Hepatitis E virus ORF2 protein over-expressed by baculovirus in hepatoma cells, efficiently encapsidates and transmits the viral RNA to naïve cells
© Parvez et al; licensee BioMed Central Ltd. 2011
Received: 10 March 2011
Accepted: 8 April 2011
Published: 8 April 2011
A recombinant baculovirus(vBacORF2) that expressed the full-length ORF2 capsid protein of a genotype 1 strain of hepatitis E virus(HEV) was constructed. Transduction of S10-3 human hepatoma cells with this baculovirus led to large amounts of ORF2 protein production in ~50% of the cells as determined by immune fluorescence microscopy. The majority of the ORF2 protein detected by Western blot was 72 kDa, the size expected for the full-length protein. To determine if the exogenously-supplied ORF2 protein could transencapsidate viral genomes, S10-3 cell cultures that had been transfected the previous day with an HEV replicon of genotype 1 that contained the gene for green fluorescent protein(GFP), in place of that for ORF2 protein, were transduced with the vBacORF2 virus. Cell lysates were prepared 5 days later and tested for the ability to deliver the GFP gene to HepG2/C3A cells, another human hepatoma cell line. FACS analysis indicated that lysates from cell cultures receiving only the GFP replicon were incapable of introducing the replicon into the HepG2/C3A cells whereas ~2% of the HepG2/C3A cells that received lysate from cultures that had received both the replicon and the baculovirus produced GFP. Therefore, the baculovirus-expressed ORF2 protein was able to trans-encapsidate the viral replicon and form a particle that could infect naïve HepG2/C3A cells. This ex vivo RNA packaging system should be useful for studying many aspects of HEV molecular biology.
Hepatitis E virus (HEV) causes acute hepatitis which has an overall fatality rate of about 2%[1, 2]: however, in developing countries, hepatitis E mortality rates may approach 20% in pregnant women[3, 4]. HEV, currently the only member of the family Hepeviridae, is classified into 4 genotypes. Genotypes 1 and 2 infect only humans and non-human primates whereas genotypes 3 and 4 are zoonotic and infect swine and some other mammals in addition to humans. The HEV genome is a linear, single-stranded, positive sense RNA of ~7.2 kb. It contains 3 open reading frames (ORFs)[6, 7]. ORF1 encodes a non-structural polyprotein essential for virus replication. ORF3 codes for a very small protein which has putative regulatory functions and which is required for release of virus from infected cells. ORF2 encodes the viral capsid protein; although the full-length capsid protein consists of 660 amino acids, the apparent susceptibility of ORF2 protein to proteolytic cleavage means the size of the protein in virions is not known. The size of ORF2 protein varies when over-expressed in insect or mammalian cells and ORF2 products of 52-84 kDa have been reported[10–13]. A truncated ORF2 protein(53 kDa) expressed in insect cells was shown to assemble into empty capsids with a T1 symmetry and an almost full-length ORF2 protein produced in insect cells was found to assemble into virus particles with T3 symmetry; these particles captured some of the ORF2-encoding mRNA. However, trans-encapsidation of infectious virion RNA by over-expressed recombinant ORF2 protein has not been reported. HEV has been difficult to grow in cell culture and although recent advances in the culturing of genotypes 3 and 4 have occurred[15, 16], each virus isolate requires adaptation by lengthy passage in cell culture to grow efficiently. Additionally, comparable systems for genotypes 1 and 2 have yet to be developed. Therefore, it would be useful to have a means of producing infectious virions of HEV by a process that did not require lengthy adaptation to cell culture
Our results are consistent with the recent report by Xing et al. that HEV virus-like particles formed in insect cells captured some of the template ORF2 RNA used to produce the particles. Whether this capture was fortuitous, specific, or efficient is unclear. In our case, although we were able to infect only 2% of the HepG2/C3A cells, this represented a reasonably efficient packaging of replicon RNA; encapsidation absolutely required co-expression of adequate levels of ORF2 protein and replicon RNA and only 16% of the S10-3 cells contained a functional replicon(Figure 3C) and an estimated 50% contained ORF2 protein. It should be possible to improve the system by further optimizing transfection or transduction parameters but in the meantime our results provide proof-of-principal for trans-encapsidation of HEV genomes by ORF2 and confirm the previous reports that ORF3 protein is not required for generation of infectious virions[18, 21]. This trans-encapsidation system should be useful for providing substrates for analysis of neutralizing antibodies or for determining parameters that are necessary for encapsidation of viral RNA, such as packaging signals in the RNA or critical regions or residues in the ORF2 protein.
This is the first demonstration that the HEV full-length ORF2 protein is efficiently expressed by baculovirus-transduced hepatoma cells. The ORF2 protein trans-complements a replicon that is deficient in capsid protein production and efficiently encapsidates the replicon viral RNA to form stable HEV particles which are infectious for naïve hepatoma cells. This ex vivo RNA packaging-system could be further used to study many aspects of HEV molecular biology.
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. The technical support of Kristina Faulk in flow cytometry and Danielle Burke in Western blot is acknowledged.
- Purcell RH: Hepatitis viruses: changing patterns of human disease. Proc Natl Acad Sci USA 1994, 91: 2401-2406. 10.1073/pnas.91.7.2401PubMed CentralView ArticlePubMedGoogle Scholar
- Aggarwal R, Naik S: Epidemiology of hepatitis E: current status. J Gastroenterol Hepatol 2009, 24: 1484-1493. 10.1111/j.1440-1746.2009.05933.xView ArticlePubMedGoogle Scholar
- Khuroo MS, Khuroo MS: Hepatitis E virus. Curr Opin Infect Dis 2008, 21: 539-543. 10.1097/QCO.0b013e32830ee08aView ArticlePubMedGoogle Scholar
- Navaneethan U, Al Mohajer M, Shata MT: Hepatitis E and pregnancy: understanding the pathogenesis. Liver Int 2008, 28: 1190-1199. 10.1111/j.1478-3231.2008.01840.xPubMed CentralView ArticlePubMedGoogle Scholar
- Meng XJ: Recent advances in hepatitis E virus. J Viral Hepat 2010, 17: 153-61. 10.1111/j.1365-2893.2009.01257.xView ArticlePubMedGoogle Scholar
- Reyes GR, Purdy MA, Kim JP, Luk KC, Young LM, Fry KE, Bradley DW: Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 1990, 247: 1335-1339. 10.1126/science.2107574View ArticlePubMedGoogle Scholar
- Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, Reyes GR: Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virol 1991, 185: 120-131. 10.1016/0042-6822(91)90760-9View ArticleGoogle Scholar
- Chandra V, Taneja S, Kalia M, Jameel S: Molecular biology and pathogenesis of hepatitis E virus. J Biosci 2008,33(4):451-464. 10.1007/s12038-008-0064-1View ArticlePubMedGoogle Scholar
- Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H: ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 2009, 90: 1880-1891. 10.1099/vir.0.010561-0View ArticlePubMedGoogle Scholar
- Zhang Y, McAtee P, Yarbough PO, Tam AW, Fuerst T: Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells. Clin Diagn Lab Immunol 1997, 4: 423-428.PubMed CentralPubMedGoogle Scholar
- Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JC, Engvall H, Hammar L, Xing L, Cheng RH: Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus. J Virol 2005, 79: 12999-3006. 10.1128/JVI.79.20.12999-13006.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Robinson RA, Burgess WH, Emerson SU, Leibowitz RS, Sosnovtseva SA, Tsarev S, Purcell RH: Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expr Purif 1998, 12: 75-84. 10.1006/prep.1997.0817View ArticlePubMedGoogle Scholar
- Jameel S, Zafrullah M, Ozdener MH, Panda SK: Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 1996, 70: 207-216.PubMed CentralPubMedGoogle Scholar
- Xing L, Li TC, Mayazaki N, Simon MN, Wall JS, Moore M, Wang CY, Takeda N, Wakita T, Miyamura T, Cheng RH: Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway. J Biol Chem 2010, 285: 33175-33183. 10.1074/jbc.M110.106336PubMed CentralView ArticlePubMedGoogle Scholar
- Lorenzo FR, Tanaka T, Takahashi H, Ichiyama K, Hoshino Y, Yamada K, Inoue J, Takahashi M, Okamoto H: Mutational events during the primary propagation and consecutive passages of hepatitis E virus strain JE03-1760F in cell culture. Virus Res 2008, 137: 86-96. 10.1016/j.virusres.2008.06.005View ArticlePubMedGoogle Scholar
- Tanaka T, Takahashi M, Takahashi H, Ichiyama K, Hoshino Y, Nagashima S, Mizuo H, Okamoto H: 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 2009, 47: 1906-1910. 10.1128/JCM.00629-09PubMed CentralView ArticlePubMedGoogle Scholar
- Emerson SU, Nguyen H, Graff J, Stephany DN, Brockington A, Purcell RH: In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 2004 78: 4838-4846. 10.1128/JVI.78.9.4838-4846.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Emerson SU, Nguyen H, Torian U, Purcell RH: ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro. J Virol 2006, 80: 10457-10464. 10.1128/JVI.00892-06PubMed CentralView ArticlePubMedGoogle Scholar
- Shukla P, Nguyen HT, Torian U, Engle RE, Faulk K, Dalton HR, Bendall RP, Keane FE, Purcell RH, Emerson SU: Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus-host recombinant. Proc Natl Acad Sci USA 2011, 108: 2438-2443. 10.1073/pnas.1018878108PubMed CentralView ArticlePubMedGoogle Scholar
- Parvez MK, Sehgal D, Sarin SK, Basir FB, Jameel S: Inhibition of hepatitis B virus DNA replication intermediate forms by recombinant IFN-γ. World J Gastroenterol 2006, 12: 3006-3014.PubMed CentralPubMedGoogle Scholar
- Emerson SU, Nguyen HT, Torian U, Burke D, Engle R, Purcell RH: Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif. J Virol 2010, 84: 9059-9069. 10.1128/JVI.00593-10PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.