title: Hepatitis E virus ORF2 protein over-expressed by baculovirus in hepatoma cells, efficiently encapsidates and transmits the viral RNA to naïve cells.
abstract: 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.
authors.affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8009, USA.
authors.forename: Mohammad K
authors.initials: MK
authors.lastname: Parvez
authors.forename: Robert H
authors.initials: RH
authors.lastname: Purcell
authors.forename: Suzanne U
authors.initials: SU
authors.lastname: Emerson
mesh_terms: D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D016752:Hepatitis E virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: C400971:ORF2 protein, Hepatitis E virus; D012367:RNA, Viral; D014764:Viral Proteins