*2.11. ReCLIP Analysis of Endogenous Proteins*

The protocol was modified from [30]. A549 cells infected with flaviviruses at MOI = 5 for 24 h were washed 2x with PBS prior to crosslinking proteins for 30 min at RT with 0.5 mM dithiobis(succinimidyl propionate) (DSP) in PBS. Crosslinked cells were subsequently quenched with incubation for 10 min at RT with TBS. Cells were then washed 1x with TBS and lysed on ice for 30 min with RIPA bu ffer followed by scraping into microcentrifuge tubes. Lysates were briefly sonicated in an ice-slurry bath (2 × 20 s pulses on the high setting with a 30 s pause). Lysate was cleared via centrifugation at 14,000 rpm for 15 min at 4 ◦C.

Lysate was pre-cleared by incubating 600 μg of total protein with 15 μL Protein G Dynabeads (ThermoFisher) in 1300 μL final volume for 30 min at 4 ◦C with rotation. For each IP, 150 μg of this cleared lysate was incubated with appropriate antibodies (2 μg mouse immunoglobulin G 2a (IgG2a) isotype control, 2 μg mouse monoclonal anti-HSP90 α/β clone F-8, or 10 μL hybridoma supernatant mouse monoclonal anti-WNV NS5 clone 5D4) in 500 μL total volume overnight at 4 ◦C with rotation. The following day, 15 μL of Protein G Dynabeads (washed 1x with 500 μL RIPA bu ffer prior to use) were added to the lysates (0.45 mg total beads added) and incubated for 3 h at 4 ◦C with rotation. Samples were subsequently applied to a magnetic rack to precipitate beads and were washed 4x with 400 μL RIPA bu ffer for 10 min each at RT with rotation. Protein complexes were eluted from beads and DSP crosslinker reduced via incubation for 5 min at ≈95 ◦C in 35 μL loading bu ffer (three parts RIPA bu ffer to one part 4X Laemmli Sample Bu ffer + 10% β-mercaptoethanol (Bio-Rad)). Samples were then analyzed by Western blot as appropriate, with 10 μL immunoprecipitate loaded per lane.

#### *2.12. Inhibition of HSP90 in Replicon Cells and During WNV and ZIKV Infection*

Seeded Huh7 WNrep cells were treated with DMSO, or with 1 μM of either geldanamycin (GA; Cayman Chemical, reconstituted in DMSO), EC144 (Tocris, Bristol, UK; reconstituted in DMSO), or NITD008 (Tocris, reconstituted in DMSO) for 12 and 24 h prior to lysis for RNA and protein analysis, or for 24 h prior to fixation and staining for immunofluorescence assay.

For HSP90 inhibition during infectious WNV and ZIKV replication, seeded Vero cells were first infected with viruses at MOI = 5 for 12 h to establish logarithmic replication. Mock- or virus-infected cells were then treated with DMSO or 1 μM HSP90 inhibitors for a further 12 h prior to lysis and analysis via Western blot.
