Geneius software

The coding sequence of ZIKV RdRp domain used in this study corresponds to the SPH2015 strain that has been optimized for codon usage using the GENEius software (Eurofins Genomics GmbH) followed by Pro codon optimization for bacterial expression (Genbank No. KY364193). The adapted gene harbored an internal BamHI recognition site that was subsequently removed and the sequence corresponding to an 8xHis tag was added in 5′ of the sequence. The corresponding DNA coding for a 72 kDa protein was obtained by gene synthesis (Eurofins Genomics GmbH, Munich) and inserted in the multi-cloning site of the pET-21a vector between BamHI and XhoI sites.

Cloning was performed using standard molecular biology techniques. PCR was performed with FailSafe PCR System (Epicentre) using the C. sporogenes genomic DNA as a template with primers PM003 (5′-GGATGTACTACAAGTGCTATTCAC-3′) and PM008 (5′-AGTGCTGGATTTAGTCTAC-3′). The PCR product was cloned into pJET1.2 (Thermo Fisher Scientific) and sequenced. For protein overexpression, the fldZ gene was codon optimised for expression in E. coli with GENEius software (Eurofins MWG Operon) and synthesised with NdeI (at the 5′-end) and NotI (at the 3’-end) restriction sites. After restriction digestion with NdeI and NotI (Thermo Fisher Scientic) the insert from the cloning vector was ligated into pET20b(+) cut with the same restriction enzymes, to form the plasmid pET20b(+):CsfldZ. Cloning of other expression plasmids is described in the Supplementary information 3.

The His-pVP2 synthetic gene (GenBank Accession Number MT780551) was obtained by fusing the nucleotide sequence encoding a poly-histidine tag (His) [13 (link)] to the 5’-end of the VP2 encoding sequence (pVP2) constructed by fusing the sequence encoding amino acids 1–452 of the IBDV VP2 protein (GenBank Accession Number JN982254) to that encoding amino acids 453–466 of the IBDV polyprotein (GenBank Accession Number AF140705.1). The nucleotide sequence of the synthetic gene (Eurofins Genomics, Ebersberg, Germany) was optimised for the expression in N. benthamiana using the GENEius software (Eurofins Genomics, Ebersberg, Germany) and cloned in the pBI-Ω plant expression vector [27 (link)] under the control of the Cauliflower Mosaic Virus 35S promoter (35S), the TMV translational enhancer sequence (Ω) and the Agrobacterium tumefaciens (A. tumefaciens) nopaline synthase gene terminator (nos-t) using the BamHI/EcoRI restriction sites, yielding the pBI-His-pVP2 construct (Fig 1A). The p35:AMCV-P19 construct encoding for the Artichoke Mottled Crinkle virus (AMCV) P19 silencing suppressor protein was previously described [28 (link)].