Identification of BPR3P0128 as an inhibitor of cap-snatching activities of influenza virus

Article abstract of DOI:10.1128/AAC.00125-11

The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin-Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of capdependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5′ viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics. Copyright

Full text of DOI:10.1128/AAC.00125-11

Identification of BPR3P0128 as an Inhibitor of Cap-Snatching
Activities of Influenza Virus
John T.-A. Hsu,a,b Jiann-Yih Yeh,a Ta-Jen Lin,c Mei-ling Li,d Ming-Sian Wu,c Chung-Fan Hsieh,c Yao Chieh Chou,c Wen-Fang Tang,c
Kean Seng Lau,e Hui-Chen Hung,a Ming-Yu Fang,a Shengkai Ko,a Hsing-Pang Hsieh,a and Jim-Tong Horngc,e,f
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 350, Taiwan,a Department of Biological Science and Technology,
National Chiao Tung University, Hsinchu 300, Taiwan,b Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan 333, Taiwan,c Department of Molecular
Genetics, Microbiology & Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway NJ 08854,d Department of Biochemistry, Chang Gung University,
Taoyuan 333, Taiwan,e Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan 333, Taiwanf
The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of
a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin–
Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50%
inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect as-
says. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of cap-
dependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA repli-
cation in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in
mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activi-
ties of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5= viral RNA to the viral
polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovi-
rus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral
PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and repli-
cation and thereby provide a potential target for antiviral chemotherapeutics.
nfluenza viruses are respiratory pathogens that affect humans tion of negative-strand vRNAs, which contrasts with the primer
I
and are responsible for substantial morbidity and mortality. The
(cap)-independent process and produces a full-length replicative
intermediate cRNA. This cRNA is then replicated to produce
more vRNA. The timing of mRNA and cRNA/vRNA synthesis
differs because replication follows mRNA transcription and de
novo protein synthesis (43). However, the tuning mechanism for
the balance between transcription and replication has remained
elusive. Hypotheses based on pieces of vital evidence of the factors
controlling the viral switch to replication have been proposed. The
switch is thought to be regulated by the availability of an NP, the
stability of cRNA mediated by the vRNP complex, and NS2/NEP
(nonstructural protein 2/nuclear export protein) (35, 48, 56). A
more recent finding identified the mechanism through which in-
fluenza virus-specific small viral RNAs regulate the switch (44).
The regions within the PB2 subunit of the influenza virus RNA
polymerase involved in cap binding have also been studied in great
detail. Early studies showed that cap binding is a function of PB2
(43). In addition, mutagenesis and cross-linking studies show that
other regions of PB2, PB1, and, possibly, PA are required for cap
binding (14, 16, 23). Attempts have been made to map the region
viral genome (viral RNA [vRNA]) comprises eight segments of
negative-sense RNA that encode up to 12 proteins (43, 60). Each
segment of RNA is encapsidated into a ribonucleoprotein (RNP)
complex containing a trimeric RNA-dependent RNA polymerase
complex comprising PA, PB1, and PB2 and multiple copies of a
nucleocapsid protein (NP). The viral polymerase activity resides
in the RNP complexes, whose replication and transcription take
place in the nucleus of the infected cells. The newly synthesized
viral RNPs (vRNPs) must be transported out of the nucleus, and
this export requires cellular and viral proteins (4).
The influenza virus polymerase transcribes cap- and poly(A)-
dependent mRNA using a cap-dependent endoribonuclease (cap-
snatching) mechanism (45). The host pre-mRNAs are bound to
the cap-binding domains of the PB2 subunit by their 5= cap. A
fragment of the first 10 to 13 nucleotides of the host mRNA is
cleaved by the endoribonuclease located in the N terminus of the
PA subunit (11, 43, 63). The production of primers is activated
only when the 5= and 3= end sequences of vRNA bind sequentially
to the PB1 subunit (33). vRNA has been used as a template to
transcribe the mRNA joined by the PB1 subunit (33, 43). Tran-
scription of influenza virus can thus be divided into the following
steps: (i) binding of the 5= and 3= vRNA sequences to the PB1
subunit, which is likely to cause a conformational change in the
polymerase complex (6, 33); (ii) binding of the 5= cap (m7GTP) of
a host pre-mRNA to the PB2 subunit (22); (iii) cleavage of a phos-
phodiester bond 10 to 13 nucleotides downstream of the cap by
the PA subunit; and (iv) activation of the transcription of viral
mRNAs at the cleaved 3= end of the capped fragment. This poly-
merase complex catalyzes both mRNA transcription and replica-
Received 30 January 2011 Returned for modification 21 March 2011
Accepted 1 August 2011
Published ahead of print 19 September 2011
Address correspondence to Hsing-Pang Hsieh (chemistry questions),
hphsieh@nhri.org.tw, or Jim-Tong Horng (virology questions),
jimtong@mail.cgu.edu.tw.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00125-11
0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p. 647–657
aac.asm.org 647

Hsu et al.
ical lab QC no. H201; collection date, January 2003; validated by antibod-
ies, catalog no. 206-84, from Argene), and adenovirus (Ad; clinical lab QC
no. Ad-PC-0812; collection date, December 2008; validated by antibodies,
catalog no. 5009, from Millipore). The human rhinovirus 2 (HRV2) strain
used was clinical lab QC no. HR201. Madin-Darby canine kidney
(MDCK), human embryonic kidney 293 (HEK293), and rhabdomyosar-
coma (RD) cells were from ATCC. MDCK cells were maintained in Dul-
becco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA)
containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Invitro-
gen), 2 mM L-glutamine (Gibco), and a nonessential amino acid mixture
(NEAA; 0.1 mM; Gibco). HEK293 and RD cells were cultivated in DMEM
containing 10% heat-inactivated FBS. Human lung carcinoma A549 cells
were cultivated in minimal essential medium (Invitrogen) supplemented
with 10% heat-inactivated FBS and 0.1 mM NEAA. Influenza viruses and
enteroviruses were propagated using MDCK and RD cells, respectively,
and viral titers were measured using a plaque assay (52, 61).
FIG 1 Chemical structures of CSV0C019002 and BPR3P0128. More than 200
analogues were synthesized on the basis of CSV0C019002 as an initial hit.
BPR3P0128 was identified as a potent anti-influenza virus agent after intensive
lead optimization, including variation of substituents, ring expansions/con-
tractions, extension of the structure, and application of ring variations.
Chemicals. Oseltamivir carboxylate (GS4071), the active metabolite
of oseltamivir, was prepared by Kak-Shan Shia (National Health Research
Institute, Miaoli, Taiwan), and a stock solution of 10 mM was prepared in
dimethyl sulfoxide (DMSO). BPR3P0128 (6-bromo-2-[1-(2,5-dimethyl-
phenyl)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylicacid),5-bromo-
isatin (1.0 mmol), 1-[1-(2,5-dimethyl-phenyl)-5-methyl-1H-pyrazol-4-
yl]-ethanone (1.0 mmol), and KOH (4 mmol) were dissolved in ethanol
(2 ml), and the mixture was refluxed for 48 h. Removal of the solvent
under vacuum produced a residue, which was dissolved in H2O (5 ml),
and the aqueous solution was washed twice with ethanol (5 ml). The
aqueous phase was cooled and acidified to pH 1 with 3 N HCl, and the
precipitate formed was collected by suction filtration, washed with H2O,
and vacuum dried to give the title compound in 38% yield.
of PB2 involved in cap binding. Two aromatic amino acids,
Phe363 and Phe404, are required for cap binding and for tran-
scriptional activity. These have been proposed to sandwich a
methylated guanosine, as in other cap-binding proteins (13, 14).
The location of the cap-binding site on PB2 has been determined
at the atomic level by crystal structure and functional analyses,
although the direct involvement of Phe363 was not confirmed
(22).
A search for influenza virus-inhibiting drugs is particularly im-
portant in the face of new pandemic strains and the high rate of
emergence of influenza virus strains resistant to several existing
influenza virus antivirals (10, 32, 59). We conducted a drug-
screening study using a large-scale anti-influenza virus cell-based
assay based on the inhibition of the virally induced cytopathic
effect (CPE) (52, 53, 62). An initial hit, CSV0C019002, with a 50%
effective (inhibitory) concentration (EC50) of 4.27 ␮M toward
A/WSN/33 was identified after screening 20,800 randomly se-
lected small compounds (molecular masses, Ͻ500 Da) from a
library. This lead compound was effective against a panel of influ-
enza A and B viruses with similar potency (data not shown). The
activity of this novel class of compounds was improved using
structure-activity relationship studies to explore the pharmaco-
phore (molecular framework responsible for a drug’s biological
activity) requirements for anti-influenza virus activity. The most
potent compound, BPR3P0128, was identified biochemically as
having potent anti-cap-snatching activity, and its chemical struc-
ture was distinct from that of two compounds identified previ-
ously in this screening (Fig. 1) (52, 53, 62) and two other viral
polymerase inhibitors, T-705 (favipiravir) and compound A3 (19,
27). Here, we characterized BPR3P0128 and identified its under-
lying antiviral mechanism.
Cytotoxicity and antiviral effectiveness assays. The compound li-
brary was obtained from ChemDiv, Inc. (San Diego, CA). The cytotoxic
effect of BPR3P0128 on cells was determined using
a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based
assay. Cells (2 ϫ 104/well) were seeded into 96-well culture plates and
incubated overnight. The medium was then removed, and the cells were
incubated with various concentrations of BPR3P0128 in the medium for
72 h. Triplicate wells were used for each treatment. The medium was then
removed, MTT was added at a concentration of 5 mg/ml, and the cells
were incubated for 3 h at 37°C. The medium was then withdrawn carefully
TABLE 1 Antiviral activities of BPR3P0128 against various viruses
Cell line or virus strain
CC50 (␮M)
EC50 (␮M)a
MDCK
A549
RD
Ͼ20
Ͼ20
Ͼ20
Ͼ20
HEK293
A/WSN/33 (H1N1)
A/TW/3355/97 (H1N1)
A/CA/07/2009 (H1N1pdm)
A/TW/3446/02 (H3N2)
B/TW/99/07
0.083 Ϯ 0.022
0.09 Ϯ 0.02
0.145 Ϯ 0.027
0.190 Ϯ 0.100
0.051 Ϯ 0.015
0.110 Ϯ 0.051
0.054 Ϯ 0.038
MATERIALS AND METHODS
Viruses, cell culture, and viral infection. Influenza virus strain
A/WSN/33 (H1N1) virus stocks purchased from the American Type Cul-
ture Collection (ATCC) were used in this study unless stated otherwise.
The clinical influenza virus strains listed in Table 1 were from the Clinical
Virology Laboratory of Chang Gung Memorial Hospital, Taiwan, except
A/CA/07/2009 (H1N1pdm), which was obtained from the Taiwanese
Centers for Disease Control. The information for other viruses is as fol-
lows: enterovirus 71 (EV71/Tainan/4643/98/MP4) was obtained from
Jen-Ren Wang, National Cheng Kung University (Tainan, Taiwan), cox-
sackievirus B3 (CVB3; clinical lab quality control [QC] no. CB3-PC-0811;
collection date, November 2008; validated by antibody; catalog no. 3306,
from Millipore, Billerica, MA), herpes simplex virus type 2 (HSV-2; clin-
B/TW/710/05
B/TW/70325/05
EV71/Tainan/4643/98/MP4
Coxsackievirus B3
Human rhinovirus 2
HSV-2
0.029 Ϯ 0.001
0.062 Ϯ 0.002
0.079 Ϯ 0.002
Ͼ20
Adenovirus
Ͼ20
a Influenza virus was tested in MDCK cells; coxsackievirus B3, enterovirus 71, and
human rhinovirus 2 were tested in RD cells; and adenovirus and HSV-2 were tested in
A549 cells. Data are presented as means Ϯ SDs of the results of at least two independent
experiments.
648 aac.asm.org
Antimicrobial Agents and Chemotherapy

Anti-Influenza Virus Activity of BPR3P0128
without touching the cells. DMSO (200 ␮l/well) was added to dissolve the The mixture was then injected subcutaneously into BALB/c mice. The
crystals, and the absorbance of each well was measured at 570 nm in a supernatants of hybridoma cells to poly(I:C) and the dsRNA of influenza
microplate reader (VICTOR3; PerkinElmer).
virus (A/WSN/33) were characterized using an enzyme-linked immu-
The anti-influenza virus activity (EC50) exerted by BPR3P0128, mea- nosorbent assay and immunofluorescence microscopy, respectively. Our
sured as the inhibition of virus-induced cell death (CPE), was measured anti-dsRNA antibodies thus have the specificity to detect dsRNA in influ-
using the crystal violet staining method. Briefly, 96-well tissue culture enza virus-infected cells, which contrasts with the commercially available
plates were seeded with 2.4 ϫ 104 cells/well and incubated for 16 to 20 h at J2 monoclonal antibodies that were screened against L-dsRNA from Sac-
37°C under 5% CO2. The cells were washed once with Dulbecco’s charomyces cerevisiae killer virus (50, 58).
phosphate-buffered saline (DPBS), infected with virus at 9 50% tissue
MeasurementofvirusRNAsynthesisbyqRT-PCR. MDCKcellswere
culture infective doses, and then overlaid with various concentrations of infected with A/WSN/33 virus at an MOI of 1, and BPR3P0128 was added
compounds in a total volume of 200 ␮l of E0 medium (DMEM with during the infection period as described in the scheme shown in Fig. 3.
penicillin [100 U/ml], streptomycin [100 ␮g/ml], L-glutamine [2 mM], Total RNA was extracted from cells at the times indicated using TRIzol
NEAA mixture [0.1 mM], and 2.5 ␮g/ml trypsin). After incubation at reagent (Invitrogen). The first strand of cDNA was synthesized using
37°C under 5% CO2 for 72 h, the cells were treated with 100 ␮l of 4% Moloney murine leukemia virus reverse transcriptase and random hex-
paraformaldehyde overnight and then stained with 0.1% crystal violet for amers (Invitrogen). Real-time quantitative reverse transcription-PCR
30 min at room temperature. The density of the cells was measured at 570 (qRT-PCR) was performed in a 20-␮l reaction mixture containing 50 nM
nm on a VICTOR3 microplate reader. The concentration of compounds forward and reverse primers (M1 forward primer, 5=-GAC CAA TCC
necessary to reduce the virus-induced cell death by 50%, relative to the TGT CAC CTC-3=; reverse primer, 5=-GAT CTC CGT TCC CAT TAA
virus control, was calculated according to the Reed-Muench method and GAG-3=), 1ϫ SYBR green master mix (Protech Technology Enterprise,
was expressed as the EC50.
Taipei, Taiwan), and various amounts of template. The fluorescence emit-
For the plaque assay, MDCK cells were seeded into six-well tissue ted by SYBR green was detected using the ABI StepOne Plus sequence
culture plates and incubated overnight. The cells were incubated with detection system (Applied Biosystems, Foster City, CA). To quantify the
influenza virus at about 60 PFU/well with serial dilutions of the test com- changes in gene expression, the change in threshold cycle (⌬CT) method
pounds. After adsorption of the virus for 1 h at 37°C, the virus suspension was used to calculate the relative changes normalized against the GAPDH
was removed and the cells were washed with Hanks’ balanced salt solution gene (forward primer, 5=-AAG AAG GTG GTG AAG CAG GC-3=; reverse
(HBSS). The cells were then overlaid with DMEM containing 0.3% aga- primer, 5=-TCC ACC ACC CTG TTG CTG TA-3=). The CT was defined as
rose with the indicated concentrations of compounds. After incubation the cycle at which fluorescence was determined to be significantly greater
for 48 h at 37°C under 5% CO2, the cells were fixed with 10% formalde- than the background. The ratio of viral RNA to the internal control was
hyde and then stained with 1% crystal violet. The numbers of plaques were normalized to the control level 0 h after infection, which was arbitrarily set
counted, and the antiviral activity of the compounds was calculated with equal to 1.0.
respect to the virus control.
Inhibition of viral polymerase activity by BPR3P0128 using a re-
Immunofluorescence microscopy and Western blotting. MDCK porter assay and primer extension assay. The production of viral RNA
cells (1 ϫ 105 cells) were seeded onto a glass coverslip and incubated was measured using a plasmid-based reverse genetics (minireplicon or
overnight. The cells were infected with influenza virus A/WSN/33 at a RNP reconstitution) system, followed by a luciferase assay (42) or primer
multiplicity of infection (MOI) of 1 for 1 h, washed three times with extension (38) assay.
DPBS, and then treated with DMEM with or without BPR3P0128 (1 ␮M).
For the luciferase assay, about 5 ϫ 104 HEK293 cells/well were trans-
The cells were harvested 9 h after infection, fixed with 4% paraformalde- fected with plasmid pPOLI-firefly luciferase (Fluc) (0.1 ␮g), pHW2000-
hyde for 20 min, and permeabilized with 0.5% Triton in PBS for 3 min at PB1 (0.1 ␮g), pHW2000-PB2 (0.1 ␮g), pHW2000-PA (0.1 ␮g), or
room temperature. The coverslips were incubated with blocking solution pHW2000-NP (0.1 ␮g) or with pTK-Renilla luciferase (Rluc; driven by
(0.5% bovine serum albumin in PBS) for 1 h at room temperature and the thymidine kinase promoter; 5 ng; Promega, Madison, WI) as the
then reacted with goat anti-NP primary antibody (ViroStat, Portland, transfection control using the calcium phosphate method. pPOLI-Fluc is
ME) diluted in blocking solution for 1.5 h. The coverslips were then a plasmid which expresses the influenza virus-like RNA driven by a trun-
washed three times with blocking solution and incubated with the appro- cated human RNA polymerase I promoter. These polymerase complex
priate Alexa-Fluor-488-labeled secondary antibody, and the fluorescence proteins originated from A/PR/8/34 virus. Tenfold serial dilutions of
was evaluated using a Zeiss Axiovert 200 M microscope.
BPR3P0128 were added 8 h after transfection, and the cells were incubated
For Western blotting, MDCK cells (5 ϫ 105) were seeded into each for a further 48 h. Cells were then harvested and subjected to the luciferase
well of a six-well plate and incubated overnight. The cells were then in- assay using a dual-luciferase assay system (Promega) and a VICTOR3
fected with influenza virus A/WSN/33 at an MOI of 1 for 1 h, washed with plate reader (PerkinElmer). Values at each dose were analyzed in tripli-
PBS, treated with DMEM with or without BPR3P0128 (1 ␮M), and har- cate, and the means and standard deviations (SDs) from three experi-
vested at the indicated times after infection. Total cell lysates were resolved ments were graphed. The y axis in Fig. 5A is the ratio of Fluc to Rluc
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- control, which was normalized to the mock treatment (arbitrarily set
PAGE), and the proteins were transferred to polyvinylidene difluoride equal to 1.0).
membranes, which were incubated with primary antibody for 1 h and
For the minireplicon assay in transfected cells, 1 ␮g of each of
then with the appropriate secondary antibody. The proteins were detected pHW2000-PB1, pHW2000-PB2, pHW2000-PA, pHW2000-NP, and
using an enhanced chemiluminescence Western blotting detection system pPOLI-CAT-RT was cotransfected into HEK293 cells in a six-well plate.
(Millipore). Antibodies directed against NP and M1 of influenza A virus pPOLI-CAT-RT possesses the chloramphenicol acetyltransferase (CAT)
were from Abcam (Cambridge, United Kingdom) and ViroStat, respec- open reading frame in negative polarity surrounded by the untranslated
tively. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regions of the NS gene of influenza virus A/WSN/33. BPR3P0128 was
from Santa Cruz Biotechnology (Santa Cruz, CA).
added 8 h after transfection, and the cells were incubated for a further 48
Monoclonal antibody against double-stranded RNA (dsRNA) was h. Total cellular RNA was then isolated using an RNeasy minikit (Qiagen,
prepared to characterize the replicative RNA by immunofluorescence mi- Hilden, Germany) and hybridized with ␥-32P-end-labeled primers at
croscopy. Briefly, methylated bovine serum albumin (Calbiochem) was 50°C for 3 h. This primer extension reaction was performed using avian
mixed with an equal volume of poly(I:C) (Sigma) dissolved in SSC buffer myeloblastosis virus (AMV) reverse transcriptase (Primer Extension Sys-
(150 mM NaCl and 15 mM sodium citrate, pH 7.2), and the resultant tem; Promega) at 42°C for 2 h. The resulting mixtures were terminated by
insoluble mixture was emulsified with one part Freund’s adjuvant (18). denaturing at 90°C for 10 min and then separated on 8% polyacrylamide
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Hsu et al.
gels containing 7 M urea. The expected sizes of mRNA, cRNA, and vRNA
were 98 to 106, 89, and 158 bp, respectively. Two CAT-specific primers
were used in the reverse transcription reactions: primer CAT(ϩ) (5=-ATG
TTC TTT ACG ATG CGA TTG GG-3=) for detecting the extension prod-
ucts of CAT mRNA (98 to 106 bp) and cRNA (89 bp) and primer CAT(Ϫ)
(5=-CGC AAG GCG ACA AGG TGC TGA-3=) for vRNA (158 bp). Primer
extension was performed on MDCK cells infected with A/WSN/33 at an
MOI of 1, and the cells were incubated with 0 to 10 ␮M BPR3P0128. Cells
were harvested 8 h after infection, and total RNA extraction and reverse
transcription were performed as described above. The primers derived
from segment 7 (M gene) were used in these reactions: 5=-ACC GTC GCT
TTA AAT ACG G-3= (for detecting vRNA) and 5=-TCA AGT CTC GGT
GCG ATC TCG-3= (for detecting mRNA and cRNA). Primer extension
assays were performed at least three times with independently transfected
or infected cells.
InhibitionofcappedRNAbindingand5=vRNAbindingtopolymer-
ase complexes by BPR3P0128. Assays to measure the binding of specific
RNAs to polymerase complexes were performed as described previously
(33, 34). Briefly, HeLa cells were cotransfected with Vac-PB1, Vac-PB2,
and Vac-PA, and nuclear extracts of the influenza virus polymerase com-
plex were prepared for subsequent experiments. For cap-binding assays,
following a 30-min reaction for the binding of nuclear extract (2.5 ␮g)
with an unlabeled oligoribonucleotide containing the chemically synthe-
sized 5=-terminal sequence of vRNA, the 13-nucleotide capped alfalfa mo-
saic virus (ALMV) RNA 4 RNA fragment containing an m7G32pppGm 5=
end (10 ng, 1 ϫ 105 cpm) was added, and the mixture was incubated in the
presence of BPR3P0128 at 30°C for 30 min. For the 5= vRNA binding
assays, nuclear extracts were incubated in binding buffer with 32P-labeled
oligonucleotide containing the 5=-terminal sequence of vRNA (10 ng, 1 ϫ
105 cpm). These protein-RNA complexes were separated from free RNA
by electrophoresis on 4% nondenaturing gels.
Inhibition of polymerase endoribonuclease assay by BPR3P0128.
Polymerase endoribonuclease assays were performed as described previ-
ously (33). The ␤-eliminated ALMV RNA 4 (4.56 ␮g) was capped and
methylated in vitro with vaccinia virus capping enzyme (guanyltransferase
and 7-methyltransferase) and 2=-O-methyltransferase in the presence of
[␣-32P]GTP (45). mRNAs labeled with m7G32pppGm at the 5= end were
gel purified, extracted by phenol-chloroform, filtered through G-25 Sep-
hadex spin columns, and then precipitated with ethanol. Following bind-
ing of 5= and 3= vRNAs, the radiolabeled cap (0.25 ng, 1 ϫ 105 cpm) was
added along with 0.1 ng of tRNA as an inhibitor of the nonspecific inhi-
bition of nuclease activity. The mixture was incubated with or without
BPR3P0128 at 30°C for 60 min. Reactions were terminated with the ad-
dition of formamide stop solution, and the mixture was analyzed on 20%
polyacrylamide-urea gels.
FIG 2 Inhibition of progeny virus production measured by a plaque reduc-
tion assay. MDCK cells were incubated with influenza virus at about 60 PFU/
well and with serial dilutions of BPR3P0128. After adsorption of the virus for 1
h at 37°C, the viral suspension was removed and the cells were washed with
HBSS. The cells were then overlaid with DMEM containing 0.3% agarose with
the indicated compound concentrations (micromolar). After incubation for
48 h, the numbers of plaques were counted and the antiviral activity of the
compound was calculated in relation to the virus-only control.
The EC50 for viral plaque formation was estimated to be 68.4 Ϯ 8.9
nM, similar to that obtained in the anti-CPE assay (Table 1).
We then investigated whether BPR3P0128 inhibits viral pro-
tein and RNA synthesis within the viral infectious (replication)
cycle. In a time course experiment, MDCK cells were infected with
influenza virus A/WSN/33 at an MOI of 1 and were then treated
with or without BPR3P0128 at a concentration of 1 ␮M, which
inhibits virus-induced CPEs. Cells were harvested at the indicated
times after infection and subjected to Western blotting using an-
tibodies specific to M1 and NP proteins. The viral proteins pro-
duced by the influenza virus were evident 3 h after infection, and
their expression persisted thereafter. The amounts of NP and M1
proteins produced by the influenza virus were reduced in
BPR3P0128-treated cells by about 2-fold (Fig. 3A, lanes 3, 6, and
9) compared with untreated cells (lanes 2, 5, and 8) at 6 and 9 h
after infection. This suggests that the reduction in viral protein
production could be attributed to inhibition of viral translation by
BPR3P0128.
RESULTS
Antiviral activity of BPR3P0128. CSV0C019002 was one of the
initial lead compounds identified from hit screening of 20,800
compounds by measuring inhibition of virus-induced CPE (Fig.
1) (52, 53, 62). The influenza virus strain A/WSN/33 was used at
an MOI of 3 ϫ 10Ϫ5 to infect MDCK cells and to induce a CPE.
Compounds that inhibited at least 50% of the CPE at a concen-
tration of 10 ␮M were selected for lead optimization and for fur-
ther investigation of their antiviral activities and the underlying
mechanism. Using an iterative structure-activity relationship
analysis, BPR3P0128 with a core structure of quinoline was then
identified to be the most potent analogue against A/WSN/33 and
had an EC50 of 0.083 Ϯ 0.022 ␮M and a 50% cell cytotoxic con-
centration (CC50) of Ͼ20 ␮M (Table 1). A plaque reduction assay
was performed to verify the antiviral activity (Fig. 2). BPR3P0128
To investigate whether BPR3P0128 might have an effect on the
subcellular distribution of viral proteins, as seen in nucleozin
treatment, in which the nuclear import of NP is abrogated and NP
accumulates in the cytoplasm (30), we performed detailed fluo-
dose-dependently reduced the yield of viral progeny produced by rescence microscopy analysis. In the presence of BPR3P0128, the
infection of MDCK cells with influenza virus A/WSN/33 (Fig. 2). immunofluorescence staining intensity declined, indicating a de-
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FIG 3 Inhibition of viral protein and vRNA synthesis by BPR3P0128. MDCK cells were infected with A/WSN/33 virus at an MOI of 1 at Ϫ1 h. BPR3P0128 (1
␮M) was added from 0 h, and cell lysates were harvested at the indicated times after infection (hours postinfection [hpi]). The data are presented as means Ϯ
SEMs of the results from at least two independent experiments. (A) Inhibition of viral protein synthesis by BPR3P0128. Aliquots of 50 ␮g lysate per lane were
subjected to SDS-PAGE separation, followed by Western blotting using anti-M1 and anti-NP antibodies. GAPDH was used as a loading control. The immunoblot
results were quantified by densitometric analysis and at 3 h after infection were normalized to those for the virus control, whose level was arbitrarily set equal to
1. The data are presented as means Ϯ SEMs of the results of two independent experiments. ء, P Ͻ 0.05 compared with the respective control. (B) BPR3P0128
(3P0128) inhibits viral protein synthesis, as indicated by immunofluorescence staining. MDCK cells were infected with A/WSN/33 at an MOI of 1, and the cells
were harvested 9 h after infection and stained with anti-NP and 4=,6-diamidino-2-phenylindole (DAPI). (C) Inhibition of replicative RNA synthesis by
BPR3P0128. The cells were harvested 9 h after infection and observed with immunofluorescence microscopy. DAPI and mouse anti-dsRNA antibody staining
show the locations of the nucleus and replicative viral RNAs, respectively. The infected cells are indicated by arrows. The right column shows the overlaid images
of dsRNA immunofluorescence and DAPI staining. (D) Inhibition of viral RNA synthesis by BPR3P0128. Viral RNA for M1 was detected by qRT-PCR. The ratio
of gene expression to that for its respective internal control (GAPDH) was normalized to the control level at 0 h after infection, which was arbitrarily set equal to
1.0. This is a representative result from two independent experiments.
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Hsu et al.
TABLE 2 Susceptibility to BPR3P0128 of recent oseltamivir-resistant
influenza virus H1N1 isolates
although the underlying mechanism requires further investiga-
tion (Fig. 4). This compound did not inhibit the phosphatidylino-
sitol 3-kinase/Akt signaling pathway, which is activated at an early
stage of infection to support influenza virus replication (data not
shown) (12).
EC50 (␮M)a
Oseltamivir
Influenza virus
BPR3P0128
Amantadine
carboxylate
BPR3P0128 inhibition of viral polymerase activity. We used
a plasmid-based minireplicon (RNP reconstitution) assay to iden-
tify the mechanism underlying the inhibition of influenza viral
polymerase by BPR3P0128 (42). Plasmids for the expression of
RNP complex of A/PR/8/34 were transfected together with
pPOLI-Fluc into HEK293 cells (Fig. 5A). Treatment with
BPR3P0128 decreased the ratio of Fluc to Rluc activity, indicating
that BPR3P0128 inhibited the RNP complex-mediated transcrip-
A/TW/147/09
A/TW/70058/09
A/TW/70066/09
0.060 Ϯ 0.001
0.075 Ϯ 0.005
0.074 Ϯ 0.003
1.47 Ϯ 0.60
0.762 Ϯ 0.28
0.96 Ϯ 0.32
Ͼ25
Ͼ25
Ͼ25
a Susceptibility was determined by inhibition of CPE in MDCK cells. Data are presented
as means Ϯ SDs of the results of at least two independent experiments.
crease in the viral protein level; this result was consistent with the tion of pPOLI-Fluc, resulting in decreased Fluc activity in this
immunoblotting data (Fig. 3B). system (Fig. 5A). No activity was detected when PA or Fluc was
We next used immunofluorescence microscopy and qRT-PCR omitted from the minireplicon system, indicating that the inhibi-
to examine the effect of BPR3P0128 on the synthesis of viral RNA. tion was specific. This inhibitory effect of BPR3P0128 might not
MDCK cells were infected with influenza virus A/WSN/33 at an result from reduced viral protein expression because the level of
MOI of 1 and incubated with or without 1 ␮M BPR3P0128 during NP was not altered in any sample (Fig. 5A, bottom).
the postinfection stages. The cells were fixed 9 h after infection,
We used a primer extension assay to study further whether
reacted with a primary anti-dsRNA antibody, and evaluated by BPR3P0128 can inhibit the synthesis of specific vRNA species (38)
immunofluorescence microscopy. The dsRNA was distributed ex- (Fig. 5B). RNA synthesis in transfected cells was subjected to
clusively in the nuclei of the infected cells, where viral RNA repli- primer extension using radiolabeled oligonucleotides comple-
cates actively (Fig. 3C; compare the middle row with the top row). mentary to positive- or negative-sense CAT RNAs. The vRNA
The expression of dsRNA was reduced markedly in the presence of detected in the absence of PA (Fig. 5B, lane 2) represents POLI-
BPR3P0128, indicating that viral replication was impaired (Fig. derived template RNA. BPR3P0128 strongly inhibited mRNA
3C, bottom row). We also used qRT-PCR to investigate total viral synthesis in a dose-dependent fashion (Fig. 5B). Surprisingly, syn-
RNA synthesis in MDCK cells infected with virus and treated with thesis of cRNA was not inhibited but was increased in a dose-
or without BPR3P0128 at 3, 6, and 9 h after infection. Random
hexamers were used in the reverse transcription reactions, and all
three RNA species were detected. The total viral RNA level was
reduced significantly by 55 to 68% when cells were treated with
this compound (Fig. 3D). In summary, BPR3P0128 inhibited viral
replication in infected cells, and this inhibition reduced the pro-
duction of viral protein and RNA.
Mechanism-of-action studies of BPR3P0128. We investi-
gated the specificity of the antiviral activity of BPR3P0128 by sub-
jecting it to bioevaluation against a variety of strains and viruses,
including different strains of influenza A and B viruses, EV71,
CVB3, HRV2, and two DNA viruses, adenovirus and HSV-2.
BPR3P0128 exhibited potency against both influenza viruses A
and B (Table 1). Interestingly, this compound also inhibited three
other RNA viruses listed in Table 1, EV71, CVB3, and HRV2.
BPR3P0128 did not show activity toward the two DNA viruses.
BPR3P0128 was also effective against recent clinical isolates of
oseltamivir-resistant viruses in an assay using oseltamivir carboxylate
as a control (Table 2). As expected, these strains were resistant to
oseltamivir carboxylate. BPR3P0128 showed similar inhibitory effi-
cacy against these resistant viruses, strongly suggesting that its inhib-
itory mechanism differs from that of oseltamivir (Table 2).
To investigate its possible time-dependent inhibitory effects on
influenza virus replication, BPR3P0128 was added to monolayers
FIG 4 Mode of action of BPR3P0128 by time-of-addition experiments.
of infected cells during or after viral adsorption, and the viral yield
MDCK cells were infected with A/WSN/33 at an MOI of 0.001 at Ϫ1 h. The
timing of treatment with the compound (1 ␮M, at 37°C) is indicated at the top.
in one infectious cycle was assessed using a plaque formation assay
(Fig. 4). In this time-of-addition assay, even when BPR3P0128 was
added 4 h after viral adsorption, there was a marked inhibition of
viral yield. There remained about a 40% reduction when the
compound was added 6 h after infection. This suggests that
BPR3P0128 affects an early step of the replication cycle after viral
BPR3P0128 was added at the time of infection (Ϫ1 h) and 0, 2, 4, and 6 h after
infection, as indicated. After treatment, the culture medium was replaced with
fresh medium, and the culture supernatant was harvested for the plaque assay
12 h after infection. Virus control, controls with no compound added. Plaque
numbers for each treatment were normalized to the virus control, which was
arbitrarily set equal to 100%. The data are presented as means Ϯ SEMs of the
entry, such as vRNA synthesis or vRNA export out of the nucleus, results of two independent experiments.
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FIG 5 Inhibition of influenza virus RNP complex activity by BPR3P0128 by minireplicon (RNA reconstitution) assay (A and B) and in virus-infected MDCK
cells (C). (A) Detection of RNP activity by a reporter assay. Various concentrations of BPR3P0128 were incubated with HEK293 cells previously transfected with
pPOLI-Fluc (Fluc), pHW2000 expression vectors for influenza virus PB1, PB2, PA, and NP proteins, and pTK-Rluc (Rluc) as a transfection control. (Bottom) NP
is a protein expression control, and DMSO was included as the vehicle control. This is a representative result of at least three independent experiments. The data
are presented as means Ϯ SEMs. (B) Inhibition of vRNA species synthesis by BPR3P0128 in a minireplicon system using a primer extension assay. HEK293 cells
were seeded overnight and transfected with plasmids for 8 h, followed by BPR3P0128 treatment for 2 days. Total cellular RNA was then isolated and analyzed by
primer extension using ␥-32P-end-labeled CAT-specific primers and hybridization at 50°C for 3 h. The resulting reactions were terminated with formamide and
the mixtures were separated on 8% polyacrylamide gels containing urea. The mRNA is shown as a wide-ranging band 10 to 13 nucleotides longer than cRNA
because of its 5=-cap-snatched sequence. The expected sizes of mRNA, cRNA, and vRNA were 98 to 106, 89, and 158 bp, respectively. (C) Inhibition of RNA
species synthesis by BPR3P0128 in virus-infected cells assessed in primer extension assays. Monolayers of MDCK cells were seeded overnight and infected with
influenza virus A/WSN/33 at an MOI of 1. After adsorption for 1 h, the culture medium containing the unbound viruses was replaced with DMEM in the presence
of BPR3P0128. Total cellular RNA was isolated 8 h after infection, and the viral RNA species were analyzed by hybridizing with viral M-gene-specific ␥-32P-
end-labeled primers for 3 h. This primer extension reaction was then subjected to reverse transcription, and the resulting mixtures were separated on 8%
polyacrylamide gels containing urea. The results shown here are single representatives of at least three independent experiments.
dependent manner, suggesting that transcription but not replica- served correspond to bona fide influenza virus replication (Fig.
tion of the polymerase is the primary molecular target of 5C, lanes 1 and 2). The solvent for the compound (DMSO) did not
BPR3P0128. This plasmid-based reverse genetics RNP reconstitu- cause any inhibition, but the synthesis of total RNA was reduced
tion assay contains only minimal components of the transcription considerably, a finding that is consistent with the minireplicon
and replication machinery and might not fully recapitulate typical assay data (Fig. 5B). Nevertheless, similar to the qRT-PCR data
viral replication inside cells. We confirmed this primer extension (Fig. 3D), total RNA synthesis was reduced in the presence of
result with virally infected MDCK cells (Fig. 5C). Cells were in- BPR3P0128 (Fig. 5C, lanes 4 to 6). Interestingly, similar to the
fected with A/WSN/33 at an MOI of 1 and were then incubated reconstituted reverse genetics assay results given above (Fig. 5B),
with BPR3P0128. Cells were harvested 8 h after infection, and mRNA was severely inhibited with a parallel increase in vRNA
total RNA extraction and reverse transcription were performed production in the presence of BPR3P0128, indicating that this
using a primer specific for the viral M gene (segment 7) as an compound specifically targets polymerase-associated functions.
indication of RNA synthesis. No vRNAs were detected in mock The reduction in the amount of cRNA by this compound might
infection controls, and the three RNA species were clearly identi- indicate that it had gone through the second cycle of replication to
fiable in virus-infected cells, demonstrating that the bands ob- make more vRNA (Fig. 5C, lanes 4 to 6).
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Hsu et al.
BPR3P0128 inhibition of viral polymerase cap-snatching ac-
tivity. The inhibition of viral mRNA synthesis by BPR3P0128 led
us to investigate the effect of this compound on the cap-
dependent mRNA transcription activity of RNP. Synthesis of
mRNAs is capped primer dependent: that is, primed by short
capped RNA fragments generated from cellular pre-mRNAs by
endonucleolytic cleavage (45). In contrast, the synthesis of cRNA
and vRNA molecules is initiated in an unprimed manner de novo
during influenza virus replication (43). Thus, the proposed mode
of action of BPR3P0128 involves targeting the viral mRNA syn-
thesis machinery, such as cap-snatching-related functions during
replication. Cap-binding activity has been shown to reside in the
PB2 subunit, and capped RNA binding can be activated only by 5=
vRNA binding to the PB1 subunit (3, 33, 55). We performed as-
says to measure the cap-binding, endoribonuclease, and 5=-
vRNA-binding activities of the polymerase complex prepared
from nuclear fractions expressing recombinant PA, PB1, and PB2
in the presence of BPR3P0128. The assay for the binding of capped
RNA fragments to polymerase complexes was performed using a
gel-shift assay (33). The nuclear extracts containing viral polymer-
ase complexes were incubated with an RNA fragment of a 13-
nucleotide capped RNA from ALMV, containing a cap structure,
in the presence of increasing concentrations of BPR3P0128. The
DMSO control had no inhibitory effect on capped RNA-
polymerase complex formation (Fig. 6A, lane 1). No capped RNA-
polymerase complex was formed when the polymerase complex
was missing from the reaction (negative control) (data not
shown). These results indicate that the binding between polymer-
ase and capped RNA was genuine. The inhibition became appar-
ent at a BPR3P0128 concentration of 0.125 ␮M and was dose
dependent. Because capped RNA binding can be stimulated only
by 5= vRNA binding to the RNP complex, we explored whether
BPR3P0128 can affect the 5= vRNA binding activity of the poly-
merase complex, which is one step before cap binding. As shown
in Fig. 6B, 5= vRNA binding to the polymerase complex was not
prevented by BPR3P0128 when the same concentrations of this
compound were used to inhibit capped RNA binding.
We further determined whether cap binding inhibited by
BPR3P0128 affects the subsequent step of endoribonuclease activ-
ity that cleaves the bound capped RNAs 10 to 13 nucleotides from
their 5= ends (45). After sequential binding of the 5=-terminal
sequence of vRNA and 3=-terminal oligoribonucleotides, full-
length ALMV RNA 4 containing an m7G32pppGm 5= end was
added. No cleavage product was detected in the absence of the
polymerase complex (Fig. 6C, lane 1). The endoribonuclease ac-
tivity was specifically and dose-dependently inhibited by
BPR3P0128 but not by the DMSO vehicle control. This suggests
that this inhibition results from the loss of cap-binding activity
exerted by BPR3P0128 because both activities were similar at an
IC50 of 0.25 ␮M (Fig. 6D). The cap-binding site of PB2 is essential
FIG 6 Inhibition of viral cap binding and endoribonuclease activity by
BPR3P0128. (A) Cap binding shown in a gel shift assay. Following a 30-min
reaction for the binding of an unlabeled oligoribonucleotide containing the
5=-terminal sequence of vRNA, the 13-nucleotide-long capped ALMV RNA 4
fragment containing an m7G32pppGm 5= end (10 ng, 1 ϫ 105 cpm) was added,
and the mixture was incubated in the presence of BPR3P0128 for 30 min at
30°C. The protein-RNA complexes were separated from free RNA by electro-
phoresis on 4% nondenaturing gels. (B) Binding of 5= vRNA to RNP. Nuclear
extracts containing polymerase complexes (2.5 ␮g) were incubated at 25°C for
30 min in the binding buffer with a 32P-end-labeled oligoribonucleotide con- presence of BPR3P0128 and separated on 20% polyacrylamide-urea gels. The
taining the 5=-terminal sequence of vRNA (10 ng). The protein-RNA com- results shown here are single representatives of three independent experi-
plexes were resolved from free RNA by electrophoresis on 4% nondenaturing ments. Control, the labeled RNA was incubated in the absence of a polymerase
gels. (C) Endoribonuclease assays. Following binding of the oligoribonucle- complex. (D) Quantification of the cap-binding and endoribonuclease exper-
otide containing the 5=-terminal sequence of vRNA and the subsequent bind- iments. The levels of viral polymerase-cap complex and the cleavage product
ing of the oligoribonucleotide containing the 3=-terminal sequence of vRNA, were normalized to the level of the vehicle control (DMSO), which was arbi-
the full-length ALMV RNA 4 fragment containing an m7G32pppGm 5= end trarily set equal to 1.0. The 50% inhibitory concentration (IC50), calculated by
(0.25 ng) was added along with 0.1 ␮g of tRNA as an additional inhibitor of Reed-Muench method, was about 0.25 ␮M for both assays. A.U., absorbance
nonspecific nucleases. The mixtures were incubated at 30°C for 60 min in the units.
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Anti-Influenza Virus Activity of BPR3P0128
for cap-dependent transcription by vRNPs in vitro and in vivo
(49). BPR3P0128 may thus target the PB2 subunit or its function-
ally associated proteins.
DISCUSSION
The polymerase complex of the influenza virus is a potential target
for the development of antiviral agents because it is responsible for
an essential and highly conserved catalytic function of the virus
FIG 7 Sequence alignment of the PB2 cap-binding domain from human and
swine influenza A viruses. The secondary structure of A/WSN/1933 is shown
and because the atomic and quasiatomic structures of its sub- over the sequence alignment of strains sensitive to BPR3P0128 (Table 1). Res-
idues His357 and Phe404 of influenza A virus indicated by underlining are
unit have become available for antiviral drug design (9, 28, 49).
principal residues in contact with the cap analogue m7GTP. Phe404 is con-
The viral polymerase of influenza virus catalyzes the reactions
served in influenza A and B viruses, whereas His357 is exclusive to influenza A
virus and is replaced by a tryptophan in influenza B virus (22). The full-length
responsible for both capped RNA-primed mRNA transcription
(vRNA ¡ mRNA) and unprimed replication of vRNAs in the
steps vRNA ¡ cRNA ¡ vRNA. How the polymerase complex
carries out these two distinct processes—transcription and rep-
lication—is poorly understood. Here, we identified a small com-
pound, BPR3P0128, that plays a role in the regulation of tran-
PB2 sequences of the influenza B virus tested (Table 1) were not available in the
public domain. Four representative sequences from 2005 and 2007 from NCBI
are shown here.
scription and replication of the influenza virus RNA genome. In play a role in the switch between transcription and replication
particular, incubation with BPR3P0128 markedly decreased the (14). This crucial residue Phe404 is also conserved in the recent
ratio between mRNA and cRNA or vRNA in RNP reconstitution pandemic swine-origin influenza virus (Fig. 7). His357, on the
and viral infection assays (Fig. 5). Our data are consistent with other side of the sandwich, is unique to influenza A virus and is
those from previous studies of the temporal changes in the replaced by the more conventional cap-stacking residue trypto-
amount of RNA (24, 31). Our subsequent results suggested that phan in influenza B virus (Fig. 7). Mutation of both Phe404 and
this compound targets a PB2-associated cap-snatching function His357 reduces the accumulation of transcription products and
(Fig. 6).
increases the accumulation of replication products (22), and these
BPR3P0128 possesses an essential backbone structure of quin- functions closely resemble those of BPR3P0128. The mode of ac-
oline (Fig. 1) and might directly target cap-binding-associated tion of BPR3P0128 might thus target these imperative sites of PB2
functions but not the endoribonuclease activity because this com- to inhibit cap snatching and subsequent transcription. We cannot
pound is not structurally akin to any known endoribonuclease exclude the possibility that inhibiting transcription provides more
inhibitors such as flutimide (54). The PB2 protein carries minimal template vRNA, which would then be available for replication.
homology to the cap-binding proteins of eukaryotes, and this en-
Interestingly, BPR3P0128 showed broad-spectrum activity
zymatic activity has no known cellular counterpart (13). However, against other RNA viruses, similar to the action of other replica-
BPR3P0128 at a concentration up to 10 ␮M did not inhibit re- tion inhibitors such as T-705 and compound A3 (19, 27). Two
combinant PB2 subunit binding to cap-Sepharose, suggesting that lines of evidence indicate that the host factor(s) associated with
BPR3P0128 is not a cap competitor (data not shown). Quinoline PB2 cap-snatching activity might also be the target of BPR3P0128
and its derivatives have been developed as anti-human immuno- in other RNA viruses. First, BPR3P0128 is also very potent in
deficiency virus compounds, but they do not target cap-related inhibiting EV71 (EC50 ϭ 29 nM), CVB3 (EC50 ϭ 62 nM), and
functions (1, 41).
HRV2 (EC50 ϭ 79 nM), although BPR3P0128 at a concentration
Our results suggest that BPR3P0128 might regulate the balance of 20 ␮M has no activity against HSV or adenoviruses. Second, no
between viral replication and transcription. Various models of drug-resistant influenza virus could be selected after more than 20
switching from transcription to replication in influenza A viral passages of viral propagation under the selection pressure of
infections have been proposed (46, 51). NP, which is involved in BPR3P0128 (data not shown). In addition, and most importantly,
encapsulation of the vRNA, was identified as a key player because identifying the target genes of BPR3P0128 could help solve the
of its RNA binding, which might modify the panhandle structure long-studied problem of the nature of the host factor(s) regulating
for the switch (17). The interaction between NP and the polymer- the switch between transcription and replication by the influenza
ase complex was also proposed to potentiate unprimed RNA rep- virus (51). This candidate host factor(s) must fulfill the following
lication via inhibiting cap-snatching activity (2, 36, 40). The NS2/ criteria. First, it must be essential for replication of the RNA vi-
NEP and PA subunits of the polymerase complex are also ruses but not the DNA viruses listed in Table 1. Second, it must
associated with the regulation of viral replication and transcrip- have both nuclear and cytosolic distributions because influenza
tion (29, 48). A new model was recently proposed in which virus- virus and the other viruses replicate in the nucleus and cytoplasm,
generated small RNAs shift the transition from transcription to respectively. Third, this factor(s) might be involved in PB2-
replication through interactions with the viral polymerase ma- associated functions through direct or indirect interactions. One
chinery (44). However, some studies have focused on the role of candidate molecule is heat shock protein 90 (Hsp90), which inter-
PB2 in tuning transcription and replication because of its cap- acts with the influenza virus PB2 protein and can stimulate vRNA
binding activity (14, 20), and we speculate that our compound synthesis (37, 39). Interestingly, inhibiting Hsp90 functions by
may target PB2.
geldanamycin, a specific Hsp90 inhibitor, reduces the replication
Two conserved aromatic amino acids, Phe363 and Phe404, activity of influenza virus and other RNA viruses, including po-
which sandwich the N7-methyl guanine of the cap structure in liovirus, CVB3, and rhinoviruses (5, 21); this inhibition is similar
influenza A and B viruses, have been shown to be important for to the broad range of inhibitory activity of our compound (Table
cap binding, but only Phe404 was shown by mutational analysis to 1). Another candidate protein is Hsp70, which is functionally re-
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Hsu et al.
13. Fechter P, Brownlee GG. 2005. Recognition of mRNA cap structures by
viral and cellular proteins. J. Gen. Virol. 86:1239–1249.
14. Fechter P, et al. 2003. Two aromatic residues in the PB2 subunit of
influenza A RNA polymerase are crucial for cap binding. J. Biol. Chem.
278:20381–20388.
15. Fislova T, Thomas B, Graef KM, Fodor E. 2010. Association of the
influenza virus RNA polymerase subunit PB2 with the host chaperonin
CCT. J. Virol. 84:8691–8699.
lated to Hsp90 and is also involved in influenza virus and poliovi-
rus replication (7, 26). Hsp70 interacts with the PB2 subunit and
vRNP of influenza virus, and it plays a role in the nuclear export of
vRNP (15, 26). Both Hsp70 and Hsp90 have nuclear and cytoplas-
mic distributions (25, 26, 39). BPR3P0128 might target such cel-
lular proteins to inhibit RNA viruses.
Although our compound is not structurally similar to any
known natural or synthetic inhibitors (47), the role of the Hsp
family as the target of BPR3P0128 is under investigation. In addi-
tion, a number of host factors have been shown to be needed for
replication of both influenza virus and poliovirus in host cells
through a direct or indirect interaction (8, 57). These communal
host factors involved in the virus replication cycle include AKT1,
ARAF1, cyclin-dependent kinase 10 (CDK10), CEL, DYRK1B,
MAP2K5, mitogen-activated protein kinase 1 (MAPK1), RAB17,
and SCN8A. Thus, it is likely that BPR3P0128 can interfere with
these crucial host functions, which are needed for influenza virus
replication. Inhibition of influenza virus could be achieved
through interfering RNA knockdown or pharmacological inhibi-
tion of the essential host factors. To our knowledge, BPR3P0128 is
the first quinoline-based anti-influenza virus compound identi-
fied and characterized to show effective antiviral activity. This
compound offers an opportunity for the development of a new
anti-influenza virus agent.
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ACKNOWLEDGMENTS
This work was supported by the National Science Council (NSC) in Tai-
wan and Chang Gung Memorial Hospital (grants NSC97-2321-B-400-
002, CMRPD160323, and CMRPD160123).
We thank Rei-Lin Kuo for his critical reading of the manuscript and
Sung-Nain Tseng for technical assistance.
The experiments were conceived and designed by J.T.-A.H., H.-P.H.,
and J.-T.H. The experiments were performed by J.-Y.Y., T.-J.L., M.L.,
Y.C.C., M.-S.W., C.-F.H., W.-F.T., K.S.L., H.-C.H., M.-Y.F., and S.K. J.T.-
A.H., H.-P.H., and J.-T.H. analyzed the data. J.T.-A.H. and J.-T.H. wrote
the paper.
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