Fig 1. Tunicamycin and thapsigargin promote g-1 HEV replication.

(A) QRT-PCR of wild type (WT HEV) and replication deficient (GAA HEV) HEV in Huh7 cells transfected with in vitro synthesized genome. TG: thapsigargin, TUN: tunicamycin. Values are mean±SEM. (B) Quantitation of viral ORF1 (Helicase) and ORF2 expression in Huh7 cells, transfected with wild type (WT HEV) or replication deficient (GAA HEV) HEV genomic RNA and treated with the indicated compounds. Ten random fields of approximately 30 cells in each field were counted for helicase, ORF2, DAPI (nuclear stain) fluorescence and percentage ±SEM calculated. (C) Measurement of secreted Gaussia luciferase activity in the culture medium of Huh7 cells expressing in vitro transcribed HEV genotype-3 replicon RNA and treated as indicated. Values are mean±SEM. (D) HEV genome organisation. Numbers indicate nucleotide position from 5’-end. Mutated bases are in bold. (E) ClustalW alignment of ORF4 protein sequence of indicated g-1 HEV isolates.