Inhibitors of endoplasmic reticulum alpha-glucosidases potently suppress hepatitis C virus virion assembly and release.
ABSTRACT α-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C.
- SourceAvailable from: Giovanni Di Pasquale[Show abstract] [Hide abstract]
ABSTRACT: Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.New England Journal of Medicine 04/2014; · 51.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. Here we report the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV Infectivity Inhibitor-1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCV with low nanomolar EC50 values. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of non-infectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its pre-fusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.Antimicrobial Agents and Chemotherapy 10/2013; · 4.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Virus infection of host cells is sensed by innate pattern recognition receptors (PRRs) and induces production of type I interferons (IFNs) and other inflammatory cytokines. These cytokines orchestrate the elimination of the viruses but are occasionally detrimental to the hosts. The outcomes and pathogenesis of viral infection are largely determined by the specific interaction between the viruses and their host cells. Therefore, compounds that either inhibit viral infection or modulate virus-induced cytokine response should be considered as candidates for managing virus infection. The aim of the study was to identify compounds in both categories, using a single cell-based assay. Our screening platform is a HEK293 cell-based reporter assay where the expression of a firefly luciferase is under the control of a human IFN-β promoter. We have demonstrated that infection of the reporter cell line with a panel of RNA viruses activated the reporter gene expression that correlates quantitatively with the levels of virus replication and progeny virus production, and could be inhibited in a dose-dependent manner by known antiviral compound or inhibitors of PRR signal transduction pathways. Using Dengue virus as an example, a pilot screening of a small molecule library consisting of 26,900 compounds proved the concept that the IFN-β promoter reporter assay can serve as a convenient high throughput screening platform for simultaneous discovery of antiviral and innate immune response modulating compounds. A representative antiviral compound from the pilot screening, 1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-methoxyphenyl) urea, was demonstrated to specifically inhibit several viruses belonging to the family of flaviviridae.Antiviral research 04/2014; · 3.61 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2011, p. 1036–1044
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 3
Inhibitors of Endoplasmic Reticulum ?-Glucosidases Potently Suppress
Hepatitis C Virus Virion Assembly and Release?
Xiaowang Qu,1Xiaoben Pan,1Jessica Weidner,1Wenquan Yu,2Dominic Alonzi,3Xiaodong Xu,2
Terry Butters,3Timothy Block,1,2Ju-Tao Guo,1and Jinhong Chang1*
Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of
Medicine, Doylestown, Pennsylvania1; Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown,
Pennsylvania2; and Glycobiology Institute, University of Oxford, Oxford, United Kingdom3
Received 27 September 2010/Returned for modification 4 November 2010/Accepted 13 December 2010
?-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan
processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that down-
regulation of the expression of ?-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology
inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of ?-glucosidases in
HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar
derivatives, which are competitive inhibitors of ?-glucosidases, did not affect intracellular HCV RNA replica-
tion and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent
degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In
searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had
a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosi-
dases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant
virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its develop-
ment as a therapeutic agent for the treatment of chronic hepatitis C.
Hepatitis C virus (HCV) chronically infects more than 170
million people worldwide. Current standard therapy for chronic
hepatitis C, the combination of pegylated alpha interferon
(IFN-?) and ribavirin, is associated with a less than 50% sus-
tained virological response in patients infected with genotype 1
virus. In the search for more effective therapeutic agents, the
development of direct-acting antiviral agents to target viral
functions, such as NS3/4A protease and NS5B RNA-depen-
dent RNA polymerase, has been the main focus during the last
2 decades (26). However, an important lesson learned from
clinical studies is that although inhibition of the essential viral
functions potently inhibited HCV replication and resulted in a
rapid drop in viremia, development of drug resistance eventually
limited the antiviral efficacy of these drugs (22, 32, 36). Therefore,
agents but rather as part of therapeutic regimens in combination
with IFN-? and/or other HCV inhibitors.
Like all other viruses, HCV relies on many host functions to
propagate. In addition, the virus needs to counteract or evade
the cellular antiviral response to colonize its host cells (23).
Therefore, an alternative approach to inhibiting HCV infec-
tion is to target host cellular functions required for HCV
replication and/or activate the host cellular antiviral re-
sponse (17, 24, 37). In fact, compared to targeting viral
functions, an obvious advantage of targeting host functions
is the low likelihood of drug resistance (28). Currently,
inhibitors targeting several cellular proteins, including 3-hy-
droxy-3-methyl-glutaryl-coenzyme A reductase (19, 41), cyclo-
philin (21, 25), phosphatidylinositol 4-kinase alpha (PI4K-?)
(2, 6), and heat shock proteins (13), have been shown to inhibit
HCV infection in cultured cells and a cyclophilin inhibitor,
alisporivir (Debio 025), has been demonstrated to reduce HCV
viremia in people (12).
?-Glucosidases I and II catalyze the sequential removal of
the three terminal glucose residues from the asparagine-linked
(N-linked) oligosaccharides on glycoprotein precursors. These
reactions are the first steps of glycan processing and are essen-
tial for the proper folding and function of certain cellular and
viral glycoproteins (10). We and others have shown previously
that treatment of cells with ?-glucosidase inhibitors such as
imino sugars that are glucose mimics and act as competitive
inhibitors of the enzymes inhibited the infection of many en-
veloped viruses (4, 5, 8, 10, 14). Although the effect of imino
sugar on HCV was achieved mainly by using bovine viral di-
arrhea virus (BVDV) as a model virus, in one recent study,
several imino sugars were being tested and their anti-HCV
effects were being confirmed using an HCV tissue culture
infection system (35). Moreover, ?-glucosidase inhibitors
have also been demonstrated to inhibit woodchuck hepatitis
virus in chronically infected woodchucks (4), several flavi-
viruses in mice (33, 38, 40), and HCV in people in a phase
II clinical trial (9).
In this study, in our effort to search for imino sugar deriva-
tives with better antiviral activity against HCV, we first for-
mally demonstrated with small interfering RNA (siRNA) tech-
nology that both ?-glucosidases I and II are essential host
factors in HCV infection. In addition, we demonstrated that
known imino sugar glucosidase inhibitors impair HCV infec-
* Corresponding author. Mailing address: Drexel Institute for Bio-
technology and Virology Research, Department of Microbiology and
Immunology, Drexel University College of Medicine, 3805 Old Easton
Road, Doylestown, PA 18902. Phone: (215) 589-6325. Fax: (215) 489-
4920. E-mail: firstname.lastname@example.org.
?Published ahead of print on 20 December 2010.
tion at the step of virion assembly and secretion. Interestingly,
we found a novel imino sugar derivative, PBDNJ0804, with
superior antiviral activity against HCV through a similar mech-
anism that is consistent with the inhibition of glucosidases.
MATERIALS AND METHODS
Compounds. Deoxynojirimycin (DNJ) and N-nonyl-DNJ (NNDNJ) were pur-
chased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada).
N-Butyl-DNJ (NBDNJ) was kindly provided by Raymond Dwek (University of
Oxford, Oxford, United Kingdom). All other imino sugar derivatives were as
Cell culture and virus. Huh7.5 cells were maintained in Dulbecco’s modified
minimal essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented
with 10% fetal bovine serum (3). A plasmid containing the full-length HCV Jc1
cDNA was cloned from chemically synthesized DNA oligomers (29) (GenScript,
Piscataway, NJ). HCV RNA was transcribed in vitro with the MEGAscript kit
(Ambion, Austin, TX) and electroporated into Huh7.5 cells (15). Generation of
a virus stock and determination of virus titers (50% tissue culture infective doses
[TCID50] per milliliter) were done as described previously (20). In general,
infection of Huh7.5 cells at a multiplicity of infection (MOI) of 0.015 for 4 days
resulted in virus yields of 0.5 ? 104to 0.5 ? 105TCID50/ml. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay; Sigma,
St. Louis, MO) was performed to measure cytotoxicity following the treatment of
Huh7.5 cells with serial doses of each compound for 4 days (Table 1).
Establishment of stable cell lines expressing shRNA. Huh7.5 cells were in-
fected with vesicular stomatitis virus G protein-pseudotyped lentivirus encoding
(i) a small hairpin RNA (shRNA) targeting human ?-glucosidase I (GCS1,
SHCLNV-NM_006302), ?-glucosidase II (GANAB, SHCLNV-NM_014610), or
PI4K-? (SHCLNV-NM_002650) or (ii) a nontargeting shRNA (Sigma, St. Louis,
MO) to establish corresponding stable cell lines.
Indirect immunofluorescence. HCV-infected cells were fixed with phosphate-
buffered saline containing 1% paraformaldehyde and then incubated with cold
methanol for 20 min at ?20°C. Cells were then blocked and incubated with HCV
NS3 antibody (clone H23; Abcam, Cambridge, MA). Bound primary antibody
was visualized by Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen,
Western blot analysis. Cells were lysed with Laemmli buffer and separated on
Tris-glycine gel, followed by transfer onto nitrocellulose membrane (Invitrogen).
Membranes were blocked and probed with antibodies against HCV NS3 (clone
H23; Abcam, Cambridge, MA), HCV E2 (clone AP33; provided by Arvind Patel
through Genentech, Inc.), ?-glucosidase I (Sigma, St. Louis, MO), ?-glucosidase
II (Sigma, St. Louis, MO), or glyceraldehyde 3-phosphate dehydrogenase
(GAPDH; Sigma, St. Louis, MO). This was followed by incubation with IRDye
secondary antibodies and imaging with the LI-COR Odyssey system (LI-COR
Biotechnology, Lincoln, NE).
TABLE 1. Structures, antiviral properties, and ?-glucosidase I-inhibitory activities of imino sugar derivatives
R in structure
?5000.68 ? 0.05
NNDNJ4.0 ? 0.587 ? 8.7 0.54 ? 0.08
OSL-95II 28.8 ? 12.3
?500 0.09 ? 0.02
52.5 ? 14.2
?5000.42 ? 0.09
27.2 ? 5.4
?500 0.46 ? 0.11
3.5 ? 1.0
?5000.15 ? 0.01
1.7 ? 0.8 208 ? 17.60.51 ? 0.09
5.4 ? 2.6
?5000.50 ? 0.16
aResults represent mean values and standard deviations from three independent experiments.
bEC50was determined by in-cell Western immunoassay as described in the legend to Fig. 4.
cCC50was determined by MTT assay.
dDetermined by using the substrate Glc3Man5GlcNAc1as previously described (1).
VOL. 55, 2011 GLUCOSIDASE INHIBITORS SUPPRESS HCV VIRION SECRETION1037
RNA quantification by qRT-PCR. Total cellular RNA or RNA in culture
medium was extracted using TRIzol reagent (Invitrogen) or a QIAamp viral
RNA minikit (Qiagen) and reverse transcribed using SuperScript III (Invitro-
gen). Quantitative reverse transcription-PCR (qRT-PCR) was performed on an
Applied Biosystems 7500 thermocycler using the probe 5?-6-carboxyfluores-
cein-CCT TGT GGT ACT GCC TGA-molecular-groove binding nonfluores-
cence quencher-3? (Applied Biosystems) and forward and reverse primers
5?-AGCGTTGGGTTGCGAAAG-3? and 5?-CACTCGCAAGCGCCCT-3?,
respectively. The standard curve was generated using serial 10-fold dilutions
of in vitro-transcribed full-length HCV RNA.
In-cell Western immunoassay. The in-cell Western immunoassay was per-
formed largely as previously described for the detection of dengue virus (DENV)
(18), except that an HCV NS3 monoclonal antibody (H23) was used. Briefly, cells
were fixed with 3.7% formaldehyde and permeabilized with 0.25% Triton X-100.
Intracellular levels of HCV NS3 protein were revealed by sequential incubation
with an HCV NS3-specific monoclonal antibody (clone H23; Abcam, Cambridge,
MA) and IRDye 800 goat anti-mouse IgG (LI-COR, Lincoln, NE). Cell viability
was determined by DRAQ5 (Biostatus Limited, Leicestershire, United King-
dom) and Sapphire 700 (LI-COR, Lincoln, NE) staining. Fluorescence signal
intensity was quantified with LI-COR Odyssey.
a substrate for glucosidase I, was incubated with purified ?-glucosidase I (from
rat liver) together with various concentrations of imino sugar derivatives as
previously described (1). Separation of the hydrolysis products was performed
using normal-phase high-performance liquid chromatography (Waters). Dose-
response curves were generated to calculate the concentration required to inhibit
enzyme activity by 50% (IC50).
?-Glucosidases I and II are essential host factors for HCV
infection. Although it has been speculated and experimentally
demonstrated with small molecular inhibitors that endoplas-
mic reticulum (ER)-resident ?-glucosidases I and II are essen-
tial for the processing of N-linked glycans of viral envelope
glycoproteins and thus required for viral particle assembly
and the secretion of many enveloped viruses, the function of
?-glucosidase II was validated only recently in a genome-
wide siRNA screening study for DENV infection (34). To
provide a solid basis for the development of ?-glucosidase
inhibitors as antiviral targets against HCV and determine the
role of both ?-glucosidases I and II in HCV infection, we first
established stable Huh7.5 cell lines expressing shRNA mole-
cules targeting ?-glucosidase I, II, or both. As shown in Fig.
1A, compared with the Huh7.5 cell line expressing a nontar-
geting shRNA, the steady-state intracellular levels of ?-gluco-
sidase I and/or II were reduced in cells expressing shRNA
targeting the desired enzymes(s). Interestingly, reduced ex-
pression of either or both of the cellular enzymes did not
apparently have an impact on cell viability and growth (Fig.
1B). However, downregulation of either glucosidase I or II
expression in Huh7.5 cells resulted in an approximately 5-fold
reduction of progeny HCV production. Not surprisingly,
knocking down both of the enzymes led to a slightly but sig-
nificantly more profound reduction of HCV yield than that
observed in cells with either one of the enzymes targeted by the
shRNA. As a positive control, ablation of a well-known HCV
host factor, PI4K-? (2, 6), reduced the HCV yield by 14-fold
(Fig. 1C). These results thus demonstrate that both ?-glucosi-
dases I and II are essential HCV host factors.
Inhibition of ?-glucosidases impairs HCV E2 protein glycan
processing and reduces HCV release. To obtain detailed
knowledge of the biological function of ?-glucosidases in HCV
replication and explore the therapeutic potential of glucosi-
dase inhibitors, we first tested the effects of a panel of three
imino sugar derivatives, OSL-95II, CM-10-18, and CM-9-78,
on HCV infection. These derivatives had been demonstrated
to inhibit ?-glucosidase I in in vitro biochemical assays (Table
1). IFN-? was used as a positive control. As shown in Fig. 2,
treatment of cells immediately after HCV infection (at an MOI
of 0.015) with either IFN-? or any one of the three imino
FIG. 1. ?-Glucosidases I and II are essential host factors for HCV
infection. (A) Levels of glucosidase I (left side) and glucosidase II
(right side) in Huh7.5-derived stable cell lines expressing nontargeting
shRNA (sh-control) or shRNA targeting glucosidase I (sh-glucosidase
I), II (sh-glucosidase II), or both (sh-glucosidase I&II) were deter-
mined by Western blot assay. An asterisk indicates a cross-reaction
band. GAPDH served as a loading control. (B) Cell lines with indi-
cated shRNA knockdown were seeded into the wells of 24-well plates.
Cell numbers were counted at the indicated time points to determine
the effect of gene knockdown on the cell growth rate. Values represent
average numbers obtained from three wells. (C) shRNA-expressing
Huh7.5 cells were infected with HCV at an MOI of 0.015. The nega-
tive-control cell line was transduced with nontargeting shRNA, and the
positive-control cell line was transduced with shRNA targeting PI4K.
Three days after infection, culture media were harvested, progeny
HCV yields in the culture media were determined by measuring
TCID50, and the results were plotted as percentages of the virus yield
of cells expressing nontargeting shRNA (sh-control). Values represent
averages and standard deviations of results obtained from four inde-
pendent experiments. P values were calculated using the Student t test.
1038QU ET AL.ANTIMICROB. AGENTS CHEMOTHER.
sugars efficiently inhibited virus spread (Fig. 2A) and accumu-
lation of intracellular viral RNA (Fig. 2B) and reduced the
yields of progeny HCV by more than 1 log (Fig. 2C). While
these results, together with the results presented in Fig. 1,
convincingly demonstrate that inhibition of host cellular ?-glu-
cosidases impairs HCV infection, the viral replication steps
requiring the host enzymes could not be deduced.
As discussed previously, the ER-resident ?-glucosidases are
presumably involved in the glycan processing of HCV envelope
glycoproteins E1 and E2 and thus required for virion assembly
and secretion. To test this hypothesis, Huh7.5 cells were in-
fected with HCV and cultured for 4 days to allow the virus to
spread and infect almost 100% of the cells in the culture, as
judged by immunofluorescent staining with an antibody against
HCV NS3 protein (Fig. 3A). At this time, cells persistently
infected with HCV were left untreated or treated with IFN-?
(positive control) or imino sugar derivatives. As expected,
treatment of HCV-infected cells with IFN-? for 4 days dra-
matically reduced the levels of intracellular HCV NS3 proteins
and HCV RNA (Fig. 3A, B, and C); as a consequence, the
HCV yield in the culture medium was reduced by more than
1,000-fold. However, treatment of HCV-infected cells with any
one of the three imino sugars did not apparently reduce the
levels of intracellular HCV NS3 protein (Fig. 3A and C) and
HCV RNA (Fig. 3B), suggesting that the ?-glucosidases are
not involved in HCV protein translation and RNA replication.
This notion was further confirmed by experiments demon-
strating that treatment of Huh7.5 cells containing HCV
subgenomic replicons (genotype 1b, Con1 strain) with the im-
ino sugars did not inhibit the replication of the replicon (data
FIG. 2. Effects of three imino sugar glucosidase inhibitors on HCV infection of Huh7.5 cells. Huh7.5 cells were infected with HCV at an MOI
of 0.015 and left untreated (Control) or treated immediately after infection with 100 IU/ml IFN-? (positive control) or 100 ?M OSL-95II,
CM-10-18, or CM-9-78 for 4 days. (A) HCV NS3 protein was detected with an indirect immunofluorescence assay. Cell nuclei were stained with
4?,6-diamidino-2-phenylindole (DAPI). (B) Cells were harvested at the indicated times postinfection, and intracellular levels of HCV RNA were
determined by qRT-PCR assay. Values represent average results obtained from four independent experiments. (C) Virus yields in culture media
between 48 and 96 h postinfection were determined and expressed as TCID50per milliliter of culture medium. Values represent averages and
standard deviations of results from four independent experiments.
VOL. 55, 2011 GLUCOSIDASE INHIBITORS SUPPRESS HCV VIRION SECRETION1039
not shown). On the contrary, in supporting the notion that
?-glucosidases are required for glycan processing of HCV en-
velope glycoproteins, imino sugar treatment resulted in an
electrophoresis mobility shift and a subsequent decay of HCV
E2 and E2-p7 proteins (an unprocessed product at E2/p7 site
), the hallmark of a failure to properly process the terminal
glucose residues of the N-linked oligosaccharide(s) of glyco-
proteins in the ER (Fig. 3C). In agreement with the observed
inhibition of E2 glycoprotein processing and degradation, pro-
duction of progeny HCV was reduced approximately 5-fold by
imino sugar treatment (Fig. 3B).
Imino sugar derivative PBDNJ0804 is a potent HCV inhib-
itor with low cytotoxicity. During the last decade, we have been
making consistent efforts to improve the antiviral efficacy of
imino sugars by generating several categories of DNJ deriva-
tives. One of the important findings from these studies is that
a DNJ derivative with a hydroxylated cyclohexyl side chain
(OSL-95II) has an improved antiviral efficacy and lower cyto-
toxicity (14). Accordingly, a family of imino sugar derivatives
containing oxygenated side chains and terminally restricted
ring structures were synthesized (as depicted in Table 1) and
demonstrated to have low cytotoxicity and superior antiviral
FIG. 3. Effects of three imino sugars on viral replication, protein expression, and virion production in persistently HCV-infected Huh7.5 cells.
Huh7.5 cells were infected with HCV at an MOI of 0.015. Four days postinfection (when approximately 100% of the cells were infected, as judged
by positive staining of HCV NS3 by indirect immunofluorescence assay), cells were seeded into the wells of 24-well plates at a density of 8 ?
104/well. Cells were left untreated (Control) or treated at 24 h postseeding with 100 IU/ml IFN-? (positive control) or 100 ?M OSL-95II,
CM-10-18, or CM-9-78 for 4 days. (A) At day 4 posttreatment, HCV NS3 protein was detected with an indirect immunofluorescence assay. Cell
nuclei were stained with DAPI. (B) Intracellular HCV RNA levels (black bar) and HCV yields in culture media (open bar) at day 4 posttreatment
were determined with qRT-PCR and titration assays, respectively. The results are expressed as percentages of the levels in control cells. Values
represent averages and standard deviations of results obtained from four independent experiments. (C) Cells were harvested at 3 and 4 days
postinfection, and intracellular levels of HCV E2 (arrowheads) and E2/E2p7 (asterisks) or NS3 proteins were determined by Western immunoblot
assay. GAPDH served as a loading control.
1040 QU ET AL.ANTIMICROB. AGENTS CHEMOTHER.
activity against several members of the Flaviviridae family,
including BVDV, DENV, and West Nile virus (WNV) (8).
Figure 4 shows the results obtained in a study that system-
atically compared the anti-HCV activities of representative
compounds from the different generations of imino sugar de-
rivatives with an in-cell Western assay. Figure 4A shows a
typical in-cell Western assay result. The amount of intracellu-
lar HCV NS3 protein was visualized by immunostaining with a
specific antibody (green color). Total cellular DNAs were
stained with DRAQ5 and Sapphire 700 (red color). Hence, the
reduced intensity of the red signal indicates the cytotoxicity of the
compounds. The accuracy of the assay was evaluated with IFN-?
as a positive control. In agreement with previous reports,
IFN-? inhibited HCV infection with an EC50of 1 IU/ml (Fig.
4B). Consistent with what we observed in previous studies with
other flaviviruses, DNJ and DNJ with a short alkyl side chain
(NBDNJ) showed little antiviral activity against HCV at a 100
?M concentration. DNJ with a nine-carbon alkyl side chain
(NNDNJ) showed strong antiviral activity, with an EC50of 4
?M, but also was relatively toxic to cells (concentration inhib-
iting cell growth by 50% [CC50], 87 ?M) (Fig. 4B and Table 1).
As demonstrated in the previous two sections and a more
complete dose-response study presented in Fig. 4B, three DNJ
derivatives, with either a hydroxylated or an oxygenated side
chain and a terminal ring structure (OSL-95II, CM-10-18, and
CM-9-78) showed antiviral activity with EC50s ranging from 27
to 52 ?M and lower cytotoxicity (CC50s of ?500 ?M). Inter-
estingly, PBDNJ0802, a stereoisomer of CM-9-78, and two
additional compounds (PBDNJ0803 and PBDNJ0804) with mod-
ifications of the terminal ring structure demonstrated much-im-
proved antiviral activity against HCV, with EC50s ranging from
1.7 to 5.4 ?M (Fig. 4B and Table 1). This represents a 10- to
30-fold improvement of antiviral efficacy over that of the pa-
rental compound, CM-9-78. More importantly, our results im-
ply that the terminal ring structure is a key moiety for antiviral
PBDNJ0804 more efficiently disrupts HCV E2 glycan pro-
cessing and potently inhibits HCV release. Compared with
CM-10-18 and other structurally similar DNJ derivatives,
PBDNJs are not actually better glucosidase I inhibitors, as
indicated by the IC50s obtained in an in vitro enzymatic assay
(Table 1). Therefore, one possible explanation for the superior
antiviral activity of PBDNJs is that the compounds are more
efficiently accumulated in the ER, which results in better inhi-
bition of the cellular enzymes and stronger antiviral activity.
Alternatively, besides inhibition of the ER ?-glucosidases, the
compounds may disrupt an additional replication step(s) of the
HCV life cycle. To distinguish these two possibilities, we first
compared the effects of PBDNJ0804 and CM-10-18 on their
abilities to inhibit HCV by adding the compounds to culture
media immediately after infection. The results showed that
PBDNJ0804 dose dependently reduced intracellular and ex-
tracellular levels of HCV RNA and infectious viral yields (a
3-log reduction at 100 ?M) (Fig. 5A). Moreover, the intra-
cellular levels of the NS3 and E2 proteins were also reduced
by PBDNJ0804 (Fig. 5B). Interestingly, consistent with the
results obtained with the in-cell Western immunoassay (Fig.
4C), treatment of cells with 10 ?M PBDNJ0804 reduced HCV
RNA levels and virus production to extents similar to those
observed in cells treated with 100 ?M CM-10-18.
To further determine if PBDNJ0804 had a stronger ability to
suppress E2 protein glycan processing and virion secretion,
Huh7.5 cells 100% persistently infected with HCV were either
left untreated or treated with 100 IU/ml IFN-?, 100 ?M CM-
10-18 (positive controls), and the indicated concentrations of
FIG. 4. Identification of novel imino sugars with more potent an-
tiviral activity against HCV. Huh7.5 cells were seeded into the wells of
a 96-well plate and mock infected or infected with HCV at an MOI of
0.01. Infected cells were either left untreated (control) or treated
with serial dilutions of IFN-? or imino sugar derivatives for 4 days.
(A) HCV NS3 protein (green) and cell density (red) were detected by
in-cell Western assay. The experiments were performed in triplicate,
and a representative image is shown with cells treated with IFN-?, the
prototype imino sugar DNJ, well-studied DNJ derivatives NBDNJ and
NNDNJ, and the new platform derivatives PBDNJ0802, PBDNJ0803,
and PBDNJ0804. (B and C) HCV NS3 protein levels were determined
with LI-COR Odyssey and normalized with cell density. The relative
fluorescence intensity represents the percentage of fluorescence sig-
nals in IFN-? (B)- or imino sugar derivative (C)-treated wells over that
in untreated control wells. Values represent averages from three wells.
VOL. 55, 2011GLUCOSIDASE INHIBITORS SUPPRESS HCV VIRION SECRETION 1041
PBDNJ0804 for 4 days. The results indicated that, as expected,
PBDNJ0804 did not reduce the intracellular levels of viral
RNA (Fig. 5C) and NS3 proteins (Fig. 5D, middle) at all of the
concentrations tested. However, PBDNJ0804 treatment re-
sulted in an electrophoretic mobility shift and degradation
of the E2 and E2-p7 proteins (Fig. 5D, top), as well as a
reduction of viral yields (Fig. 5C) in a dose-dependent man-
ner. Similar to the results obtained in the de novo infection
assay presented above (Fig. 5A and B), it appeared that 10
?M PBDNJ0804 demonstrated a potency in the inhibition of
HCV E2 glycoprotein processing and a viral yield reduction
similar to those of 100 ?M CM-10-18. Our results thus imply
that the novel DNJ derivative PBDNJ0804 more potently
inhibited HCV glycoprotein processing and virion secretion
but did not disrupt additional steps of HCV replication. This
is consistent with the proposed mechanism of action of
We show in this study that ER-resident ?-glucosidases I and
II are essential host factors for HCV infection by using either
(i) RNA interference (RNAi) technology to knock down the
cellular protein expression of these enzymes or (ii) imino sugar
derivatives known to inhibit their enzymatic activity. Mecha-
nistically, we demonstrate that the ER ?-glucosidases play an
essential role in glycan processing and the function of HCV
Despite the great potential of imino sugars as broad-spec-
trum antiviral drugs, their clinical development has been ham-
pered by their relatively low efficacy. For example, NBDNJ, an
imino sugar that has been approved by the U.S. and European
FDAs for use in the treatment of Gaucher’s disease, had been
terminated for development as an antiviral agent against HIV
due to the failure to achieve a therapeutic concentration in
FIG. 5. PBDNJ0804 is a potent HCV inhibitor. (A) Huh7.5 cells were infected with HCV at an MOI of 0.015 and left untreated (control) or
treated with 100 IU/ml IFN-? or the indicated concentrations of PBDNJ0804 or CM-10-18 for 4 days. The intracellular and extracellular amounts
of HCV RNA and virus yields in culture media at day 4 postinfection were determined and expressed as percentages of the untreated-control
values. Values represent averages and standard deviations from four independent experiments. (B) Intracellular levels of HCV E2/E2p7 and NS3
proteins at day 4 postinfection were determined by Western immunoblot assay. GAPDH served as a loading control. (C and D) Persistently
HCV-infected Huh7.5 cells (as described in the legend to Fig. 3) were left untreated (control) or treated with 100 IU/ml IFN-? or the indicated
concentrations of PBDNJ0804 or CM-10-18 for 4 days. (C) Intracellular and extracellular amounts of HCV RNA and progeny virus yields were
quantified as described above. Values represent averages and standard deviations from four independent experiments. (D) Intracellular HCV
E2/E2p7 and NS3 proteins were detected by Western blotting. GAPDH served as a loading control.
1042 QU ET AL.ANTIMICROB. AGENTS CHEMOTHER.
vivo (11). Similarly, another glucosidase inhibitor, Celgosivir
(a prodrug of the natural product castanospermine), showed
only a modest antiviral effect in chronically HCV-infected pa-
tients (5% of the patients tested experienced a ?1-log10re-
duction in viremia) as a monotherapeutic agent in a phase II
clinical trial, most likely due to the relatively low plasma drug
concentration and poor antiviral activity achieved (9).
Our studies reported herein discovered three novel DNJ
derivatives with a six-carbon side chain, represented by
PBDNJ0804, with more potent antiviral activity against HCV.
Mechanistic analyses revealed that although PBDNJ0804 is
not a more potent ?-glucosidase I inhibitor, the compound
appears to more efficiently inhibit the glycan processing of E2
glycoprotein and thus the assembly and secretion of virions.
Although the possibility cannot be ruled out that PBDNJ0804
inhibits additional viral functions, such as the p7 ion channel,
as suggested by others for NNDNJ (35), or other, unknown,
host functions to achieve improved antiviral activity, the fact
that the compound is also a better inhibitor of DENV and
WNV (8), which do not encode a p7-like protein, favors the
hypothesis that the improved antiviral effect of PBDNJ0804 is
caused by enhanced cellular uptake and accumulation of com-
pounds to a higher concentration in the ER. Indeed, it was
recently shown that encapsulation of imino sugar into ER-
targeting liposomes allowed enhanced delivery, as well as dra-
matically improved antiviral efficacy against HIV (30). There-
fore, in future efforts to improve the antiviral activity of imino
sugars, we should take into consideration that the cell-perme-
ating ability and subcellular distribution of the compounds are
important factors in determining their biological activity.
Interestingly, we showed that reducing ?-glucosidase I, II, or
both by approximately 50% using RNAi technology signifi-
cantly reduced HCV production but did not apparently inter-
fere with host cell growth. These results suggest that HCV and
possibly other viruses are more dependent on these host en-
zymes than the host cell itself and thus provide a solid basis for
inhibition of ?-glucosidases as a practical antiviral strategy.
However, prolonged treatment of cells with ?-glucosidase in-
hibitors may also disrupt the glycan processing and maturation
of certain host cellular glycoproteins, including viral receptors,
which could, in turn, inhibit virus entry. In fact, it was reported
previously that treatment of cells with NNDNJ could modestly
inhibit HCV entry (35). In agreement with this observation, we
have also demonstrated that pretreatment of MDBK cells with
imino sugars for 24 h reduced the permissiveness of the cells to
BVDV infection by 50% (data not shown). Moreover, we con-
sistently observed a stronger antiviral activity of the imino
sugars in de novo infection assays than in persistently infected
cells (Fig. 2, 3, and 5). Hence, it is possible that inhibition of
HCV entry contributes, at least in part, to the antiviral activity
of imino sugars against HCV.
Experiences in antiviral therapies of HIV and hepatitis B
virus infections and more recently in clinical trials of antiviral
drugs against chronic HCV infection have shown that although
drugs targeting viral functions initially induce a profound drop
in viremia, development of drug resistance eventually limits
their antiviral efficacy. A strategy of combination therapy with
antivirals targeting multiple steps of viral replication has been
proved to induce sustained suppression of HIV replication and
prevent the emergence of drug resistance (22, 32, 36). It has
also been demonstrated in recent clinical trials that a triple
combination of PEGylated IFN-?2b, ribavirin, and an NS3/4A
protease inhibitor (telaprevir or boceprevir) significantly im-
proved the sustained virological response rate in treatment-
naïve HCV patients (27, 31). Moreover, it was demonstrated
previously that NNDNJ treatment of HCV-infected Huh7 cells
was able to eliminate the virus after only five passages without
emergence of drug-resistant viruses (35). The absence of se-
lection of ?-glucosidase inhibitor-resistant mutants was also
observed in a BVDV infection model (39). These results
strongly argue that, unlike drugs targeting viral functions, in-
hibition of host cellular functions essential for HCV replica-
tion, such as ?-glucosidases, has a low likelihood of developing
resistant mutants. This unique property, in combination with
much-improved antiviral efficacy, makes PBDNJ0804 a prom-
ising antiviral candidate for combination therapy with IFN-?
or drugs that inhibit viral functions (7).
This work was supported by an NIH grant (AI061441) and by the
Hepatitis B Foundation through an appropriation from the Common-
wealth of Pennsylvania.
We thank Lijuan Wang for technical support, Tina Gill for critical
reading of the manuscript, Robert Moriarty (University of Illinois at
Chicago) for chemistry consultation, Raymond Dwek for providing
NBDNJ, and Arvind H. Patel (University of Glasgow, Glasgow, United
Kingdom) for providing HCV E2 AP33 antibody.
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