High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.
The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.
Torresi
J
J
Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Victoria, Australia.
Meanger
J
J
Lambert
P
P
Li
F
F
Locarnini
S A
SA
Anderson
D A
DA
D000818:Animals; D002213:Capsid / Q000235:genetics* / Q000378:metabolism*; D002415:Cats; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D016752:Hepatitis E virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D006982:Hypertonic Solutions; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011233:Precipitin Tests; D012093:Replicon; D012672:Semliki forest virus / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014775:Virus Activation; D019065:Virus Assembly
D006982:Hypertonic Solutions