For the reversal-of-silencing assay in Drosophila S2 and mosquito Aag2 cells, the EGFP reporter gene, FHV B2 and DENV1-4 viral proteins were constructed into the insect expressing vector pAc5.1/V5-HisB as previously described (16). The 400-nt dsRNA for EGFP silencing in S2 or Aag2 cells were generated by in vitro transcription as described (16). The 500-nt dsRNAs for knockdown of Dicer-2 and AGO2 of S2 and Aag2 cells were described previously (16, 44). Full-length complementary DNA (cDNA) of FHV RNA1 and RNA1-ΔB2 (T2739C and C2910A) encoded in CuSO4-induced pMT vector were provided by S.-w. Ding (Riverside, CA, USA). Mutations were introduced into the DENV2 NS2A coding region by polymerase chain reaction (PCR)–mediated mutagenesis with appropriate primers containing the desired nucleotide changes.

For the reversal-of-silencing assay in mammals, the plasmid pEGFP-C1 and the EGFP-specific shRNA were used, as described (16). The flaviviral (DENV1-4, JEV, ZIKV, and WNV) NS2A were cloned into mammalian expression vector pRK-Flag/His (provided by H.-B. Shu, Wuhan, China). Plasmids for the purification of MBP fusion protein NS2A and its mutants were constructed by inserting NS2A ORF into a pMAL-c2X vector. The primers used in this study are shown in table S2.

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