*2.7. Conditioned Media Experiments*

A549 cells were mock infected or infected with WNV-TX at multiplicity of infection (MOI) = 5 for 24 h. Cell-free culture media was harvested, divided into two equal volumes per condition, and half subjected to UV inactivation for 30 min in a Spectrolinker XL-1000 UV crosslinker (Spectronics Corporation, Westbury, NY, USA) at full power on ice. Normal and UV-inactivated conditioned media was then aliquoted and stored at −80 ◦C. Thawed conditioned media was subsequently used neat in the treatment of seeded A549 cells for 24 h prior to acute, 30 min cytokine treatment. Treated cells were lysed post-stimulation and analyzed via Western blot.

## *2.8. Generation of Recombinant DNA Constructs*

Plasmids pCAGGS-HA and pCAGGS-WNV-NS4B were kindly provided by Adolfo Garcia-Sastre (Munoz-Jordan et al., 2005). Each gene from WNV strain TX02 was amplified from cDNA and cloned into the mammalian expression vector pCAGGS-HA in-frame with the 3'-HA tag. The 5' end of amplified WNV genes included either EcoRI, NsiI, or SacI restriction sites followed by an AUG start codon while 3' end contained KpnI or NsiI restriction sites for insertion into the multiple cloning site of pCAGGs-HA. Because the amino acid sequence of WNV strain NY99 NS4B from pCAGGS-WNV-NS4B (Munoz-Jordan et al., 2005) was identical to that of TX02 NS4B, the NY99-based construct pCAGGS-WNV-NS4B was used in these studies.

Plasmid pTwist-CMV-HSP90-HA was designed in-house and custom synthesized by Twist Bioscience (USA). The vector plasmid pTwist-CMV was created via excision of the HSP90-HA coding sequence with a Not-I/Nhe-I digestion, followed by blunting of the terminal ends via digestion with Mung Bean Nuclease (New England Biolabs, Ipswich, MA, USA), and finally blunt-end ligation. pTwist-CMV-FLAG-JAK1 was created via amplification of human JAK1 from a cDNA open reading frame (ORF) cloning vector (Sino Biological, Beijing, China) with gene-specific primers incorporating an N-terminal FLAG tag. This PCR product was then digested with Not-I/Nhe-I and ligated into the pTwist–CMV vector.

The vector pcDNA3.1(+) was purchased from Thermo Fisher Scientific (USA) and the plasmids pcDNA3.1-ZIKV(C)-FLAG and pcDNA3.1-ZIKV(NS5)-FLAG were the kind gift of Tom C. Hobman [29].
