Roles of the genomic sequence surrounding the stem-loop structure in the junction region including the 3' terminus of ORF1 in hepatitis E virus replication.

Hepatitis E virus (HEV), a member of the family Hepeviridae, causes both acute and chronic viral hepatitis. We have previously demonstrated that the stem-loop structure in the junction region (JR) of HEV genome plays a critical role in HEV replication. However, the function of the sequence bordering the JR, including the 3 terminus of open reading frame (ORF1), in HEV replication is unknown. In this study, a panel of HEV Renilla luciferase (Rluc) replicons containing various deletions at 5 or 3 termini of the JR was constructed to determine the effect of the deletions on HEV replication in Huh7 human liver cells. We showed that even a single nucleotide deletion at the 5 terminus of the JR abolished HEV replication, whereas deletions at the 3 terminus of the JR also decreased virus replication efficiency. Furthermore, we also constructed firefly luciferaseand Rluc dual-reporter HEV replicons containing the 3 terminal ORF1 of various lengths and the JR inserted upstream of the Rluc reporter. A higher level of HEV replication was observed in cells transfected with replicons containing the 3 terminal ORF1 than that of the JR only replicon. We also showed that the ORF3 noncoding sequence along with the JR promoted a higher level of translation activity than that promoted by JR and the ORF2 noncoding sequence.