The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA
Fig 2
Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone.
(A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A. (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.