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. 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.

RNA samples were diluted to a final concentration of 20 ng/μL and hybridized to reporters and capture probes from a custom-generated innate immunity probe set (NanoString, Seattle, WA, USA). Following hybridization for 16 h at 65°C, sample preparation was completed on an nCounter MAX prep station (NanoString) according to the manufacturer’s protocol. RNA transcript counts were quantified on a NanoString nCounter (NanoString) according to the manufacturer’s instructions.

Raw reads were assessed for technical quality control flags using the nSolver software (NanoString) and transferred into RStudio (Boston, MA, USA). As the design of our probe set was nonrandom, we utilized a custom approach for data analysis as previously described to account for large differences in the number of observed counts between mock-infected and infected samples (114). Briefly, we used count data within each sample vector as an internal reference, against which we compared genes of ...

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 ...