Virus Infection Assay in Cell Lines

HEK293-MOV10 or vector cells were plated in 8-well chamber slides (4×10⁴ cells/well). Cells were infected with VSV for up to 16h and fixed with paraformaldehyde (4% v/v), following permeabilization with Triton-X100 0.1%. Nuclei were stained with DAPI Vectashield (Vector Laboratories, Inc. Burlingame, CA). Human primary foreskin fibroblasts were plated in 8-well chamber slides (1×10⁴ cells/well) and transfected with siRNA using Lipofectamine RNAiMAX with either siRNA 10 nM or control siRNA for 72 h. Subsequently, cells were infected with EMCV for 16 h and fixed with paraformaldehyde (4% v/v), following permeabilization with Triton-X100 1%. Infected cells were stained using anti-dsRNA sera (J2) previously described (27 (link)) for 16 h at 4 °C. Immunofluorescence detection was carried out with conjugated anti-rabbit Alexa Fluor 488 and conjugated anti-mouse Alexa Fluor 594 (Invitrogen). Representative micrographs were obtained followed by quantitation of the % of infected cells by counting over 500 DAPI positive cells for each condition.