*2.3. Validation of Y2H Interactions*

Protein–protein interactions identified in Y2H screens were validated by expression in human embryonic kidney (HEK) 293FT cells and protein pulldowns. HEK293FT cells were co-transfected with pDEST27 containing prey fusions to GST, and pNTAP containing bait fusions to SBP and CBP. Cells were collected after 24 h and lysed in 1% 3-[(3-Cholamidopropyl)dimethylammonio]-1-Propanesulfonate (CHAPS) lysis bu ffer (1% CHAPS, 150 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4) with protease and phosphatase inhibitors). Whole cell lysates were subjected to affinity purification of the TAP-tagged NS constructs using streptavidin-conjugated agarose beads, which were washed four times with 1% CHAPS lysis bu ffer, and then analyzed by Western blot using anti-GST (GE27–4577-01; Sigma Aldrich) and anti-CBP tag antibodies (GenScript; Cat.no. A00635).

#### *2.4. Tandem A*ffi*nity Purification Coupled to Mass Spectrometry*

HEK293FT cells were transfected using the calcium phosphate method with the SBP-CBP-tagged NS or control (Green Fluorescent Protein; GFP) vectors (Figure 1A). HEK cells have been previously used as a model for characterizing host–pathogen protein–protein interactions (PPIs) [20,24,25].

About 1 × 10<sup>8</sup> cells were used for the purification of the protein complexes using the InterPlay TAP purification kit (Stratagene) as described previously [23].

A nanoflow ultra-high-performance liquid chromatograph (RSLC, Dionex) coupled to an electrospray bench top orbitrap mass spectrometer (Q-Exactive plus, Thermo) was used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing experiments. Samples were loaded onto a pre-column and washed for 8 min with aqueous 2% acetonitrile and 0.04% trifluoroacetic acid. Trapped peptides were eluted onto an analytical column (C18 PepMap100, Thermo) and separated using a 90-min gradient delivered at 300 nl/min. Sixteen tandem mass spectra were collected in a data-dependent manner following each survey scan using a 15 s exclusion for previously sampled peptide peaks (QExactive, Thermo).
