A specific EMC subunit supports Dengue virus infection by promoting virus membrane fusion essential for cytosolic genome delivery
Fig 3
EMC4 supports cytosol escape of the DENV genome.
A. Huh 7.5.1 cells were transfected with the indicated siRNAs followed by infection with luc-DENV2 (MOI 0.1) for 48 h, fixed, stained with the indicated antibody, and imaged under widefield epifluorescence microscope. Zoomed-in images of the white box regions are also shown. B. Quantification of dsRNA signal/cell from A. C. Huh 7.5.1 cells were transfected with the indicated siRNAs followed by infection with luc-DENV2 (MOI 0.05) for 48 h and the total RNA were extracted followed by RT-qPCR to monitor viral RNA level (normalized to scrambled). D. Whole cell lysate (WCL), endosomal, and cytosolic fractions derived from Huh 7.5.1 cells were subjected to SDS-PAGE and immunoblotted with indicated antibodies. 7% of WCL and 8% of each the endosomal and cytosolic fractions was loaded for western. E. Huh 7.5.1 cells were transfected with the indicated siRNAs followed by infection with luc-DENV2 for 1 h (MOI 6) with or without chloroquine and the RNA was extracted from the cytosolic fraction followed by RT-qPCR to determine viral RNA level (normalized to scrambled). The data values representing means and standard deviations (SD) (n ≥ 3). F. Huh 7.5.1 cells were transfected with the indicated siRNAs followed by infection with luc-DENV2 for 1 h (MOI 6) at either 37°C or 4°C, treated with Proteinase K for 45 min in ice and the RNA was extracted followed by RT-qPCR to determine viral RNA level (normalized to scrambled). The data values representing means and standard deviations (SD) (n ≥ 3). ***P ≤ 0.001.