Assays

A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. Extracellular RNA was extracted from the clarified supernatants using the QIAmp viral RNA minikit to quantify extracellular viral copy numbers. Cells were washed with ice-cold stringent wash buffer (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) for 3 min to remove cell surface-associated viruses. After ice-cold 1× PBS washes, ...

A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. The clarified supernatants were used for plaque assays to quantify extracellular virions. Cells were washed with ice-cold stringent wash buffer as described above and then detached with 0.05% trypsin-EDTA (Fisher Scientific). Cells were resuspended in 1 mL cDMEM and centrifuged at 300 × g for 5 min at 4°C. Cell pellets were ...

Vero cells were seeded at a density of 1.85 × 105 cells per well in 6-well plates. The next day, supernatants from infected samples were serially diluted in DMEM containing 1% FBS. Vero cells were incubated with dilutions in duplicate at 37°C for 1 h with rocking. Following incubation, cells were overlaid with 1% low-melt agarose (Fisher Scientific), 5% sodium bicarbonate (Fisher Scientific), and 5% FBS in 2× DMEM (Fisher Scientific). Plaques were visualized 4 to 5 days later with a 1% low-melt ...

cDNA was synthesized from isolated RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). cDNA was diluted to a final concentration of 2.5 ng/μL for TaqMan qRT-PCR on the QuantStudio 5 real-time PCR system (Thermo Fisher) using TaqMan universal PCR master mix (Fisher Scientific) and ZIKV NS3-specific primers and probe (Table 4; IDT, Coralville, IA). The ZIKV NS3-specific probe has a 5′ 6-carboxyfluorescein (FAM) reporter dye and a 3′ Iowa Black FQ quencher. ZIKV NS3-specific ...

cDNA was synthesized as described above. For each sample, cDNA was diluted to a final concentration of 2.5 ng/μL for SYBR green qRT-PCR on the QuantStudio 5 real-time PCR system using the SYBR green master mix (Thermo Fisher) and gene-specific primers (Table 4; Qiagen; IDT). Each sample was run in triplicate.

Cells were washed with 1× PBS and lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing freshly added phosphatase inhibitors (1:100; VWR), protease inhibitors (1:100; Sigma-Aldrich), and 250 nM okadaic acid (1:1,000; Fisher Scientific). Cell lysates were collected and stored at −80°C. Lysates were subsequently thawed on ice and sonicated in a water bath containing ice slurry for three 30-s bursts on ...

Cells were seeded, infected, or transfected and treated with IFN-β as described above. Following IFN-β treatment, A549 RIG-I KO and HEK293T cells were pelleted and fixed with 4% formaldehyde in 1× PBS at room temperature for 15 min. Cells were then washed with excess 1× PBS, pelleted, and resuspended in 1× PBS for storage at 4°C. Fixed cells were permeabilized with 90% ice-cold methanol in 1× PBS for at least 10 min on ice. Cells were washed with excess 1× PBS and incubated for 1 h at room ...