*2.1. cDNA Constructs*

We generated the cDNA of seven individual NS proteins (NS1, NS2A, NS3, NS2B, NS4A, NS4B, and NS5) corresponding to a ZIKV Brazilian isolate from the state of Pernambuco (GenBank AMD16557.1) [22]. We used the virus isolate as template for polymerase chain reaction (PCR) to generate cDNAs for NS1, NS2A, NS2B, NS4A, and NS4B. We were unable to obtain the correct product corresponding to NS3 and NS5 cDNAs using the virus isolate. We generated the Brazilian genotype cDNAs for NS3 and NS5 using nucleotide substitutions introduced by site-directed mutagenesis on the Asian lineage (PRVABC59 strain) cDNAs for NS3 and NS5 (pLV\_Zika\_Flag\_NS3 and pLV\_Zika\_NS5\_Flag plasmids; Addgene #79634 and #79639, respectively).

ZIKV cDNAs coding for NS proteins were cloned into pGBKT7 (Clontech) in frame with the GAL4 DNA binding domain (DBD) for yeast-two hybrid (Y2H) assays, and into pNTAP (Agilent) in frame with the Streptavidin-binding peptide (SBP) and Calmodulin-binding peptide (CBP) epitope tags for the tandem a ffinity purification coupled to mass spectrometry (TAP-MS) assays. The glutathione-S-transferase (GST)-tagged baits used for Y2H validations were generated by subcloning the cDNA from the isolated pGADT7 (Clontech) plasmid into the pDEST27 vector using Gateway recombination cloning according to the manufacturer's instructions (ThermoFisher).

To validate Y2H interactions, recovered Y2H plasmids containing prey cDNAs were amplified by PCR using primers containing attb sites. PCR products were cloned into pDONR221 for Gateway recombination cloning (Invitrogen) and subsequently into pDEST27, to produce an N-terminal GST fusion.

PIAS1 cDNA (NM 001320687.1) was obtained via PCR amplification using a human leukocyte cDNA library and was cloned into pGEX-6P1 using *EcoRI* and *SalI* sites. For expression in mammalian cells, PIAS1 cDNA was subcloned into the pEBG vector using *Bam*HI and *NotI* sites. All constructs were confirmed by Sanger sequencing.
