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.
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.
Thakral
Deepshi
D
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
Nayak
Baibaswata
B
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
Rehman
Shagufta
S
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
Durgapal
Hemlata
H
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
Panda
Subrat Kumar
SK
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India.
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
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins