*2.7. ZIKV RdRP Expression and Purification*

The RdRP domain of ZIKV NS5 (amino acids 2772-3423 of accession number AMB18850) was fused to an N-terminal 6×-histidine tag containing a TEV cleavage site, and the cDNA was codon-optimized and synthesized by IDT (San Diego, CA, USA). This construct was then inserted into a pET-17b vector (Invitrogen) and heterologously expressed in the Rosetta strain of *Escherichia coli*. Cultures transfected with the RdRP construct were grown at 37 ◦C in LB media containing 1% ethanol and 100 μg/mL amplicillin until an OD600 of 0.6–0.7 was reached. Protein expression was then induced with 100 μM IPTG prior to an additional 5 h incubation at 20 ◦C and lower speed shaking (130 RPM). Cells were then harvested by centrifugation at 9000 RPM for 30 min at 4 ◦C, and pellets were stored at −80 ◦C. For purification, pellets were resuspended in lysis buffer (50 mM HEPES at pH 7.5, 20 mM NaCl, 20% glycerol, 0.1% octyl-glucoside, 5 mM β-mercaptoethanol) supplemented with a protease inhibitor tablet (Thermo Fisher). Cells were lysed using a microfluidizer (Microfluidics International Corporation, Newton, MA, USA) and clarified via centrifugation at 12,000 RPM for 30 min at 4 ◦C. The supernatant was loaded onto TALON cobalt affinity beads (Clontech, Mountain View, CA, USA) at 4 ◦C. The unbound protein was washed with 50 mL each of the following buffers: wash buffer (50 mM HEPES at pH 7.5, 5 mM β-mercaptoethanol) containing 100 mM NaCl and 10 mM imidazole, wash buffer containing 500 mM NaCl and 20 mM imidazole, and finally wash buffer containing 200 mM NaCl and 30 mM imidazole. The protein was eluted in wash buffer containing 200 mM NaCl and a linear imidazole gradient of 30–300 mM. Protein fractions were pooled and dialyzed overnight against 50 mM HEPES at pH 7.0, 100 mM NaCl, 10% glycerol, 0.01% CHAPS, and 5 mM dithiothreitol at 4 ◦C. The protein was concentrated to ≥6 μM and stored in single-use aliquots at −80 ◦C.
