Fig. 7.

Subcellular localization of double-stranded RNA. (A) Hot-phenol-extracted RNA from intracellular membrane fractions of mock-treated or JEV-infected LLC-MK2 cells was incubated with nonspecific or dsRNA-specific antibody on immunosorbent plates or left untreated (input). Antibody-bound RNA was harvested by proteinase K/SDS treatment, reextracted, and quantified by real-time RT-PCR using JEV-specific primers. The results are expressed as log₁₀ copies of viral RNA. The error bars indicate standard deviations of the means. (B and C) Membrane-specific permeabilization of LLC-MK2 cells using NP-40 to permeabilize all membranes or SLO to permeabilize only the plasma membrane. Nuclear staining was achieved using DAPI. (B) Immunodetection of IRF-3 or calregulin in NP-40- or SLO-permeabilized LLC-MK2 cells. (C) Immunodetection of dsRNA and E protein in NP-40- or SLO-permeabilized LLC-MK2 cells at various times postinfection by JEV (MOI, 1). (D) Colocalization of dsRNA and calregulin in LLC-MK2 cells mock treated or infected with JEV (MOI, 1) 24 h postinfection. Nuclear staining was achieved using DAPI.