*2.2. Y2H Library Screening*

To identify direct human brain protein targets of ZIKV NS proteins, we used the MATCHMAKER Gold Y2H system (Clontech). Seven ZIKV viral proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) were transformed into the Saccharomyces cerevisiae strain Y2HGold (Clontech) alone or co-transformed together with an empty pGADT7 vector and tested for auto-activation and toxicity (defined by low transformation e fficiency, small colony phenotype, or inability to grow in liquid culture), as previously described by our group [23].

All bait proteins were expressed in Y2HGold and did not induce toxic e ffects on the yeas<sup>t</sup> cell cycle or survival (Figure S3A,B). Y2HGold transformants expressing each bait were mated to Y187 strain expressing a pre-transformed human brain normalized cDNA library (Matchmaker ® Gold Yeast Two-Hybrid System; Cat.no. 630486; Clontech) for 20 h. The mated cultures were then plated on quadruple dropout medium (SD -Trp/-Leu/-His/-Ade) and incubated for 8–12 days (NS5 was screened twice). For every screen, more than 1 × 10<sup>6</sup> transformants were screened (Table S1). Yeast miniprep DNA was used to recover pGADT7 fusions from each positive clone (Clontech Yeast Plasmid Isolation Kit), amplified by KOD polymerase chain reaction (PCR) and Sanger sequenced using a T7 primer. Out of frame clones were discarded and in-frame clones were kept for further analysis (Table S2).
