VIROLOGICA SINICA, October 2008, 23 (5):339-344
-CLC number: Q786 Document code: A Article ID: 1674-0769 (2008) 05-0339-06
Silencing of UBP43 by shRNA Enhances the Antiviral Activity of
Interferon against Hepatitis B Virus*
He-bin FAN 1 , Bao-ju WANG 1, Yin-ping LU 1, You-hua HAO 1,
Xin-xing YANG 1, Meng-ji LU 2 and Dong-liang YANG 1**
(1. Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan 430030, China; 2. Institute of Virology, School of Medicine, University of Essen, Essen,
Abstract: Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43
is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We
hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated
whether vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell
culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels
were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by
H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43
modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV
Key words: UBP43; shRNA; Hepatitis B virus; Interferon
Hepatitis B virus (HBV) is a serious public health
problem. The best antiviral therapy at present is IFN
and nucleoside analogues. But high resistance mutations
to nucleoside analogues and low response to IFN
makes it difficult to eradicate hepatitis B.
widespread non-response to exogenous IFN-α, novel
therapeutic approaches that either augment or
complement the antiviral activity of IFN-α would be
Several factors could be associated with the low
response to IFN, such as age, sex, HBV DNA levels
and transaminase (ALT) level in hepatitis B patients.
Recently, some studies have shown that when
compared with responder’s liver tissue, non-res-
ponders have increased expression of a number of
IFN-sensitive genes (3). The expression of a subset of
8 genes accurately predicted treatment response in
more than 90% of patients as well as viral load, ALT
level, or hepatic fibrosis. Two of the 8 genes, UBP43
and IFN-sensitive gene ISG15, are linked biochemi-
cally. ISG15 is a ubiquitin-like molecule that is post-
translationally attached to the lysine residues of more
Received: 2008-03-24, Accepted: 2008-07-20
* Foundation items: National Science Foundation of China
(30271170); National Hish Technology Research and
Douelopment program of China (2006AA02Z128)
** Corresponding author.
Tel: +86-27-83662894, E-mail: email@example.com
340 Virol. Sin. (2008) 23: 339-344
than 150 target proteins (6, 17). Protein targets include
other IFN-stimulated genes, in addition to proteins in
a diverse set of unrelated pathways. However, the
function of the ISG15 conjugation remains unknown.
UBP43 is a ubiquitin-specific protease that cleaves the
ubiquitin-like (and IFN-induced) ISG15 protein from
its cellular targets in vitro (12). Together these data
suggest that non-responder patients have a disordered
host IFN response, and the up-regulation of UBP43
can be correlated with non-response to IFN treatment.
Multiple lines of evidence previously have linked
ISG15, UBP43, and the cellular response to IFN. Both
genes are induced by type I IFN in many cell types (4,
and cells isolated from UBP43 knockout mice
are hypersensitive to IFN, with prolonged Jak–Stat
These proteins also have been linked to
the innate immune response to viruses. Overex-
pression of ISG15 enhances the antiviral activity of
IFN against human immunodeficiency virus and
Sindbis virus replication in vitro (10, 11, 13). In-
fluenza B virus NS1 protein binds ISG15 and prevents
host protein ISGylation, a function that correlates with
influenza B resistance to IFN (15).
Based on these data,
we hypothesized that UBP43 may be a critical determi-
nant of the human IFN antiviral response to HBV.
MATERIALS AND METHODS
Restriction enzymets Pst I, EcoR I, Bgl II, Sal I, T4
polynucleotide kinase and T4 DNA ligase were
obtained from Takara (Japan). Plasmid purification
kits were purchased from Omega (USA). Lipofec-
tamineTM was obtained from Invitrogen and the
ELISA kit from the Dakewei Company (China).
DMEM and fetal calf serum were purchased from
Hyclone and Sijiqing Biotech Company (China)
respectively. pSUPER vector was kindly provided by
Professor Xin-hua Feng (Department of Molecular
and Cellular Biology, Baylor College of Medicine
Houston, United States). HepG2.2.15 cells and
Escherichia coli DH5α were preserved in our
pSuper plasmid construction
shRNAs were cloned downstream of the human H1
promoter in the vector pSUPER as described
previously (1). The target sites for siRNA were
recommended by the website (www.ambion.com and
www.dharmacon.com). The selected sequences were
submitted to a BLAST search to avoid targeting to the
Human genome. The sequence was as follows: 5'-
TCCGGA-3' and irrelevant sequences was 5'-GATC
GGA-3'. The phosphorylation, regeneration and annealing
of single siDNA were performed as follows: after
mixture of water, polynucleotide kinase buffer, single
siDNA, ATP and T4 polynucleotide kinase, then 37
℃ for 45 min, 95 ℃ for 5 min, and 55℃ for 10 min.
The product was ligated into purified pSUPER
plasmid from 1% agarose gel which was enzymatic
digested with Sal I and Bgl II. The recombinant
plasmid was used to transform Escherichia coli DH5α
cells. The positive clones were selected and verified
by DNA sequence.
Cells culture, preparation of total RNAs
HepG2.2.15 cells, a human hepatoblastoma HepG2
Virol. Sin. (2008) 23: 339-344 341
cell line stably transfected by the HBV genome, were
cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum. Then
HepG2.2.15 cells were transfected in 6-well plates
using LipofectamineTM. The cells were divided into 3
groups, namely the interference group (transfected
with psiUBP43), the negative control group (trans-
fected with pSuper plasmid by inserting a nonspecific
sequence, Nonspecific sequence means that the nucleotide
bases were the same as the target seqeuence but the
order was different.So we also refer to the negative
control as the irrelevant control.) and the blank control
group (without transfection). Transfection of cells was
performed with lipofectamine following the manu-
facturer’s guidelines. The plate was incubated at 37
with 5%CO2 for 4 h, after which 2mL DMEM
medium containing 10% FBS were added into the
well, with a final concentration of IFN equal to
3000IU/mL. After further incubation for 48 h, the
cells were taken for observation.
Total RNAs were extracted 48h after transfection.
The cells were collected from wells in each group for
total RNA extraction with Trizol according to the
manufacturer's instructions. After being dissolved in
DEPC, the RNA was accurately quantified with an
ultraviolet (UV) spectrophotometer.
Assay of UBP43 mRNA expression after RNA
The primers for GAPDH, UBP43 were as follows:
UBP43-s: 5’-CAGACCCTGACAATCCACCT-3’; UBP-
43-as: 5’-AGCTCATACTGCCCTCCAGA-3’; GAPDH
-s: 5’-CGGATTTGGTCGTATTGGG-3’; GAPDH-as:
5’-CTCGCTCCTGGAAGATGG-3’. We used a RQ-
PCR assay based on Sybergreen fluorescence
methodology to quantify the full range of UBP43
mRNA copy numbers (2, 9). The number of PCR
cycles required to reach the fluorescence threshold
was the cycle threshold (Ct). The Ct value for each
sample was proportional to the log of the initial
amount of input cDNA. By plotting the Ct value of an
unknown sample on the standard curve, the amount of
target sequence in the sample could be calculated. To
normalize the UBP43 mRNA expression for sample to
sample differences in RNA input, RNA quality, and
reverse transcriptase efficiency, we amplified the
housekeeping gene GAPDH. From their respective
standard curves, we obtained the copy numbers of
GAPDH and UBP43. The ratio between copy
numbers of UBP43 and GAPDH represented the
normalized (nUBP43) for each sample and could be
compared with that of other samples and were calcu-
lated according to nUBP43 = (UBP43 mRNA copies
sample/GAPDH mRNA copies sample) ×10 000 (1).
ELISA and Southern blot
Following the protocol provided with the antigen
detection kit, the concentration of HBsAg and HBeAg
in the supernatant were detected by ELISA. The
results were presented as S/CO values. Extraction of
HBV DNA replicative intermediates and Southern
blotting were performed as previously described (8,
pSuper plasmid construction
The result of 2.5% agarose gel electrophoresis
showed that the plasmid psiUBP43 and irrelevant
control cut by EcoR I and Sal I was about 60 bp
longer than the pSUPER blank control as shown in
Fig.1 and was verified by sequencing. Because the
psimIFNAR1 and irrelevant plasmid had inserted
342 Virol. Sin. (2008) 23: 339-344
Fig. 1. Agrose gel electrophoresis of double enzyme digestion
of plasmid. 1, DL2000 marker; 2, psiUBP43 digested by EcoR
I and Sal I; 3, pSuper plasmid by inserting a nonspecific
sequence ; 4, pSuper digested by EcoR I and Sal I.
shRNA into plasmid pSUPER, so they were about 60
Effect of shRNA on UBP43 mRNA expression
We found that the expression of UBP43 was
decreased compared to the control. For each experi-
mental sample, the amount of UBP43 and GAPDH
was determined from the appropriate standard curve.
Then, the UBP43 amount was divided by the GAPDH
amount to obtain the nUBP43 ratio. The nUBP43 ratio
was significantly lower (P<0.05, also significant after
Fig. 2. Realtime fluorescence quantiative RT-PCR assay of
UBP43 mRNA (n=3 Mean±SD). Negative control (with
irrelevant pSuper) versus interference group (transfected with
psiUBP43) (P=0.035) and blank control (without plasmid)
versus interference group (P=0.003). nUBP43 was calculated
according to equation 1.
Bonferroni correction for multiple comparisons) in the
interference group than in the negative control and
blank control (Fig. 2).
Effects of silencing UBP43 on secreted HBsAg and
The HepG2.2.15 cells were divided into three
groups: the interference group, the negative control
and the blank control. They were treated with IFN
(3000IU/mL) for 48h; the HBeAg and the HbsAg were
Fig. 3. Inhibition of viral gene expression by shRNA in
sequence specific fashion. After transfection of HepG2.2.15
cells with shRNA expression plasmids and addition of IFN, the
HBeAg expression was substantially suppressed, but not for
HBsAg and HepG2.2.15 cells which were more sensitive to
IFN by psiUBP43 relative to blank control (without plasmid)
and negative control (with irrelevant pSuper) (P＜0.05).
Fig. 4. Inhibition of viral replication by IFN after trasfection of
shRNA. After transfection of HepG2.2.15 cells with psiUBP43,
the intracellular viral replicative intermediates were detected by
Southern blotting. RC DNA: relaxed-circular DNA; DL: Double-
stranded linear DNA. 1, Negative control (with irre- levant
pSuper plasmid); 2, Blank control (without plasmid); 3, Inter-
ference group ( transfected with psiUBP43).
Virol. Sin. (2008) 23: 339-344 343
detected in the medium by ELISA and HBV DNA
isolated from treated HepG2.2.15 cells was measured
by Southern Blot. The secretion of HBeAg, but not
HBsAg, was significantly suppressed compared to
controls (Fig. 3). and it was found that the HBV
replication was significantly inhibited compared to the
controls (Fig. 4).
In a recent study, it was found that expression of
UBP43 was disordered in the pretreatment liver tissue
of patients with chronic HCV who do not respond to
subsequent treatment with pegylated IFN-2-α and
ribavirin (3). The study also showed that silencing the
UBP43 increases the antiviral activity of IFN against
hepatitis C virus infection. So, we hypothesized that
silencing UBP43 might enhance the antiviral activity
of IFN-α to inhibit HBV replication.
The pSUPER plasmid used in this study is a good
tool for generating small hairpin RNAs (shRNAs) to
interfere with the expression of the targeted gene. First,
we constructed the recombinant shRNA plasmid
(psiUBP43) by inserting the oligonucleotides down-
stream of the H1 promoter. When we transfected
psiUBP43 into the HepG2.2.15 cells, it significantly
suppressed the expression of UBP43 compared with
negative or blank controls. We then tested the effect
of UBP43 on IFN-α biological activity. We show
that UBP43 also plays an important role in the
anti-HBV type I IFN response using HepG2.2.15 cells.
The silencing of UBP43 altered the cellular response
to IFN-α, resulting in increased inhibition of gene
expression (HBeAg) and HBV replication. The
increased biochemical effect of IFN-α was mirrored
by a potent inhibition of HBV DNA in vitro. So our
results suggest that UBP43 plays an important role in
UBP43 may have a broader role in human disease.
In animal models, UBP43 knockout mice are resistant
to otherwise fatal intracerebral infection by lym-
phocytic choriomeningitis virus and vesicular sto-
matitis virus. This effect was associated with decreased
viral replication and increased cellular ISGylation by
However, the mechanism by which UBP43
modulates the antiviral activity of IFN remains
unclear. Most scientists agree that it play an important
role in the negative regulation of IFN signaling.
Because UBP43 is itself induced by IFN-α, it appears
to be a critical regulator in a classic negative feedback
loop. UBP43 would appear to regulate a process
upstream of STAT1 phosphorylation, such as IFN-α–
receptor turnover or the interactions between the
receptor and signaling molecules. Prolonged STAT1
activation is associated with a general increase in
IFN-stimulated gene expression in UBP43-silenced
We believe that the question of how UBP43
contributes to IFN-α antiviral activity in human HBV
is a critical one. Not only is IFN-α the major
component of antiviral therapy for chronic HBV
infection, but non-response to IFN-α is an important
and unresolved issue in HBV treatment. IFN-α has
been shown to target multiple stages of viral life
cycles, notably translation, but also viral entry and
capsid assembly (7). The HBV infectious cell culture
system used in this study models the entire viral life
cycle, and therefore provides the most advanced
system for studying the antiviral activity of IFN-α
against HBV. UBP43 silencing in this system
produced a robust and consistent enhancement of the
344 Virol. Sin. (2008) 23: 339-344 Download full-text
ability of IFN-α to inhibit HBV replication.
There fore, enhancing the antiviral activity of IFN
by modulation of UBP43 may represent a new
strategy for improving responses to HBV treatment.
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