lower affinity for template/primer
substrate. Nucleic Acids Res. 32:422431.
Munoz-Jordan, J. L., M. Laurent-Rolle, J. Ashour, L. Martinez-Sobrido, M.
Ashok, W. I. Lipkin, and A. Garcia-Sastre. 2005. Inhibition of alpha/beta
interferon signaling by the NS4B protein of flaviviruses. J. Virol. 79:8004
8013.
Munoz-Jordan, J. L., G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-Sastre. 2003. Inhibition of interferon signaling by dengue virus. Proc.
Natl. Acad. Sci. U. S. A. 100:1433314338.
Ogawa, A., T. Murate, M. Suzuki, Y. Nimura, and S. Yoshida. 1998. Lithocholic acid, a putative tumor promoter, inhibits mammalian DNA polymerase beta. Jpn. J. Cancer Res. 89:11541159.
Olsen, D. B., A. B. Eldrup, L. Bartholomew, B. Bhat, M. R. Bosserman, A.
Ceccacci, L. F. Colwell, J. F. Fay, O. A. Flores, K. L. Getty, J. A. Grobler,
R. L. LaFemina, E. J. Markel, G. Migliaccio, M. Prhavc, M. W. Stahlhut,
J. E. Tomassini, M. MacCoss, D. J. Hazuda, and S. S. Carroll. 2004. A
7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C
virus replication with excellent pharmacokinetic properties. Antimicrob.
Agents Chemother. 48:39443953.
Patkar, C. G., and R. J. Kuhn. 2008. Yellow Fever virus NS3 plays an
essential role in virus assembly independent of its known enzymatic functions. J. Virol. 82:33423352.
Pauwels, F., W. Mostmans, L. M. Quirynen, L. van der Helm, C. W. Boutton,
A. S. Rueff, E. Cleiren, P. Raboisson, D. Surleraux, O. Nyanguile, and K. A.
Simmen. 2007. Binding-site identification and genotypic profiling of hepatitis
C virus polymerase inhibitors. J. Virol. 81:69096919.
Pauwels, R., K. Andries, Z. Debyser, M. Kukla, D. Schols, J. Desmyter, E. De
Clercq, and P. A. Janssen. 1992. TIBO derivatives: a new class of highly
potent and specific inhibitors of HIV-1 replication. Biochem. Soc. Trans.
20:509512.

J. VIROL.
36. Puig-Basagoiti, F., M. Tilgner, B. Forshey, S. Philpott, N. Espina, Wentworth, S. Goebel, P. S. Masters, B. Falgout, P. Ren, D. M. Ferguson, and
P. Y. Shi. 2006. Triaryl pyrazoline compound inhibits flavivirus RNA replication. Antimicrob. Agents Chemother. 50:13201329.
37. Ray, D., A. Shah, M. Tilgner, Y. Guo, Y. Zhao, H. Dong, T. Deas, Y. Zhou,
H. Li, and P.-Y. Shi. 2006. West Nile virus 5-cap structure is formed by
sequential guanine N-7 and ribose 2-O methylations by nonstructural protein 5. J. Virol. 80:83628370.
38. Schul, W., W. Liu, H. Y. Xu, M. Flamand, and S. G. Vasudevan. 2007. A
dengue fever viremia model in mice shows reduction in viral replication and
suppression of the inflammatory response after treatment with antiviral
drugs. J. Infect. Dis. 195:665674.
39. Tan, B. H., J. Fu, R. J. Sugrue, E. H. Yap, Y. C. Chan, and Y. H. Tan. 1996.
Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli
exhibits RNA-dependent RNA polymerase activity. Virology 216:317325.
40. Tscherne, D. M., M. J. Evans, T. von Hahn, C. T. Jones, Z. Stamataki, J. A.
McKeating, B. D. Lindenbach, and C. M. Rice. 2007. Superinfection exclusion in cells infected with hepatitis C virus. J. Virol. 81:36933703.
41. Voshol, H., N. Brendlen, D. Muller, B. Inverardi, A. Augustin, C. Pally, G.
Wieczorek, R. E. Morris, F. Raulf, and J. van Oostrum. 2005. Evaluation of
biomarker discovery approaches to detect protein biomarkers of acute renal
allograft rejection. J. Proteome Res. 4:11921199.
42. Wengler, G., and G. Wengler. 1991. The carboxy-terminal part of the NS 3
protein of the West Nile flavivirus can be isolated as a soluble protein after
proteolytic cleavage and represents an RNA-stimulated NTPase. Virology
184:707715.
43. Yap, T., T. Xu, Y. Chen, H. Malet, M. Egloff, B. Canard, S. Vasudevan, and
J. Lescar. 2007. Crystal structure of the dengue virus RNA-dependent RNA
polymerase catalytic domain at 1.85- resolution. J. Virol. 81:47534765.
44. Yin, Z., Y.-L. Chen, W. Schul, Q.-Y. Wang, F. Gu, J. Duraiswamy, K. R.
Reddy, P. Niyomrattanakit, S. B. Lakshminarayana, A. Goh, H. Y. Xu, W.
Liu, B. Liu, J. Y. H. Lim, C. Y. Ng, M. Qing, C. C. Lim, A. Yip, G. Wang,
W. L. Chan, H. P. Tan, K. Lin, B. Zhang, G. Zou, K. A. Bernard, C. Garrett,
B. Beltz, M. Dong, W. M., H. He, X. Han, A. Pichota, V. Dartois, T. H. Keller,
and P.-Y. Shi. 2009. An adenosine nucleoside inhibitor of dengue virus. Proc.
Natl. Acad. Sci. U. S. A. 106:2043520439.
45. Yin, Z., Y. L. Chen, R. R. Kondreddi, W. L. Chan, G. Wang, R. H. Ng, J. Y.
Lim, W. Y. Lee, D. A. Jeyaraj, P. Niyomrattanakit, D. Wen, A. Chao, J. F.
Glickman, H. Voshol, D. Mueller, C. Spanka, S. Dressler, S. Nilar, S. G.
Vasudevan, P. Y. Shi, and T. H. Keller. 2009. N-Sulfonylanthranilic acid
derivatives as allosteric inhibitors of dengue viral RNA-dependent RNA
polymerase. J. Med. Chem. 52:79347937.
46. Zhang, B., H. Dong, Y. Zhou, and P. Y. Shi. 2008. Genetic interactions
among the West Nile virus methyltransferase, the RNA-dependent RNA
polymerase, and the 5 stem-loop of genomic RNA. J. Virol. 82:70477058.
47. Zhang, C., Z. Cai, Y. C. Kim, R. Kumar, F. Yuan, P. Y. Shi, C. Kao, and G.
Luo. 2005. Stimulation of hepatitis C virus (HCV) nonstructural protein 3
(NS3) helicase activity by the NS3 protease domain and by HCV RNAdependent RNA polymerase. J. Virol. 79:86878697.

Downloaded from http://jvi.asm.org/ on December 22, 2014 by guest

29.

NIYOMRATTANAKIT ET AL.