*2.13. Proteasome Inhibition with MG-132*

A549 cells were mock-infected or infected with WNV-TX at MOI = 5. At 18 h post-infection (hpi), cells were treated with either DMSO or 40 μM MG-132 (Sigma) for a further 6 h prior to lysis and analysis via Western blot.

## *2.14. Generation of CRISPR-Targeted IFNAR1-*/*- PH5CH8 Cells*

For clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 targeting of IFNAR1, we generated the plasmid pRRL-MND-IFNAR1-2A-Puro by in-fusion cloning of the IFNAR1 genomic RNA (gRNA) sequence: 5'-GACCCTAGTGCTCGTCGCCGTGG-3' into pRRL-MND-2A-Puro. PH5CH8 cells were transfected using the Amaxa 96-well Nucleofector Kit SF according to manufacturer's instructions. Then, 48h post-transfection, cells were selected in growth medium with 2 μg/mL of puromycin. Gene targeting was confirmed by T7 endonuclease I assay, loss of IFNAR1 cell surface expression by flow cytometry, and loss in the response to type I IFN treatment.

#### *2.15. Generation of Huh7 WNrep Cells and Replicon-Cured Cells*

BHK-21 cells harboring the WNV subgenomic replicon Rluc/NeoRep were kindly provided by Pei-Yong Shi (Lo et al., 2003). Rluc/NeoRep contains a dual reporter system and was derived from the parental WNV Replicon by replacing most of the structural gene region (nt 190 to 2379) with an in-frame Renilla luciferase (Rluc) reporter gene. A neomycin phospho-transferase (Neo) gene under the control of an EMCV IRES was placed just downstream from the NS5 gene in the 3'-untranslated region (UTR). Total RNA was isolated from these cells with TRIzol according to the manufacturer's instructions and stored at −80 ◦C. Rluc/NeoRep RNA was treated with DNaseI (Ambion, Austin, TX, USA). Huh7 cells were transfected with 1, 2, or 4 μg of DNaseI-treated Rluc/NeoRep RNA using Transmessenger Transfection Reagent (QIAGEN) according to the manufacturer's instructions. After 3 h, transfection mix was replaced with cDMEM for 16-20 h at 37 ◦C. Cells were then washed in 1X PBS, trypsinized, transferred to new plates in complete Dulbecco's Modified Eagle Medium (cDMEM), and allowed to

recover for 48 h at 37 ◦C. After recovery, cDMEM was replaced with cDMEM containing G418 (G418 DMEM, 400 μg/mL) for selection of resistant colonies. Resistant colonies were transferred to new plates via colony selection discs and expanded in the presence of G418 (400 μg/mL). Approximately 11 weeks after RNA transfection, WNV Rluc/NeoRep protein expression was examined by Western blot analysis. Renilla luciferase activity of cells was also confirmed. Several distinct clones of WNV Rluc/NeoRep with variable protein and luciferase expression were recovered in Huh7 cells, with clone #8 used in these experiments.

To create a Huh7-derived control cell line for WNV Rluc/NeoRep clone #8, this line was subjected to curing with IFN. Briefly, the replicon cell line was passaged in the presence of pegylated IFN α-2b (PEG-INTRON, Schering, Berlin, Germany) at a concentration of 100 U/mL in cDMEM. After 11–25 days of maintenance in PEG-INTRON, cells were collected and analyzed for Renilla luciferase activity. When Renilla luciferase activity was below the background level of Huh7 control cells, the cured replicon cells were switched to cDMEM. Loss of the WNV replicon was confirmed by re-exposing the cells to lethal G418 to confirm death, and by RT-PCR analysis of RNA.
