Hepatitis E virus replication requires an active ubiquitin-proteasome system.
ABSTRACT The mechanism of hepatitis E virus (HEV) replication remains largely unknown. Here we demonstrate that HEV replication requires an active ubiquitin-proteasome system and that proteasome inhibitors affect HEV replication, possibly by inhibition of viral transcription or/and translation without a significant effect on cellular translation. Overexpression of ubiquitin in inhibitor-treated cells partially reverses the inhibitor effect on HEV replication. The results suggest that HEV replication requires interactions with proteasome machinery, which could be a potential therapeutic target against HEV.
- [Show abstract] [Hide abstract]
ABSTRACT: Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occuring during infections of solid-organ transplant (SOT) recipients are essentially unknown. The polyproline region (PPR) of strains isolated from SOT patients were sequenced during the acute infection phase (n=59) and during follow-up of patients whose infections became chronic (n= 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48 or 88 months after infection. In one strain parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second a fragment of human tyrosine aminotransferase gene (TAT) was inserted first, followed by a fragment of PPR. A fragment of the human inter-α-trypsin inhibitor gene (ITI) was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insert promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination and phosphorylation sites. We find that recombinant events occur in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed-up in Toulouse University Hospital. These inserted fragments come from the HEV genome or a human gene and could enhance virus replication.Journal of Virology 08/2014; · 4.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is still a serious threat to the swine industry. However, the pathogenic mechanism of HP-PRRSV remains unclear. We infected host porcine alveolar macrophages (PAMs) with the virulent HuN4 strain and the attenuated HuN4-F112 strain and then utilized fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) to screen for intracellular proteins that were differentially expressed in host cells infected with the two strains. There were 153 proteins with significant different expression (P<0.01) observed, 42 of which were subjected to mass spectrometry, and 24 proteins were identified. PAM cells infected with the virulent strain showed upregulated expression of pyruvate kinase M2 (PKM2), heat shock protein beta-1 (HSPB1), and proteasome subunit alpha type 6 (PSMA6), which were downregulated in cells infected with the attenuated strain. The upregulation of PKM2 provides sufficient energy for viral replication, and the upregulation of HSPB1 inhibits host cell apoptosis and therefore facilitates mass replication of the virulent strain, while the upregulation of PSMA6 facilitates the evasion of immune surveillance by the virus. Studying on those molecules mentioned above may be able to help us to understand some unrevealed details of HP-PRRSV infection, and then help us to decrease its threat to the swine industry in the future.PLoS ONE 01/2014; 9(1):e85767. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD). It has been shown that the ubiquitin-proteasome system (UPS) is correlated with viral infection, but its role in PCV2 replication remains unknown. In the present study, we explored the interplay between PCV2 replication and the UPS in PK15 cells and found that treatment with a proteasome inhibitor (MG132 and lactacystin) significantly decreased the PCV2 titer at the early infection stage. We further revealed that inhibition of the UPS did not affect virus entry but decreased viral protein expression and RNA transcription potentially in a cell cycle-dependent manner. PCV2 infection has little effect on the chymotrypsin-like activity, and the gene-silencing of ubiquitin reduced the PCV2 titer, which indicates that the effective replication of PCV2 may be related to protein ubiquitination. Taken together, our data suggested that PCV2 replication requires the UPS machinery, which may represent a potential antiviral target against PCV2.Virology 05/2014; s 456–457:198–204. · 3.28 Impact Factor
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 Virologyp. 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 10 jvi.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.
1. Agrawal S, Gupta D, Panda SK. 2001. The 3= end of hepatitis E virus
(HEV) genome binds specifically to the viral RNA polymerase (RdRp).
2. Arankalle VA, et al. 1994. Seroepidemiology of water-borne hepatitis in
India and evidence for a third enterically-transmitted hepatitis agent.
Proc. Natl. Acad. Sci. U. S. A. 91:3428–3432.
3. Burch AD, Weller SK. 2004. Nuclear sequestration of cellular chaperone
and proteasomal machinery during herpes simplex virus type 1 infection.
J. Virol. 78:7175–7185.
4. Cao D, Huang YW, Meng XJ. 2010. The nucleotides on the stem-loop
RNA structure in the junction region of the hepatitis E virus genome are
critical for virus replication. J. Virol. 84:13040–13044.
5. Chandra V, Kar-Roy A, Kumari S, Mayor S, Jameel S. 2008. The
hepatitis E virus ORF3 protein modulates epidermal growth factor recep-
tor trafficking, STAT3 translocation, and the acute-phase response. J. Vi-
6. Chandra V, Kalia M, Hajela K, Jameel S. 2010. The ORF3 protein of
interacting with CIN85 and blocking formation of the Cbl-CIN85 com-
plex. J. Virol. 84:3857–3867.
7. Chen D, Frezza M, Schmitt S, Kanwar J, Dou QP. 2011. Bortezomib as
the first proteasome inhibitor anticancer drug: current status and future
prospective. Current Cancer Drug Target 11:23953.
8. Cordoba L, et al. 2011. Three amino acid mutations (F51L, T59A, and
to virus attenuation. J. Virol. 85:5338–5349.
9. Delboy MG, Roller DG, Nicola AV. 2008. Cellular proteasome activity
facilitates herpes simplex virus entry at a postpenetration step. J. Virol.
10. Edelmann MJ, Benjamin N, Kessler BM. 2011. Pharmacological targets
tive disorders and infectious diseases. Expert Rev. Mol. Med. 13:e35.
11. Emerson SU, Purcell RH. 2003. Hepatitis E virus. Rev. Med. Virol. 13:
12. Emerson SU, et al. 2004. In vitro replication of hepatitis E virus (HEV)
genomes and of an HEV replicon expressing green fluorescent protein. J.
13. Emerson SU, Nguyen H, Torian U, Purcell RH. 2006. ORF3 protein of
hepatitis E virus is not required for replication, virion assembly, or infec-
tion of hepatoma cells in vitro. J. Virol. 80:10457–10464.
14. Emerson SU, et al. 2010. Release of genotype 1 hepatitis E virus from
frame 3 protein and requires an intact PXXP motif. J. Virol. 84:9059–
15. Graff J, Torian U, Nguyen H, Emerson SU. 2006. A bicistronic sub-
virus. J. Virol. 80:5919–5926.
16. Graff J, et al. 2008. Mutations within potential glycosylation sites in the
particles. J. Virol. 82:1185–1194.
17. Hershko A, Ciechanover A. 1998. The ubiquitin system. Annu. Rev.
18. Ichiyama K, et al. 2009. Determination of the 5=-terminal sequence of
19. Isaacson MK, Ploegh HL. 2009. Ubiquitination, ubiquitin-like modifiers,
and deubiquitination in viral infection. Cell Host Microbe 5:559–570.
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
May 2012 Volume 86 Number 10 jvi.asm.org 5951
20. Kabrane-Lazizi Y, Meng XJ, Purcell RH, Emerson SU. 1999. Evidence
that the genomic RNA of hepatitis E virus is capped. J. Virol. 73:8848–
21. Karpe YA, Lole KS. 2010. NTPase and 5= to 3= RNA duplex-unwinding
activities of hepatitis E virus helicase domain. J. Virol. 84:3595–3602.
22. Karpe YA, Lole KS. 2010. RNA 5= triphosphatase activity associated with
hepatitis E virus helicase domain. J. Virol. 84:9637–9641.
23. Karpe YA, Lole KS. 2011. Deubiquitinating activity associated with hep-
24. Kar-Roy A, Korkaya H, Oberoi R, Lal SK, Jameel S. 2004. The hepatitis
E virus open reading frame 3 protein activates ERK through binding and
inhibition of the MAPK phosphatase. J. Biol. Chem. 279:28345–28357.
25. Koonin EV, et al. 1992. Computer-assisted assignment of functional
of an additional group of positive-strand RNA plant and animal viruses.
Proc. Natl. Acad. Sci. U. S. A. 89:8259–8263.
26. Korkaya H, et al. 2001. The ORF3 protein of hepatitis E virus binds to Src
homology 3 domains and activates MAPK. J. Biol. Chem. 276:42389–
27. Kouroukis CT, et al. 2011. A phase II study of bortezomib and gemeit-
abine in relapsed mantle cell lymphoma from the National Cancer Insti-
tute of Canada clinical trial group (IND 172). Leuk. Lymphoma 52:394–
28. Lopez T, Silvia-Ayala D, Lopez S, Arias CA. 2011. Replication of the
rotavirus genome requires an active ubiquitin-proteasome system. J. Vi-
29. Magden J, et al. 2001. Virus-specific mRNA capping enzyme encoded by
hepatitis E virus. J. Virol. 75:6249–6255.
30. Meng XJ. 2010. Recent advances in hepatitis E virus. J. Viral Hepat. 17:
31. Meng XJ. 2011. From barnyard to food table: the omnipresence of hepa-
titis E virus and risk for zoonotic infection and food safety. Virus Res.
32. Meng XJ, et al. 2011. Hepeviridae, p 1021–1028. In King AMQ, Adams
MJ, Carstens EB, Lefkowitz EJ (ed), Virus taxonomy, 9th report of the
International Committee on Taxonomy of Viruses. Elsevier Academic
Press, London, England.
34. Moin SM, Chandra V, Arya R, Jameel S. 2009. The hepatitis E virus
tional activity through p300/CBP. Cell. Microbiol. 11:1409–1421.
35. Ratra R, Kar-Roy A, Lal SK. 2008. The ORF3 protein of hepatitis E virus
interacts with hemopexin by means of its 26 amino acid N-terminal hy-
drophobic domain II. Biochemistry 47:1957–1969.
36. Satheshkumar PS, Anton LC, Sanz P, Moss B. 2009. Inhibition of the
ubiquitin-proteasome system prevents vaccinia virus DNA replication
and expression of intermediate and late genes. J. Virol. 83:2469–2479.
37. Schmitt AP, Leser GP, Morita E, Sundquist WI, Lamb RA. 2005.
Evidence for a new viral late-domain core sequence, FPVI, necessary for
budding of a paramyxovirus. J. Virol. 79:2988–2997.
38. Schubert U, et al. 2000. Proteasome inhibition interferes with gag poly-
protein processing, release, and maturation of HIV-1 and HIV-2. Proc.
Natl. Acad. Sci. U. S. A. 97:13057–13062.
39. Tam AW, et al. 1991. Hepatitis E virus (HEV): molecular cloning and
sequencing of the full-length viral genome. Virology 185:120–131.
40. Tanahashi N, Kawahara H, Murakami Y, Tanaka K. 1999. The protea-
some-dependent proteolytic system. Mol. Biol. Rep. 26:3–9.
41. Thakral D, Nayak B, Rahman S, Durgapal H, Panda SK. 2005. Repli-
and generation of short-term cell line producing viral RNA and proteins.
J. Gen. Virol. 86:1189–1200.
42. Tran K, Mahr JA, Spector DH. 2010. Proteasome subunits relocalize
essary for efficient viral gene transcription. J. Virol. 84:3079–3093.
43. Tyagi S, Korkaya H, Zafrullah M, Jameel S, Lal SK. 2002. The phos-
phorylated form of the ORF3 protein of hepatitis E virus interacts with its
non-glycosylated form of the major capsid protein, ORF2. J. Biol. Chem.
44. Tyagi S, Surjit M, Roy AK, Jameel S, Lal SK. 2004. The ORF3 protein of
cursor ?1-microglobulin/bikunin precursor (AMBP) and expedites their
export from the hepatocyte. J. Biol. Chem. 279:29308–29319.
45. Tyagi S, Surjit M, Lal SK. 2005. The 41-amino-acid C-terminal region of
the hepatitis E virus ORF3 protein interacts with bikunin, a kunitz-type
serine protease inhibitor. J. Virol. 79:12081–12087.
46. Voges D, Zwickl P, Baumeister W. 1999. The 26S proteasome: a molec-
ular machine designed for controlled proteolysis. Annu. Rev. Biochem.
47. Widjaja I, et al. 2010. Inhibition of the ubiquitin proteasome system
affects influenza A virus infection at a postfusion step. J. Virol. 84:9625–
48. Yamada K, et al. 2009. ORF3 protein of hepatitis E virus is essential for
virion release from infected cells. J. Gen. Virol. 90:1880–1891.
49. Zafrullah M, Ozdener MH, Panda SK, Jameel S. 1997. The ORF3 protein
of hepatitis E virus is a phosphoprotein that associates with the cytoskel-
eton. J. Virol. 71:9045–9053.
50. Zafrullah M, Ozdener MH, Kumar R, Panda SK, Jameel S. 1999.
Mutational analysis of glycosylation, membrane translocation, and cell
51. Zhang Z, et al. 2000. Structural and functional characterization of inter-
Biol. Chem. 275:15157–15165.
Karpe and Meng
jvi.asm.org Journal of Virology