title: Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.
abstract: Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
authors.forename: Deepshi
authors.initials: D
authors.lastname: Thakral
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
authors.forename: Baibaswata
authors.initials: B
authors.lastname: Nayak
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
authors.forename: Shagufta
authors.initials: S
authors.lastname: Rehman
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
authors.forename: Hemlata
authors.initials: H
authors.lastname: Durgapal
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
authors.forename: Subrat Kumar
authors.initials: SK
authors.lastname: Panda
mesh_terms: D045744:Cell Line, Tumor; D017930:Genes, Reporter / Q000235:genetics*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016752:Hepatitis E virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016366:Open Reading Frames; D012367:RNA, Viral / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins