Immunofluorescence analysis

A549 cells were seeded on no. 1 glass coverslips in a 24-well plate at a density of 1 × 105 cells per well. The next day, cells were mock infected or infected with ZIKV strains and fixed with 4% formaldehyde (Fisher Scientific) in filtered 1× TBS for 30 min at room temperature. Cells were then washed with filtered 1× TBS and permeabilized and blocked in 5% NGS in 1× TBS (5% NGS/TBS) containing 0.1% Triton X-100 for 30 min at 4°C. For pSTAT1 staining, cells were permeabilized with 100% ice-cold methanol (Fisher Scientific) for 10 min at −20°C and washed in 1× TBS for 5 min before blocking in 5% NGS/TBS for 30 min at room temperature. For all immunofluorescence staining, cells were stained for 1 h at room temperature or overnight at 4°C with the following primary antibodies diluted in 5% NGS/TBS: dsRNA (J2, no. 10010500; 1:800; Scicons, Budapest, Hungary), ZIKV NS5 (GTX133327; 1:500; GeneTex), and pSTAT1 (no. 7649; 1:400; CST). Cells were washed with filtered 1× TBS and probed for 1 h at room temperature with isotype-specific and fluorophore-conjugated secondary antibodies (A-21131, A-11036, A-11008, or A-21241; Thermo Fisher-Life Technologies) diluted 1:1,000 in 5% NGS/TBS containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:10,000; Thermo Fisher-Life Technologies) to stain cell nuclei. After staining with secondary antibodies, cells were washed with filtered 1× TBS followed by dH2O. Excess water was carefully removed from coverslips, and coverslips were mounted onto glass slides (VWR) with ProLong gold mounting medium (Thermo Fisher-Life Technologies) and dried overnight at room temperature. Images and Z-stacks with 2.5-μm step sizes were acquired on an Eclipse Ti confocal microscope (Nikon, Tokyo, Japan) with a 60× oil immersion objective. Images were merged and processed using the Nikon confocal analysis software (NIS Elements AR 5.11.03). Further image analyses were performed in Fiji as indicated in the figure legends.