Hepatitis E Virus Replication Requires an Active Ubiquitin-
Yogesh A. Karpe and Xiang-Jin Meng
Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA
46). The UPS is composed of ubiquitination and substrate-de-
grading machinery. Ubiquitination is the conjugation of proteins
tions of an E1 activating enzyme, E2 conjugation enzyme, and E3
of their host cell to effect their own survival. UPS has been impli-
cated in the infection cycle and virus-host interplay of several vi-
ruses (3, 9, 28, 36, 51). Bortezomib is an FDA-approved protea-
some inhibitor that has demonstrated clinical efficacy in the
(HEV) and evaluated the potential use of UPS inhibitors as ther-
apeutic agents against HEV infection.
HEV, a nonenveloped single-strand positive-sense RNA virus
ied human pathogen (2, 11, 31, 33). The genome of HEV is ?7.2
(ORFs) (39). The ORF1 protein possesses domains for replicase
enzymes (25) and among these, functional activities of RdRp (1),
Hel (21, 22), and MetT (29) have been experimentally verified.
ORF2 encodes the viral capsid protein (16, 50). ORF3 encodes a
pathways (5, 6, 13, 24, 26, 33–35, 43–45, 48, 49). The ORF2 and
18). The expression of the ORF3 protein is not required for virus
replication, virion assembly, or infection in vitro (12, 14).
It has been reported that proteasome inhibitors affect the rep-
lication of herpesviruses (9), vaccinia virus (36), influenza virus
(47), human immunodeficiency virus (38), and cytomegalovi-
ruses (42). Many viruses encode proteins that can modify the
host’s ubiquitin machinery, (19). Recently, a papain-like cysteine
cinoma cell line, Huh7-S10-3, which is permissive for HEV repli-
cation, was used, and the cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine
maintained under the same conditions except at 34.5°C.
he cellular ubiquitin-proteasome system (UPS) is important
for intracellular protein degradation in eukaryotic cells (40,
HEV replication, we tested the effects of proteasome inhibitors
(Invitrogen), and we showed that there was ?80% cell survival
(Fig. 1A). Thus, for all further experiments in this study, the con-
centration of inhibitors we used was 1 ?M or less.
tem (designated pSK-HEV-2RLuc) was developed previously us-
ing the genotype 1 human HEV infectious clone pSK-HEV-2 (4).
The capped RNA transcript of the pSK-HEV-2RLuc clone was
transfected into Huh7-S10-3 cells by using the DMRIE-C reagent
(Invitrogen). UPS inhibitors were added to culture medium at 24
h posttransfection. The luciferase activities were measured with a
dual luciferase reporter assay system (Promega) at 5 days post-
transfection. Firefly luciferase RNA was cotransfected with HEV
Rluc replicon RNA to normalize the Renilla luciferase signal. All
for HEV replication. The MG132 inhibitor had a more pro-
Furthermore, we found that this inhibition of HEV replication
was concentration dependent (Fig. 1C).
To investigate which specific step(s) of the HEV replication
cycle might be affected by lack of proteasome activity, we per-
formed an immunofluorescence assay (IFA) to detect viral capsid
protein synthesis, and we performed negative-strand-specific re-
verse transcription-PCR (RT-PCR) to detect replicative negative-
strand viral RNA. Briefly, Huh7 cells were transfected with the
full-length capped RNA transcripts of the pSK-HEV2 infectious
clone, and capsid protein synthesis was monitored by IFA (8).
h posttransfection, no capsid protein synthesis was detected by
active ubiquitin proteasome system.
Received 7 December 2011 Accepted 6 March 2012
Published ahead of print 21 March 2012
Address correspondence to Xiang-Jin Meng, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virology p. 5948–5952
For the detection of negative-strand replicative viral RNA, a
strand-specific anchored RT-PCR was carried out essentially as
described previously (41). RNA was reverse transcribed with a
forward primer (5=-GGGGGGGGGGGGCCGCGCCCATACTT
CGGAACGCAG-3=). As for a positive control, a negative-strand
HEV RNA was prepared by in vitro transcription of a PCR DNA
AAAGACC-3=) and a reverse primer (5=-GATCATCTCCCTA
polymerase promoter sequence, underlined). Huh7 cells were
transfected with the capped full-length RNA transcripts of the
pSK-HEV2 infectious clone, and cells were treated with 1 ?M
MG132 inhibitor at 1 day posttransfection and harvested on the
fifth day posttransfection. No negative-strand viral RNA was de-
tected when cells were treated with MG132 inhibitor (Fig. 2B),
suggesting that the proteasome activity is needed for the replica-
tion of the HEV genome, possibly by inhibition of viral transcrip-
tionortranslationorboth.We believe that inhibition of early or
multiple stages of virus replication will result in little or no
synthesis of negative-strand RNA, thus explaining our obser-
vation of the absolute negative result on the detection of the
of inhibitors. It is possible that inhibitors may impair cellular trans-
lation and thus could attribute to the inhibition of viral replication.
Therefore, we subsequently tested the effects of the inhibitor drug
in another set of experiments we expressed part of the viral capsid
ence and absence of MG132. Huh7 cells were transfected, inhibitor
treatment was the same as described above, and pAcGFP N1 and
pTrix-neo-ORF2 (with deletion of amino acids 1 to 111 [?1-111])
The concentrations of inhibitors are indicated. Mean values from three independent experiments are plotted. (B) HEV replication is reduced by treatment with
values from six independent experiments are plotted. Statistical analysis was performed using analysis of variance followed by Dunnett’s procedure, and
posttransfection, and the concentration of MG132 used is indicated. A luciferase assay was performed on the fifth day posttransfection. Mean values from three
independent experiments are plotted.
Ubiquitin-Proteasome System and HEV Replication
May 2012 Volume 86 Number 10jvi.asm.org 5949
anti-GFP rabbit polyclonal antibody (1:500), anti-HEV chimpanzee
polyclonal serum (1:200), and anti-actin goat polyclonal antibody
(1:200) (all from Santa Cruz Biotechnology) and with appropriate
secondary antibody. Comparable levels of GFP, capsid protein, and
B). Also, when the full-length RNA genome of HEV was transfected
into Huh7 cells in the presence of MG123, no capsid protein was
It has been shown that MG132 reduces the pool of free ubiq-
uitin in cells (28). MG132 inhibits budding of human parainflu-
enza virus 5 by depletion of free ubiquitin in cells by blocking the
26S proteasomal degradation of polyubiquitinated proteins (37).
Therefore, to determine whether the observed inhibition of HEV
replication by MG132 was due to depletion of free ubiquitin, we
cotransfected plasmid pRK5-HA-ubiquitin (kindly provided by
Ted Dawson [Addgene plasmid]) with capped viral RNA tran-
script, and the effect on viral replication was monitored. An in-
crease in viral replication was observed when the cells were
cotransfected with capped HEV RNA transcript with pRK5-HA-
ubiquitin compared to cells cotransfected with capped HEV RNA
transcript with the pTrix neo plasmid (Fig. 4A). Immunoblotting
protein by using chimpanzee 1313 anti-HEV immune serum. (B) Detection of HEV replication by strand-specific anchored RT-PCR. A subclone of Huh7 cells was
posttransfection. For detection of replicative negative-sense viral RNA, a strand-specific anchored RT-PCR was carried out. Lane 1, 100-bp marker; lane 2, RT-PCR
results for positive-control negative-strand RNA transcript generated by in vitro transcription; lane 3, PCR performed with positive-control negative-strand RNA
5, PCR performed with RNA isolated from full-length capped RNA transfected cells (reaction without RT); lane 6, RT-PCR performed with RNA isolated from
FIG 3 The inhibitory effect of MG132 on HEV replication does not result
from the inhibition of translation. (A) Effect of MG132 treatment on GFP
synthesis. A subclone of Huh7 cells was transfected with a similar amount of
pAcGFP N1 plasmid in six-well plates. Inhibitor treatment started 1 day post-
pAcGFP N1-transfected cells with 1 ?M MG132 treatment. (B) Effect of
MG132 treatment on ORF2 protein synthesis. A subclone of Huh7 cells were
transfected with a similar amount of pTrix-neo-ORF2 (?1–111) plasmid in
six-well plates. Inhibitor treatment started 1 day posttransfection, cells were
harvested on the fifth day posttransfection, and immunoblotting was per-
neo-ORF2 (?1–111)-transfected cells treated with 1 ?M MG132.
Karpe and Meng
jvi.asm.org Journal of Virology
was performed to detect the expression of hemagglutinin (HA)-
ubiquitin in the transfected cells by using an anti-HA tag mono-
overexpression of HA-ubiquitin and MG132 treatment, recycling
of ubiquitin molecules may be affected; in this case, the pool of
HA-ubiquitin but sufficient to restore virus replication. There-
fore, we believe that the reason that there was no difference be-
tween lanes 4 (without MG132) and 5 (with MG132) is likely due
to the overexpression of HA-ubiquitin. Viral replication was not
efficiency. Nevertheless, the results suggest that depletion of free
ubiquitin may be important for inhibition of viral replication.
In summary, in this study we demonstrated an important role
of UPS in the life cycle of HEV. Proteasome inhibitors affected
viral replication, possibly by inhibition of viral transcription or/
and translation. The results strongly suggested that an active pro-
teasome system is essential for HEV replication, and therefore
proteasome inhibitors could be useful as therapeutics against
This study was supported by grants from the U.S. National Institutes of
Health (R01 AI074667 and R01 AI050611).
We thank Laura Cordoba, Scott Kenny, Dianjun Cao, and Barbara
H. Purcell for kindly providing the chimpanzee 1313 anti-HEV antisera,
the pSK-HEV-2 infectious clone, and the Huh7-S10-3 subclone of the
hepatocellular carcinoma cell line.
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FIG 4 Overexpression of HA-ubiquitin partially restores virus replication. (A) Effect of HA-ubiquitin overexpression on HEV replication in the context of
MG132 treatment. In six-well plates, cotransfection of capped RNA transcripts of the pSK-HEV2RLuc clone and pRK-HA-Ub/pTrix-neo was carried out.
MG132 treatment started 1 day posttransfection. A luciferase assay was performed on the fifth day posttransfection. Mean relative light unit (RLU) values from
at a P level of ?0.05 (indicated with an asterisk). Data analysis was performed using JMP9 software. (B) Representative results from an immunoblot assay
performed with the anti-HA tag monoclonal antibody of the experiment shown in panel A.
Ubiquitin-Proteasome System and HEV Replication
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