| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi-Ken 329-0498, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Present address: Division of Infectious Diseases, Department of Social and Environmental Medicine, Institute of Scientific Research, Oita University, Oita 879-5593, Japan. 
The nucleotide sequences reported in this study have been assigned DDBJ/EMBL/GenBank accession numbers AB437316 (pJE03-1760F/wt) and AB437319 (pJE03-1760F/
ORF1).
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). HEV is classified as the sole member of the genus Hepevirus (Emerson et al., 2004a) in the family Hepeviridae. HEV is a non-enveloped virus and its genome is a single-stranded, positive-sense RNA, which is capped and polyadenylated (Kabrane-Lazizi et al., 1999; Tam et al., 1991). The genome is approximately 7.2 kb and contains three open reading frames (ORFs), ORF1, ORF2 and ORF3 (Tam et al., 1991). The ORF1 encodes non-structural proteins including RNA helicase and RNA-dependent RNA polymerase (Agrawal et al., 2001; Koonin et al., 1992; Magden et al., 2001). ORF2 and ORF3 proteins are translated from a single subgenomic RNA (Graff et al., 2006; Huang et al., 2007). The ORF2 protein is the viral capsid protein, consisting of 660 aa. The ORF3 protein is a small protein (113–114 aa) that is essential for viral infectivity in animals (Graff et al., 2005; Huang et al., 2007). However, the replication mechanism and protein functions of HEV are not fully understood.
Propagation of HEV in vitro has been attempted in various cell lines (Divizia et al., 1999; Huang et al., 1992; Kazachkov et al., 1992; Meng et al., 1997; Wei et al., 2000), but an efficient cell culture system has not been developed. Several research groups have established infectious cDNA clones of HEV, which were observed to replicate in non-human primates or pigs (Emerson et al., 2001, 2004b; Graff et al., 2005; Huang et al., 2005, 2007; Panda et al., 2000). However, efficient HEV propagation in cultured cells has not yet been observed in any of these infectious cDNA clone systems, due to the inability of the virus progeny to spread to other cells in culture (Emerson et al., 2004b). Recently, we developed an efficient cell-culture system for HEV in PLC/PRF/5 and A549 cells using a genotype 3 HEV (strain JE03-1760F) that had been obtained from a faecal specimen from a Japanese hepatitis E patient (Lorenzo et al., 2008; Takahashi et al., 2007; Tanaka et al., 2007).
To develop a full-length infectious cDNA clone of HEV, RNA was extracted from the faecal specimen containing the JE03-1760F strain (Tanaka et al., 2007) using TRIzol LS (Invitrogen) and cDNA was synthesized using SuperScriptII (Invitrogen) with primers ENDr and 4927r (Supplementary Table S1, available in JGV Online). Using the synthesized cDNA as template, three fragments covering the entire JE03-1760F genome were amplified by PCR with KOD plus ver. 2 (Toyobo) (Fig. 1). Fragment 1 (f1), which contains the T7 RNA polymerase promoter sequence upstream of the extreme 5'-end of the JE03-1760F genome, was amplified with primers f1-f and f1-r (Supplementary Table S1). The f2 fragment was amplified with primers f2-f and f2-r, and the f3 fragment with primers f3-f and f3-r; an AflII site was introduced as a genetic marker at the 3' or 5' end of the f2 and f3 fragments, respectively, by replacing T with C at nt 4784 and G with T at nt 4786, without amino acid substitution (Fig. 1). Fragment polyAT7
, consisting of 31 nt of adenine and a T7 terminator sequence, was amplified by PCR with primers polyAT7-f and polyAT7-r using pTnT vector (Promega) as a template. An A overhang was added to the amplified blunt-ended fragments using A-Addition kit (Qiagen) and these were cloned into pT7 Blue T vector (Novagen). After confirming the sequence of the cloned fragments, the four fragments were ligated stepwise at NotI, AflII and SapI sites, and inserted into the HindIII–BamHI site of the pUC19AatIISapI vector (kindly provided by Dr N. Ito, Gifu University, Japan). The full-length cDNA of the JE03-1760F strain that was constructed was designated pJE03-1760F/wt. In addition, a recombinant plasmid was constructed as a negative control. To achieve this, pJE03-1760F/wt plasmid DNA was digested with AatII, followed by blunting with T4 DNA polymerase (TaKaRa Bio) and self-ligation, resulting in the formation of a frameshift mutant of ORF1, pJE03-1760F/ORF1 (Fig. 1).
|
), in a well of six-well plates (Iwaki) in duplicate, using the TransIT-mRNA Transfection kit (Mirus Bio); the transfected cells were incubated at 37 °C. At 2 days post-transfection (p.t.), the culture medium was replaced with 2 ml growth medium and incubated at 35.5 °C. Then, every other day, half of the culture medium (1 ml) was replaced with fresh maintenance medium consisting of 50 % DMEM and 50 % medium 199 (Invitrogen) containing 2 % FCS, 30 mM MgCl2, 100 U penicillin G ml–1, 100 µg streptomycin ml–1 and 2.5 µg amphotericin B ml–1. The collected medium was centrifuged at 800 g at 4 °C for 5 min and the supernatant was stored at –80 °C until use.
To monitor virus production in transfected cells (two wells each), HEV RNA levels in the culture supernatants of the transfected cells were serially quantified by real-time RT-PCR using the QuantiTect Probe RT-PCR kit (Qiagen) in a LightCycler apparatus (Roche Diagnostics) as described previously (Takahashi et al., 2008a). The HEV RNA titre decreased to approximately 106 copies ml–1 in all samples tested at 4 days p.t., due to the medium change on day 2 p.t. (Fig. 2a). Thereafter, a gradual decrease in HEV load was observed in the culture medium of the ORF1 mutant RNA-transfected cells, whose titre probably reflects residual amounts of the introduced RNA transcripts in the culture medium. On the other hand, in the pJE03-1760F/wt RNA-transfected cells, the viral RNA levels started to increase at 6 days p.t. and reached greater than 107 copies ml–1 at 28 days p.t. The viral RNA load in the medium was maintained in the order of 107 copies ml–1 in the pJE03-1760F/wt RNA transfection through to 60 days p.t.
|
). Briefly, on days 5, 7, 11 and 15 p.t., pJE03-1760F/wt RNA-transfected PLC/PRF/5 cells were fixed with 4 % paraformaldehyde, permeabilized with 0.2 % Triton X-100, incubated with a mouse monoclonal antibody (mAb) against the ORF2 protein (H6225) (Takahashi et al., 2008a) and stained with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen). ORF2 protein expression was detectable in the transfected cells at 5 days p.t., and increasing levels of ORF2 antigens on days 7, 11 and 15 p.t. suggested the spread of HEV infection in cell culture (Fig. 2b
).
For detection of ORF2 protein in the culture supernatant, Western blot analysis was performed using the 60 days p.t. samples. The culture supernatant of HEV RNA-transfected cells was directly mixed with the same amount of 2x SDS-PAGE sample buffer [125 mM Tris/HCl (pH 6.8), 4 % SDS, 10 % (w/v) sucrose, 10 % (v/v) 2-mercaptoethanol and 0.01 % (w/v) bromophenol blue]. Proteins in the samples were separated by SDS-PAGE on an 8 % polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane (0.45 µm; Millipore). The membrane was incubated with anti-HEV ORF2 mAb (H6210) conjugated with horseradish peroxidase (HRP) (Takahashi et al., 2008a) and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). As shown in Fig. 2(c), a single 83 kDa band was detected in the culture medium of the pJE03-1760F/wt RNA transfection (lane 1) but not in that of the ORF1 mutant RNA transfection (lane 2). In addition, cell lysates prepared after DNA or RNA transfection were subjected to detection of ORF2 protein by Western blotting with H6210 mAb and enhanced chemiluminescence HRP-conjugated anti-mouse IgG from sheep (GE Healthcare). For preparation of cell lysates, DNA- or RNA-transfected cells on six-well plates were lysed in 100 µl lysis buffer [50 mM Tris/HCl (pH 8.0), 1 % NP-40 and 150 mM NaCl] and the lysate was mixed with the same amount of 2x SDS-PAGE sample buffer. The 83 kDa single band was also detected in the lysate of the pJE03-1760F/wt RNA-transfected cells (lane 5) but not in the lysate of ORF1 mutant RNA-transfected cells (lane 6) or mock-transfected cells (lane 4) (Fig. 2c). For comparison, an expression plasmid, pCI-HEVORF2, for the ORF2 protein of strain JE03-1760F was constructed in the following way. The full-length ORF2 sequence of the JE03-1760F genome was amplified with primers ORF2-f and ORF2-r (Supplementary Table S1). The amplified DNA was cloned into the NheI–XbaI site of pCI vector (Promega), and the resulting plasmid, pCI-HEVORF2, was transfected into subconfluent PLC/PRF/5 cells in wells of a six-well plate using TransIT-LT1 Transfection reagent (Mirus Bio). The transfected cells were incubated at 37 °C for 3 days and then analysed by Western blotting. Interestingly, the molecular mass of the ORF2 protein expressed by the pCI expression plasmid (Fig. 2c, lane 3) was slightly less than that detected in the pJE03-1760F/wt RNA-transfected cells.
The genomic region containing the AflII site (Fig. 1) was amplified by RT-PCR using primer 5811r for cDNA synthesis and primers 4511f and 5522r for PCR (Supplementary Table S1). In contrast with the original JE03-1760F strain in faeces, the amplified fragment from the culture supernatant of pJE03-1760F/wt RNA transfection was digestable with AflII (Fig. 2d), confirming that the propagated cDNA-derived virus (pJE03-1760F/wt) possessed the introduced genetic marker.
The pJE03-1760F/wt and original JE03-1760F strains were inoculated into PLC/PRF/5 or A549 cells (ATCC no. CCL-185), which were grown in maintenance medium on six-well plates at 1.0x105 or 1.0x104 copies per well, respectively. Virus inoculation and maintenance of inoculated cells were carried out as described previously (Tanaka et al., 2007). Fig. 3 indicates that the cDNA-derived pJE03-1760F/wt grew as efficiently as the original faeces-derived virus in both PLC/PRF/5 and A549 cells, reaching 106 or 105 copies ml–1 at 30 days post-inoculation, respectively.
|
), suggesting that strain JE03-1760F has heightened replication activity. Therefore, the use of the JE03-1760F genome in the present study may have been the greatest factor contributing to the successful development of an infectious HEV cDNA clone with robust infection and propagation in vitro. The JE03-1760F genome has 29 unique nucleotide substitutions that were not seen in any of the 25 reported HEV isolates of the same genotype (Takahashi et al., 2007). One or some of these substitutions may be responsible for the high replication capability of HEV.
Interestingly, the ORF2 protein was detectable as a single band of 83 kDa following Western blot analysis of the supernatant and lysate of the pJE03-1760F/wt RNA-transfected cells, and the molecular mass was slightly greater than that of the protein transcribed by the pCI-HEVORF2 plasmid vector (Fig. 2c). In the reported transient overexpression systems, both glycosylated and non-glycosylated forms of ORF2 proteins have been detected. Jameel et al. (1996) reported that the ORF2 protein was detected in three molecular forms, including a 74 kDa non-glycosylated form and 82 and 88 kDa glycosylated forms. It was reported that the glycosylated form of ORF2 protein gradually shifted to the non-glycosylated form in the cytoplasm through the retrotranslocation pathway (Surjit et al., 2007) and that only the non-glycosylated form was stable in the cytoplasm of mammalian cells (Torresi et al., 1999). However, the 83 kDa ORF2 protein detected in the culture supernatants and lysates of pJE03-1760F/wt RNA-transfected cells (Fig. 2c) and cells infected with cell culture-generated HEV (data not shown) is likely to be glycosylated. In support of our speculation that the glycosylated form of ORF2 protein has a role in the HEV replication cycle, Graff et al. (2008) recently reported that the formation of infectious virus particles was prevented by mutations within the potential glycosylation sites in the ORF2 protein. In addition, it was shown that the recombinant replicase domain of HEV ORF1 protein is localized on the endoplasmic reticulum membrane (Rehman et al., 2008). HEV particles in the culture supernatant banded at a low buoyant density of 1.15–1.16 g ml–1 in sucrose, suggesting that HEV virions released from infected cells associate with lipids (Takahashi et al., 2008b). Therefore, the ORF2 capsid protein existing as a glycoprotein may be advantageous for virion assembly if cellular membranes play an important role in HEV particle maturation.
In conclusion, we established a reverse genetics system for HEV that is utilizable in a robust cell-culture system. This system will be useful for further elucidation of the mechanism of HEV replication and the functional roles of HEV proteins.
|
ACKNOWLEDGEMENTS |
|---|
AatIISapI. This study was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from the Ministry of Health, Labour, and Welfare of Japan, and from the Research Award in Jichi Medical University (to K. Y.). |
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Divizia, M., Gabrieli, R., Degener, A. M., Renganathan, E., Pillot, J., Stefanoni, M. L., el Ghazzawi, E., Kader, O. A., Gamil, F. & other authors (1999). Evidence of hepatitis E virus replication on cell cultures. New Microbiol 22, 77–83.[Medline]
Emerson, S. U., Zhang, M., Meng, X. J., Nguyen, H., St Claire, M., Govindarajan, S., Huang, Y. K. & Purcell, R. H. (2001). Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element. Proc Natl Acad Sci U S A 98, 15270–15275.
Emerson, S. U., Anderson, D., Arankalle, A., Meng, X. J., Purdy, M., Schlauder, G. G. & Tsarev, S. A. (2004a). Hepevirus. In Virus Taxonomy, VIIIth Report of the International Committee on Taxonomy of Viruses, pp. 853–857. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego, CA: Elsevier.
Emerson, S. U., Nguyen, H., Graff, J., Stephany, D. A., Brockington, A. & Purcell, R. H. (2004b). In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 78, 4838–4846.
Graff, J., Nguyen, H., Yu, C., Elkins, W. R., St Claire, M., Purcell, R. H. & Emerson, S. U. (2005). The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques. J Virol 79, 6680–6689.
Graff, J., Torian, U., Nguyen, H. & Emerson, S. U. (2006). A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. J Virol 80, 5919–5926.
Graff, J., Zhou, Y. H., Torian, U., Nguyen, H., St Claire, M., Yu, C., Purcell, R. H. & Emerson, S. U. (2008). Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles. J Virol 82, 1185–1194.
Huang, R. T., Li, D. R., Wei, J., Huang, X. R., Yuan, X. T. & Tian, X. (1992). Isolation and identification of hepatitis E virus in Xinjiang, China. J Gen Virol 73, 1143–1148.
Huang, Y. W., Haqshenas, G., Kasorndorkbua, C., Halbur, P. G., Emerson, S. U. & Meng, X. J. (2005). Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs. J Virol 79, 1552–1558.
Huang, Y. W., Opriessnig, T., Halbur, P. G. & Meng, X. J. (2007). Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 81, 3018–3026.
Jameel, S., Zafrullah, M., Ozdener, M. H. & Panda, S. K. (1996). Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 70, 207–216.[Abstract]
Kabrane-Lazizi, Y., Meng, X. J., Purcell, R. H. & Emerson, S. U. (1999). Evidence that the genomic RNA of hepatitis E virus is capped. J Virol 73, 8848–8850.
Kazachkov, Yu. A., Balayan, M. S., Ivannikova, T. A., Panina, L. I., Orlova, T. M., Zamyatina, N. A. & Kusov, Yu. (1992). Hepatitis E virus in cultivated cells. Arch Virol 127, 399–402.[CrossRef][Medline]
Koonin, E. V., Gorbalenya, A. E., Purdy, M. A., Rozanov, M. N., Reyes, G. R. & Bradley, D. W. (1992). Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. Proc Natl Acad Sci U S A 89, 8259–8263.
Lorenzo, F. R., Tanaka, T., Takahashi, H., Ichiyama, K., Hoshino, Y., Yamada, K., Inoue, J., Takahashi, M. & Okamoto, H. (2008). Mutational events during the primary propagation and consecutive passages of hepatitis E virus strain JE03-1760F in cell culture. Virus Res 137, 86–96.[Medline]
Magden, J., Takeda, N., Li, T., Auvinen, P., Ahola, T., Miyamura, T., Merits, A. & Kaariainen, L. (2001). Virus-specific mRNA capping enzyme encoded by hepatitis E virus. J Virol 75, 6249–6255.
Meng, J., Dubreuil, P. & Pillot, J. (1997). A new PCR-based seroneutralization assay in cell culture for diagnosis of hepatitis E. J Clin Microbiol 35, 1373–1377.[Abstract]
Okamoto, H. (2007). Genetic variability and evolution of hepatitis E virus. Virus Res 127, 216–228.[CrossRef][Medline]
Panda, S. K., Ansari, I. H., Durgapal, H., Agrawal, S. & Jameel, S. (2000). The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. J Virol 74, 2430–2437.
Rehman, S., Kapur, N., Durgapal, H. & Panda, S. K. (2008). Subcellular localization of hepatitis E virus (HEV) replicase. Virology 370, 77–92.
Surjit, M., Jameel, S. & Lal, S. K. (2007). Cytoplasmic localization of the ORF2 protein of hepatitis E virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum. J Virol 81, 3339–3345.
Takahashi, M., Tanaka, T., Azuma, M., Kusano, E., Aikawa, T., Shibayama, T., Yazaki, Y., Mizuo, H., Inoue, J. & Okamoto, H. (2007). Prolonged fecal shedding of hepatitis E virus (HEV) during sporadic acute hepatitis E: evaluation of infectivity of HEV in fecal specimens in a cell culture system. J Clin Microbiol 45, 3671–3679.
Takahashi, M., Hoshino, Y., Tanaka, T., Takahashi, H., Nishizawa, T. & Okamoto, H. (2008a). Production of monoclonal antibodies against hepatitis E virus capsid protein and evaluation of their neutralizing activity in a cell culture system. Arch Virol 153, 657–666.[Medline]
Takahashi, M., Yamada, K., Hoshino, Y., Takahashi, H., Ichiyama, K., Tanaka, T. & Okamoto, H. (2008b). Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces. Arch Virol 153, 1703–1713.
Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C. C., Bradley, D. W., Fry, K. E. & Reyes, G. R. (1991). Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185, 120–131.[CrossRef][Medline]
Tanaka, T., Takahashi, M., Kusano, E. & Okamoto, H. (2007). Development and evaluation of an efficient cell-culture system for Hepatitis E virus. J Gen Virol 88, 903–911.
Torresi, J., Li, F., Locarnini, S. A. & Anderson, D. A. (1999). Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. J Gen Virol 80, 1185–1188.[Abstract]
Wei, S., Walsh, P., Huang, R. & To, S. S. (2000). 93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. J Med Virol 61, 311–318.[CrossRef][Medline]
Received 25 September 2008;
accepted 27 October 2008.
This article has been cited by other articles:
|
|
 |
|
  T. Tanaka, M. Takahashi, H. Takahashi, K. Ichiyama, Y. Hoshino, S. Nagashima, H. Mizuo, and H. Okamoto Development and Characterization of a Genotype 4 Hepatitis E Virus Cell Culture System Using a HE-JF5/15F Strain Recovered from a Fulminant Hepatitis Patient J. Clin. Microbiol., June 1, 2009; 47(6): 1906 - 1910. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
