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, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virology p. 5948–5952