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Journal of General Virology (2010), 91, 382–388
DOI 10.1099/vir.0.015388-0
ISG15, a ubiquitin-like interferon-stimulated gene,
promotes hepatitis C virus production in vitro:
implications for chronic infection and response to
treatment
Limin Chen,1,2 Jing Sun,1 Larry Meng,1 Jenny Heathcote,3
Aled M. Edwards1,2,4 and Ian D. McGilvray5
Correspondence
Ian D. McGilvray
[email protected]
1
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario,
Canada
2
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
3
Department of Medicine, University of Toronto, Toronto, Ontario, Canada
4
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
5
Department of Surgery, University of Toronto, Toronto, Ontario, Canada
Received 17 August 2009
Accepted 15 October 2009
Upregulation of interferon (IFN)-stimulated genes (ISGs), including IFN-stimulated gene 15
(ISG15) and other members of the ISG15 pathway, in pre-treatment liver tissue of patients
chronically infected with hepatitis C virus (HCV) is associated with subsequent treatment failure
(pegylated IFN-a/ribavirin). This study assessed the effect of ISG15 on HCV production in vitro.
The levels of ISG15 and of its conjugation to target proteins (ISGylation) were increased by
plasmid transfection, but ISGylation was inhibited by small interfering RNA directed against the
E1 activating enzyme, Ube1L, in Huh7.5 cells. Cells were infected with HCV FL-J6/JFH virus, and
HCV RNA and viral titres were determined. Levels of both HCV RNA and virus increased when
levels of ISG15 and ISGylation were increased, and decreased when ISGylation was inhibited.
The effects of ISGylation on HCV were independent of upstream IFN signalling: IFN-a-induced
ISG expression was not altered by Ube1L knockdown. Thus, although ISG15 has antiviral activity
against most viruses, ISG15 promotes HCV production. HCV might exploit ISG15 as a host
immune evasion mechanism, and this may in part explain how increased expression of ISGs,
especially ISG15, correlates with subsequent IFN-based treatment failure.
INTRODUCTION
Hepatitis C virus (HCV) is adept at evading host antiviral
mechanisms and is often resistant to the current standard
of care – combination treatment with pegylated interferon
(IFN)-a and ribavirin (PegIFN/Rib). This regimen eradicates the virus in only 50 % of cases. A number of
mechanisms contribute to evasion and treatment resistance, including cleavage of the RIG-I adaptor protein IPS1/
MAVS/Cardif by the HCV NS3/NS4A protease and
modulation of the host response by the HCV core protein
(Li et al., 2005; Loo et al., 2006). However, none of these
mechanisms has consistently been demonstrated to play a
role in the clinical disease and thus cannot explain the
ability of the virus to escape the host response in patients.
A supplementary table of primer sequences is available with the online
version of this paper.
382
Response to treatment can be predicted by levels of
expression of IFN-stimulated genes (ISGs) in the liver prior
to initiation of PegIFN/Rib treatment. Non-responders
have increased expression of a number of ISGs (Chen et al.,
2005; Feld et al., 2007; Asahina et al., 2008; Asselah et al.,
2008; Sarasin-Filipowicz, et al., 2008). Three of these ISGs
are components of the ISG15 ubiquitin-like pathway.
ISG15 was the first ubiquitin-like protein to be described
and, like its homologue ubiquitin, is conjugated to proteins
in a tightly regulated process called ISGylation. The ISG15
E1 activating protein, Ube1L, coordinates with the E2
conjugating enzyme (UbcH8) and the E3 ligase (CEB1) to
join the C terminus of ISG15 to a wide variety of proteins
(Loeb & Haas, 1992). ISG15 can be removed from its target
proteins by USP18, an ISG15 protease (Malakhov et al.,
2002). ISG15, CEB1 and USP18 are upregulated in the liver
tissue of patients infected with HCV who have not
responded to treatment with PegIFN/Rib (Chen et al.,
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ISG15 stimulates HCV production
2005). A functional association between this pathway and
the regulation of HCV production has been established:
knockdown of USP18 increases the anti-HCV potency of
IFN-a (Randall et al., 2006), and ISG15 protein expression
is highly upregulated in the hepatocytes of treatment nonresponders, but is increased in the macrophages of
treatment responders. The ISG15 pathway is thus likely
to be important in clinical HCV disease and in determining
treatment outcomes.
ISGylation is implicated in different cellular processes, but
its role in viral biology is the one that is best established
(Dao & Zhang, 2005). ISG15 is targeted by a number of
viruses in animal model and cell culture systems. For
example, non-structural protein 1B of influenza B virus
binds to the free form of ISG15, preventing ISGylation
(Yuan & Krug, 2001). Overexpression of ISG15 in IFN-a/b
receptor knockout mice protects them from Sindbis virusinduced lethality and decreases Sindbis virus replication in
multiple organs (Lenschow et al., 2005). ISG152/2 mice are
more susceptible to influenza A and B viruses, herpes
simplex virus type 1 (HSV-1), Sindbis virus and murine
gammaherpesvirus 68 infection; for Sindbis virus, this
effect is dependent on ISGylation (Lenschow et al., 2007).
Whilst these studies suggest a general role for ISG15 as an
antiviral agent, a recent report found that ISG15 can
inhibit IFN responses after infection by Newcastle disease
virus (Kim et al., 2008a). ISGylation of the antiviral RIG-I
enzyme inhibited IFN signalling in MEF cells (Kim et al.,
2008b). Thus, ISG15 inhibits virus production for many
viruses, but may promote production of some.
In this study, we examined the role of ISG15 and ISGylation
in HCV production in vitro, using the FL-J6/JFH HCV
infectious model. Unexpectedly, increasing the level of
ISG15/ISGylation promoted HCV production, whilst blocking ISGylation decreased HCV RNA and viral titres. This
work therefore suggests a new context for the host ISG
response to HCV: some aspects of the host ISG response to
HCV foster viral production, rather than inhibiting it.
RESULTS
Increasing and decreasing ISGylation in Huh7.5
cells
In order to test whether ISG15 conjugation plays a role in
HCV replication/production, we developed ways of
increasing
and
decreasing
ISG15
conjugation
(ISGylation). Inducing ISGylation can be difficult in
certain cells: for example, in HeLa cells, ISGylation could
only be induced by overexpression of ISG15 in combination with its E1 activating enzyme Ube1L and its E2
conjugating enzyme UbcH8 (Zhao et al., 2005). However,
in Huh7.5 cells, overexpression of ISG15 alone led to
pronounced protein ISGylation in Huh7.5 cells (Fig. 1a).
Combining overexpression of ISG15 with overexpression
of Ube1L and/or UbcH8 did not appreciably increase
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Fig. 1. Modulation of ISGylation in Huh7.5 cells. (a) Western blot
for ISG15 after transfection of Huh7.5 cells with ISG15 or empty
vector. U, Untreated; V, empty vector. The presence or absence of
IFN-a (100 U ml”1, 24 h) is indicated. (b) Comparison of protein
ISGylation (Western blot for ISG15) in the presence or absence of
IFN-a (100 U ml”1, 24 h) following electroporation of irrelevant
siRNA (siIrr) or siRNA specific to Ube1L (siUbe1L). Molecular
mass markers are shown on the left (kDa).
protein ISGylation beyond that observed with overexpression of ISG15 alone (data not shown). In order to inhibit
ISGylation, the ISG15 E1 Ube1L enzyme was knocked
down with small interfering RNA (siRNA). Ube1L mRNA
was successfully knocked down, even in the presence of
increasing concentrations of IFN-a (0–1 U ml21) (Fig. 2).
This method abolished ISGylation, even in the presence of
high levels of IFN-a (100 IU ml21) (Fig. 1b). Thus, in
Huh7.5 cells, ISGylation could be increased and decreased
relatively easily.
HCV RNA and virus are increased in parallel with
ISGylation
We next asked whether ISGylation (and ISG15) modulates
HCV production. As seen in Fig. 3, increasing ISGylation
by overexpression of ISG15 significantly increased the
production of both HCV RNA and virus, even in the
presence of increasing IFN-a, suggesting that increased
ISG15/ISGylation decreases IFN-a anti-HCV activity
(Fig. 4) in the FL-J6/JFH HCV in vitro culture system. In
contrast, silencing of the ISG15 Ube1L E1 enzyme
decreased the levels of HCV RNA and virus both at
baseline (in the absence of IFN-a) and in the presence of
IFN-a, an effect that was more pronounced for HCV viral
titres (Fig. 5). To ensure that the siRNA was selective, we
tested the effects of four individual Ube1L siRNAs and
compared these with the effects of pooled Ube1L siRNA. As
shown in Fig. 6, all four individual siRNAs had a similar
inhibitory effect on HCV viral particle secretion when
compared with the pooled siRNA, indicating that the effect
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L. Chen and others
important role in IFN signalling (Malakhova et al., 2003).
To test this hypothesis in the HCV model, we assessed the
effect of decreasing ISGylation by Ube1L knockdown on
downstream ISG expression in the presence of IFN-a. As
shown in Fig. 7, the expression of a number of ISG
transcripts was not affected following Ube1L siRNA
knockdown in the presence of IFN-a.
DISCUSSION
Fig. 2. Ube1L mRNA expression was silenced by siRNA. Huh7.5
cells were electroporated with irrelevant siRNA (shaded bars) or
siRNA specific to Ube1L (filled bars). Thirty hours after electroporation, cells were treated with increasing amounts of IFN-a for
15 h before being harvested to determine the levels of Ube1L
mRNA by real-time PCR (normalized to b-actin). Data represent
the means±SD of three replicates; the results shown are
representative of three similar experiments. *P,0.05 (versus
irrelevant siRNA).
we observed was specific to the knockdown of Ube1L.
Taken together, these data suggested that ISGylation is
important for baseline HCV production.
Silencing Ube1L does not affect upstream IFN-a
signalling
Increased ISGylation has been shown to prolong STAT1
phosphorylation, suggesting that ISGylation might play an
Fig. 3. ISG15 promotes HCV production in vitro. Huh7.5 cells
were transfected with empty vector (V) or ISG15 plasmid DNA for
48 h before the cells were infected with FL-J6/JFH as described in
Methods. HCV RNA was quantified by real-time PCR (a), and HCV
virus particle titres were assessed by serial dilution of culture
supernatants (b). Data represent the means±SD of three
replicates; the results shown are representative of three similar
experiments. U, Untreated control. ***P,0.001; *P,0.05 (versus
empty vector).
384
ISG15 is one of the most abundant ISGs induced after virus
infection and type I IFN treatment, and we and others have
found that increased pre-treatment ISG15 expression in the
livers of HCV-infected patients predicts subsequent
treatment failure (Chen et al., 2005; Feld et al., 2007;
Asahina et al., 2008; Asselah et al., 2008; Sarasin-Filipowicz
et al., 2008). Although ISG15 is generally considered to be
antiviral, we have presented evidence here that ISGylation
promotes HCV production, decreases the anti-HCV effect
of IFN-a and is particularly relevant for steps downstream
of HCV RNA replication. Thus, aspects of the host ISG
response favour, rather than inhibit, HCV persistence. The
action of ISG15 is a novel mechanism for virus persistence,
and ISGylation is a possible target for therapy of HCV
infection.
As noted in the Introduction, the effect of ISG15 on virus
production may be specific to the virus. For example,
ISG15 can have an antiviral effect on Sindbis virus,
influenza virus, HSV, human immunodeficiency virus
(HIV) and Ebola virus (Lenschow et al., 2005, 2007;
Okumura et al., 2006; Zhang et al., 2007), but ISGylation
does not contribute to murine susceptibility to lymphocytic choriomeningitis virus and vesicular stomatitis virus
(Knobeloch, et al. 2005), nor to hepatitis B virus
replication in ISGylation-deficient mice (Ube1L2/2)
(Kim et al., 2008a). Although ISG15 may also promote
viral production by acting as a negative regulator of the
innate immune response through its conjugation to RIG-I
(Kim et al., 2008b), this mechanism is unlikely to
contribute to our observed effects, as Huh7.5 cells are
deficient in RIG-I (Sumpter et al., 2005). Our data suggest
that HCV exploits the ISG15/ISGylation pathway to
increase HCV production: overexpression of ISG15, which
increases ISGylation in Huh7.5 cells (Fig. 1), increased
HCV RNA 3-fold and viral titres 2.2-fold (Fig. 3). Blocking
ISGylation by knockdown of Ube1L decreased HCV RNA
and largely abolished the production of infectious virus
(Fig. 5).
Another approach to examining the role of ISG15 in HCV
production would be to decrease ISG15 mRNA using
specific siRNA. We have not employed this method in the
current study – in preliminary work, we found that ISG15specific siRNA was not able to decrease ISG15 mRNA
consistently in Huh7/7.5 cells, particularly in the presence
of IFN-a (data not shown). However, others have
demonstrated that knockdown of ISG15 in two HCV
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Journal of General Virology 91
ISG15 stimulates HCV production
Fig. 4. Increased ISG15/ISGylation decreases
IFN-a anti-HCV activity. Huh7.5 cells were
transfected with empty vector (shaded bars)
or ISG15 plasmid DNA (filled bars) for 48 h
before the cells were treated with increasing
amounts of IFN-a, as indicated, for 16 h
followed by infection with FL-J6/JFH as
described in Methods. HCV RNA was quantified by real-time PCR (a), and HCV virus
particle titres were assessed by serial dilution of
culture supernatants (b). Data represent the
means±SD of three replicates; the results
shown are representative of three similar
experiments. *P,0.05 (versus empty vector).
replicon models (Con1 and murine MH1 cells) resulted in
decreased HCV production both with and without IFN-a
(Broering et al., 2008). Although this study does not
directly address the role of ISGylation, it adds evidence for
the permissive role of the ISG15 pathway in the HCV life
cycle.
Our data provide a mechanistic insight into how ISG15
affects HCV production. ISG15 exists in three forms: (i) a
free, unconjugated intracellular protein, (ii) conjugated to
viral and/or host target proteins, and (iii) an extracellular
cytokine (Recht et al., 1991; D’Cunha et al., 1996a, b; Lai et
al., 2009). All three forms could potentially affect HCV
viral production. In other systems, the free form of ISG15
has been shown to inhibit the release of Ebola virus-like
particles by interfering with the activity of Nedd4
(Malakhova & Zhang, 2008; Okumura et al., 2008).
ISGylation is critical to the effect of ISG15 on Sindbis
virus, and ISGylation is targeted by the human influenza
virus NS1 protein (Yuan & Krug, 2001; Lenschow et al.,
2007). As a cytokine, purified ISG15 can activate natural
killer and cytotoxic T-cells, stimulate IFN-c production,
and induce dendritic cell maturation and neutrophil
recruitment (Recht et al., 1991). Our data argue that
ISGylation is the predominant mechanism through which
ISG15 affects HCV production.
Blocking ISGylation by Ube1L knockdown did not
decrease free ISG15 but dramatically reduced HCV viral
titres and significantly reduced HCV RNA levels. In order
to test for a direct cytokine role of ISG15, we exposed
Huh7.5 cells to a high dose of purified ISG15 (2 mg ml21)
for 36 h before cells were infected with FL-J6/JFH virus
(m.o.i.50.3) as before. Although the dose we used is
considerably higher than that used by D’Cunha et al.
(1996b) (100 ng ml21) to define the cytokine effect of
ISG15, we were unable to find any inhibition of HCV
production, nor did we find any reduction in the ability of
IFN-a to stimulate ISG expression (data not shown).
The current study demonstrates that increasing ISGylation
promotes HCV production and decreases the anti-HCV
effect of IFN-a. However, previous work from our group
has demonstrated that decreasing the expression of USP18,
the ISG15 protease, increases ISGylation yet potentiates
IFN-a anti-HCV activity (Randall et al., 2006). These data
initially appear conflicting, but only if one assumes that
USP18 and ISG15 work entirely through the same pathway.
In fact, USP18 clearly has additional targets beyond ISG15,
and manipulating USP18 expression has effects on protein
expression that are independent of ISG15. For example,
epidermal growth factor receptor synthesis is regulated by
USP18 (Duex & Sorkin, 2009). USP18 has both protease-
Fig. 5. Silencing Ube1L inhibits HCV production. Huh7.5 cells were electroporated with
irrelevant siRNA (shaded bars) or Ube1L
siRNA (filled bars) and the cells were treated
with different concentrations of IFN-a as
indicated. HCV RNA (a) and the number of
infectious particles (b) were determined as
described in Methods. Data represent the
means±SD of three replicates; the results
shown are representative of three similar
experiments. *P,0.05 (versus siIrr-transfected
cells).
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385
L. Chen and others
the data from our group suggest that ISGylation is
necessary but not sufficient for HCV production.
Fig. 6. Comparison of individual versus pooled Ube1L siRNAs.
siRNA was electroporated into Huh7.5 cells, after which the cells
were infected with FL-J6/JFH as described in Methods. Viral
particles were titrated 48 h after infection. Data represent the
means±SD of three replicates; the results shown are representative of three similar experiments.
dependent and -independent functions (Malakhova et al.,
2006). Our preliminary data would support USP18 having
a role in HCV production that is independent of ISG15 and
ISG15 protease activity (Chen et al., 2008). Taken together,
Blocking ISGylation enhances the anti-HCV effect of IFN-a
(Fig. 5). This effect is not likely to be mediated at the level
of IFN signalling, as Ube1L knockdown did not promote
(or inhibit) IFN-dependent ISG expression, which is the
indicator of activation of the IFN pathway (Fig. 7).
Although ISGylation may play an important role in
regulation of the JAK/STAT pathway and IFN signalling
in some cells (Malakhov et al., 2002; Malakhova et al.,
2002; Ritchie et al., 2002), the IFN signalling pathway is
intact in ISG152/2 and Ube1L2/2 mice (Osiak et al., 2005;
Kim et al., 2006). In our work, IFN signalling appeared to
be unaffected, despite knockdown of Ube1L, and Ube1L
knockdown inhibited HCV production, even in the
absence of IFN-a. These data suggest that ISGylation of
viral (or host) proteins is directly important for the viral
life cycle. The conjugation of ISG15 to cellular or viral
proteins might alter their function, or compete for
ubiquitination. For example, in order for HIV to be
secreted from infected cells, the Gag protein must be
ubiquitinated and then recruited to the endosomal
transport complex. ISG15 conjugation to Gag prevents its
ubiquitination and thus inhibits HIV release (Okumura
et al., 2006). In another example, ISG15 conjugation to
interferon regulatory factor 3 (IRF3), a key signaltransducing factor for IFN-dependent immune responses,
Fig. 7. IFN-a-induced ISG expression following Ube1L siRNA knockdown. Huh7.5 cells
were electroporated with irrelevant (shaded
bars) or Ube1L (filled bars) siRNA, after which
the cells were exposed to different concentrations of IFN-a as indicated. Cells were lysed
24 h after the introduction of IFN-a, and ISG
expression was quantified using real-time PCR
normalized to b-actin expression levels. Data
represent the means±SD of three replicates;
the results shown are representative of three
similar experiments.
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Journal of General Virology 91
ISG15 stimulates HCV production
protects IRF3 from ubiquitin-mediated degradation (Lu et
al., 2006). Thus, ISGylation of HCV proteins or host
proteins important for the HCV life cycle may alter their
function or protect them from degradation; the specific
steps involved in the ISG15 effect remain to be defined.
In summary, our in vitro data strongly argue that ISG15,
and specifically ISGylation, is important to the HCV life
cycle in an infectious cell culture model of HCV. This study
offers one explanation for how increased baseline expression of some ISGs, including ISG15, correlates with
treatment failure in HCV-infected patients. Targeting
ISGylation – or specific targets of ISGylation – may
identify new antiviral therapies for HCV.
METHODS
Cells and HCV FL-J6/JFH virus. Huh7.5 cells and HCV FL-J6/JFH
were kindly provided by Dr Charles Rice (Rockefeller University, NY,
USA) (Lindenbach et al., 2005). Cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with non-essential amino
acids, 10 % fetal bovine serum, 100 U ampicillin ml21 and 100 mg
streptomycin ml21 at 37 uC in a 5 % CO2 humidified incubator.
ISG15 expression plasmid. Human full-length ISG15 was generated
using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as
template, and the resulting PCR product was cloned into a pcDNA4/
HisMax TOPO TA expression vector (Invitrogen). The primers used
were 59-ATGGGCTGGGACCTGACGGTG-39 (forward) and 59TTAGCTCCGCCCGCCAGGCTC-39 (reverse). Plasmid DNA for transfection studies was prepared using a Plasmid Maxiprep kit (Qiagen).
ISG15 transfection and detection of ISG15 expression by
Western blotting. ISG15 plasmid DNA (8 mg) or empty vector
was transfected into Huh7.5 cells (2.56105 ml21, 5 ml per 6 cm
culture dish) with Lipofectamine 2000 (Invitrogen) following the kit
protocol. At 48 h post-transfection, the cells were harvested, washed
in PBS and lysed in 200 ml lysis buffer [50 mM HEPES (pH 7.8),
500 mM NaCl, 1 % Triton X-100, 1 mM EDTA, protease inhibitor
cocktail (Sigma)]. Proteins were separated by NuPAGE 4–12 % Bis/
Tris gels and transferred onto nitrocellulose membrane using a TransBlot SD Semi-dry transfer cell (Bio-Rad). ISG15 expression was
assessed using a polyclonal anti-ISG15 antiserum raised in rabbits
against purified human ISG15 protein (Cedarlane Laboratories). Blots
were developed using an OptiBlaze WEST femtoLUCENT kit (GBiosciences).
For studies of the effect of ISG15/ISGylation on HCV production,
0.3 mg ISG15 plasmid DNA or empty vector was transfected into
Huh7.5 cells in each well of a 96-well plate for 48 h, after which the
cells were treated in the absence or presence of IFN-a (0–1 U ml21)
for 16 h, followed by infection with HCV FL-J6/JFH virus
(m.o.i.50.3) for 6 h. The cells were then washed and overlaid with
fresh medium. At 2 days post-infection (p.i.), the cells were harvested
for assessment of HCV replication (RNA and infectious particles, see
below).
recommended by the manufacturer (Dharmacon). One nanomole
of RNA duplexes was electroporated into 2.56106 Huh-7.5 cells as
described previously (Randall et al., 2006). Thirty hours after
electroporation, cells were treated with IFN-a (0–1 U ml21) for
15 h, and then either harvested for determination of siRNA knockdown efficiency (Ube1L mRNA by real-time PCR) or washed,
infected with HCV FL-J6/JFH (m.o.i.50.3) for 6 h, rinsed and
overlaid with fresh medium. At 48 h p.i., cells were harvested for
assessment of HCV production (RNA and infectious particles). A
similar experiment was performed using higher dosages of IFN-a (0–
1000 U ml21) to investigate whether silencing Ube1L and/or
decreased ISGylation had any effect on IFN downstream ISG
mRNA expression (real-time PCR).
Quantification of infectious HCV virion and HCV RNA. Viral
titres were determined by limiting dilution analysis of culture
supernatants as described previously (Lindenbach et al., 2005). For
HCV RNA quantification, total cellular RNA was harvested and
purified with 96-well RNeasy columns (Qiagen), reverse transcribed
(Superscript II; Invitrogen) and the cDNA constructed (AnCT
primer; Invitrogen). Real-time PCR was performed using SYBR
Green mix and either the primers listed in Supplementary Table S1
(available in JGV Online) or the HCV-specific primers 59TGAGTGTCGTACAGCCTCCA-39 and 59-ACGCTACTCGGCTAGCAGTC-39 (Platinum Quantitative RT-PCR ThermoScript One-Step
System; Invitrogen, Life Technologies) as described previously
(Randall et al., 2006).
Statistics. Where appropriate, Student’s t-test was used to compare
two categorical values and one-way analysis of variance was used to
compare more than two categorical values. For Western blot studies,
the experiments were repeated at least three times.
ACKNOWLEDGEMENTS
This work was funded by a grant from the Canadian Institute of
Health Research (no. 62488 to I. D. M). L. C. was supported by the
National Canadian Research Training Program in Hepatitis C
(NCRTP-HepC) and Canada Graduate Scholarship (CGS) from the
Canadian Institute of Health Research.
REFERENCES
Asahina, Y., Izumi, N., Hirayama, I., Tanaka, T., Sato, M., Yasui, Y.,
Komatsu, N., Umeda, N., Hosokawa, T. & other authors (2008). Potential
relevance of cytoplasmic viral sensors and related regulators involving
innate immunity in antiviral response. Gastroenterology 134, 1396–1405.
Asselah, T., Bieche, I., Narguet, S., Sabbagh, A., Laurendeau, I.,
Ripault, M. P., Boyer, N., Martinot-Peignoux, M., Valla, D. & other
authors (2008). Liver gene expression signature to predict response to
pegylated interferon plus ribavirin combination therapy in patients
with chronic hepatitis C. Gut 57, 516–524.
Broering, R., Trippler, M., Gerken, G. & Schlaak, J. F. (2008). The
interferon stimulated gene 15 is an important proviral factor for the
hepatitis C virus. Hepatology 48, 758A.
Ube1L siRNA knockdown. Four Ube1L siRNAs were designed as
Chen, L., Borozan, I., Feld, J., Sun, J., Tannis, L. L., Coltescu, C.,
Heathcote, J., Edwards, A. M. & McGilvray, I. D. (2005). Hepatic gene
follows (the sense strand sequence is described): siUbe1L#1, 59CAUCUUUGCUAGUAAUCUA-39; siUbe1L#2, 59-CGAAUUGUGGGCCAGAUUA-39; siUbe1L#3, 59-AUAGAGCGCUCCAAUCUCA-39; and siUbe1L#4, 59-GCAUGGAGUUUGCUUUCUG-39. The
irrelevant siRNA was 59-GGCGCUUGUGGACAUUCUGTT-39.
Chemically synthesized RNA oligonucleotides were prepared as
Chen, L., Sun, J., Meng, X., Edwards, A. M., Heathcote, J. & McGilvray,
I. D. (2008). USP18 promotes HCV replication and blunts the antiviral
effect of IFNa in a protease-independent fashion. Hepatology 48, 399A.
http://vir.sgmjournals.org
expression profiling discriminates responders and non-responders in
treatment of chronic hepatitis C viral infection. Gastroenterology 128,
1437–1444.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 08:01:09
387
L. Chen and others
Dao, C. T. & Zhang, D. E. (2005). ISG15: ubiquitin-like enigma.
Regulation of ISG15 expression and conjugation. Front Biosci 10,
2701–2722.
D’Cunha, J., Knight, E., Haas, A. L., Truitt, R. L. & Borden, E. C.
(1996a). Immunoregulatory properties of ISG15, an interferon-
induced cytokine. Proc Natl Acad Sci U S A 93, 211–215.
D’Cunha, J., Ramanujam, S., Wagner, R. J., Witt, P. L., Knight, E. &
Borden, E. C. (1996b). In vitro and in vivo secretion of human ISG15.
J Immunol 157, 4100–4108.
the innate antiviral response by inhibition of IRF-3 degradation. Cell
Mol Biol (Noisy-le-grand) 52, 29–41.
Malakhov, M. P., Malakhova, O. A., Kim, K. I., Ritchie, K. J. & Zhang,
D. E. (2002). UBP43 (USP18) specifically removes ISG15 from
conjugated proteins. J Biol Chem 277, 9976–9981.
Malakhova, O. A. & Zhang, D. E. (2008). ISG15 inhibits Nedd4
ubiquitin E3 activity and enhances the innate antiviral response. J Biol
Chem 283, 8783–8787.
Duex, J. E. & Sorkin, A. (2009). RNA interference screen identifies
Malakhova, O. A., Malakhov, M. P., Hetherington, C. & Zhang, D. E.
(2002). Lipopolysaccharide activates the expression of ISG15-specific
Usp18 as a regulator of epidermal growth factor receptor synthesis.
Mol Biol Cell 20, 1833–1844.
protease UBP43 via interferon regulatory factor 3. J Biol Chem 277,
14703–14711.
Feld, J. J., Nanda, S., Huang, Y., Chen, W., Cam, M., Pusek, S. N.,
Schweigler, L. M., Theodore, D., Zacks, S. L. & other authors (2007).
Malakhova, O. A., Yan, M., Malakhov, M. P., Yuan, Y., Ritchie, K. J.,
Kim, K. I., Peterson, L. F., Shuai, K. & Zhang, D. E. (2003). Protein
Hepatic gene expression during treatment with peginterferon and
ribavirin: identifying molecular pathways for treatment response.
Hepatology 46, 1548–1563.
Kim, K. I., Yan, M., Malakhova, O., Luo, J. K., Shen, M. F., Zou, W., de la
Torre, J. C. & Zhang, D. E. (2006). Ube1L and protein ISGylation are not
ISGylation modulates the JAK-STAT signaling pathway. Genes Dev
17, 455–460.
Malakhova, O. A., Kim, K. I., Luo, J. K., Zou, W., Kumar, K. G., Fuchs,
S. Y., Shuai, K. & Zhang, D. E. (2006). UBP43 is a novel regulator of
essential for alpha/beta interferon signaling. Mol Cell Biol 26, 472–479.
interferon signaling independent of its ISG15 isopeptidase activity.
EMBO J 25, 2358–2367.
Kim, J. H., Luo, J. K. & Zhang, D. E. (2008a). The level of hepatitis B
Okumura, A., Lu, G., Pitha-Rowe, I. & Pitha, P. M. (2006). Innate
virus replication is not affected by protein ISG15 modification but is
reduced by inhibition of UBP43 (USP18) expression. J Immunol 181,
6467–6472.
Kim, M. J., Hwang, S. Y., Imaizumi, T. & Yoo, J. Y. (2008b). Negative
feedback regulation of RIG-I-mediated antiviral signaling by interferon-induced ISG15 conjugation. J Virol 82, 1474–1483.
Knobeloch, K. P., Utermöhlen, O., Kisser, A., Prinz, M. & Horak, I.
(2005). Reexamination of the role of ubiquitin-like modifier ISG15 in
the phenotype of UBP43-deficient mice. Mol Cell Biol 25, 11030–
11034.
Lai, C., Struckhoff, J. J., Schneider, J., Martinez-Sobrido, L., Wolff, T.,
Garcı́a-Sastre, A., Zhang, D. E. & Lenschow, D. J. (2009). Mice
lacking the ISG15 E1 enzyme UbE1L demonstrate increased
susceptibility to both mouse-adapted and non-mouse-adapted
influenza B virus infection. J Virol 83, 1147–1151.
Lenschow, D. J., Giannakopoulos, N. V., Gunn, L. J., Johnston, C.,
O’Guin, A. K., Schmidt, R. E., Levine, B. & Virgin, H. W., IV (2005).
Identification of interferon-stimulated gene 15 as an antiviral molecule
during Sindbis virus infection in vivo. J Virol 79, 13974–13983.
Lenschow, D. J., Lai, C., Frias-Staheli, N., Giannakopoulos, N. V.,
Lutz, A., Wolff, T., Osiak, A., Levine, B., Schmidt, R. E. & other
authors (2007). IFN-stimulated gene 15 functions as a critical
antiviral molecule against influenza, herpes, and Sindbis viruses. Proc
Natl Acad Sci U S A 104, 1371–1376.
Li, K., Foy, E., Ferreon, J. C., Nakamura, M., Ferreon, A. C., Ikeda, M., Ray,
S. C., Gale, M., Jr & Lemon, S. M. (2005). Immune evasion by hepatitis C
virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3
adaptor protein TRIF. Proc Natl Acad Sci U S A 102, 2992–2997.
Lindenbach, B. D., Evans, M. J., Syder, A. J., Wölk, B., Tellinghuisen,
T. L., Liu, C. C., Maruyama, T., Hynes, R. O., Burton, D. R. & other
authors (2005). Complete replication of hepatitis C virus in cell
antiviral response targets HIV-1 release by the induction of ubiquitinlike protein ISG15. Proc Natl Acad Sci U S A 103, 1440–1445.
Okumura, A., Pitha, P. M. & Harty, R. N. (2008). ISG15 inhibits Ebola
VP40 VLP budding in an L-domain-dependent manner by blocking
Nedd4 ligase activity. Proc Natl Acad Sci U S A 105, 3974–3979.
Osiak, A., Utermöhlen, O., Niendorf, S., Horak, I. & Knobeloch, K. P.
(2005). ISG15, an interferon-stimulated ubiquitin-like protein, is not
essential for STAT1 signaling and responses against vesicular
stomatitis and lymphocytic choriomeningitis virus. Mol Cell Biol 25,
6338–6345.
Randall, G., Chen, L., Panis, M., Fischer, A. K., Lindenbach, B. D.,
Sun, J., Heathcote, J., Rice, C. M., Edwards, A. M. & other authors
(2006). Silencing of USP18 potentiates the antiviral activity of
interferon against hepatitis C virus infection. Gastroenterology 131,
1584–1591.
Recht, M., Borden, E. C. & Knight, E. (1991). A human 15-kDa IFNinduced protein induces the secretion of IFN-c. J Immunol 147, 2617–
2623.
Ritchie, K. J., Malakhov, M. P., Hetherington, C. J., Zhou, L., Little, M. T.,
Malakhova, O. A., Sipe, J. C., Orkin, S. H. & Zhang, D. E. (2002).
Dysregulation of protein modification by ISG15 results in brain cell
injury. Genes Dev 16, 2207–2212.
Sarasin-Filipowicz, M., Oakeley, E. J., Duong, F. H., Christen, V.,
Terracciano, L., Filipowicz, W. & Heim, M. H. (2008). Interferon
signaling and treatment outcome in chronic hepatitis C. Proc Natl
Acad Sci U S A 105, 7034–7039.
Sumpter, R., Jr, Loo, Y. M., Foy, E., Li, K., Yoneyama, M., Fujita, T.,
Lemon, S. M. & Gale, M., Jr (2005). Regulating intracellular antiviral
defense and permissiveness to hepatitis C virus RNA replication
through a cellular RNA helicase, RIG-I. J Virol 79, 2689–2699.
culture. Science 309, 623–626.
Yuan, W. & Krug, R. M. (2001). Influenza B virus NS1 protein inhibits
Loeb, K. R. & Haas, A. L. (1992). The interferon-inducible 15-kDa
conjugation of the interferon-induced ubiquitin-like ISG15 protein.
EMBO J 20, 362–371.
ubiquitin homolog conjugates to intracellular proteins. J Biol Chem
267, 7806–7813.
Loo, Y. M., Owen, D. M., Li, K., Erickson, A. K., Johnson, C. L., Fish, P. M.,
Carney, D. S., Wang, T., Ishida, H. & other authors (2006). Viral and
therapeutic control of IFN-b promoter stimulator 1 during hepatitis C
Zhang, Y., Burke, C. W., Ryman, K. D. & Klimstra, W. B. (2007).
Identification and characterization of interferon-induced proteins
that inhibit alphavirus replication. J Virol 81, 11246–11255.
virus infection. Proc Natl Acad Sci U S A 103, 6001–6006.
Zhao, C., Denison, C., Huibregtse, J. M., Gygi, S. & Krug, R. M.
(2005). Human ISG15 conjugation targets both IFN-induced and
Lu, G., Reinert, J. T., Pitha-Rowe, I., Okumura, A., Kellum, M.,
Knobeloch, K. P., Hassel, B. & Pitha, P. M. (2006). ISG15 enhances
constitutively expressed proteins functioning in diverse cellular
pathways. Proc Natl Acad Sci U S A 102, 10200–10205.
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