To generate replicon RNA, the full replicon cassette from CMV-driven clones was amplified using primers annealing to the backbone of the plasmid, and in which the forward primer contained an SP6 sequence. RNA was in vitro transcribed from the PCR product (mMESSAGE mMACHINE SP6 Transcription kit, Thermo Fisher), and purified using the RNeasy mini kit (QIAGEN). RNA was quantified using a Qubit fluorometer (Invitrogen) and used for transfection of HEK-293T or Vero cells. Briefly, sub-confluent cells in 24-well plates were transfected with 100 ng of replicon RNA using TransIT-mRNA transfection kit (Mirus Bio). Over a 72 h time course, the supernatant was removed and cells lysed in passive lysis buffer (Promega). Luciferase expression was measured using the Nano-Glo luciferase assay system (Promega) in a Tecan infinite M200 pro plate reader (Tecan group).

Assays in which the effect of non-structural proteins on DVG replicon activity was studied were performed as described above, except HEK-293T cells were transfected with E30-NS, E30-NS*NS1, E30-NS*NS2A, E30-NS1 plasmids using LT-1 transfection reagent (Mirus Bio) 24 h prior to reporter RNA transfection. E30-NS is a CMV-driven clone encoding the Zika virus C-terminal 30 amino acids of E and all nonstructural proteins. E30-NS*NS1, E30-NS*NS2A plasmids are identical to E30-NS, except a stop codon introduced as the first amino acid of NS1 or NS2A, respectively. E30-NS1 encodes only E30 and NS1. We performed these assays in HEK-293T cells because of their high transfection efficiency, which proved to be important for the successful transfection of both DNA and RNA molecules. To confirm successful expression from the clones, we generated a C-terminal, V5-tagged version of E30-NS1. Cell lysates of transfected cells were collected at 24 h p.t. in RIPA buffer (Sigma). V5-tagged NS1 was detected by conventional western blotting, using an anti-V5 antibody (Abcam; ab27671) and anti-mouse HRP linked antibody (GE Healthcare Life Sciences).

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