Pfn11a

The cDNA coding for each ZIKV protein, including prM, NS4A and NS4A-Flag (with a C-terminal Flag epitope tag), was PCR amplified from ZG-01 strain viral cDNA as a template and cloned into pFN11A (Promega, San Luis Obispo, CA) or pcDNA3.1 vectors. Plasmids including pcDNA3.1-DENV NS4A, -IVA PB1-F2, -MAVS, -RIG-I (N), pFN10A (ACT)-RIG-I, -MAVS, -TBK1, and-IKKε were constructed as previously described [22 (link)]. Truncated forms of MAVS (aa 1–77, 74–173 and 174–540) with N-terminal Flag epitope tags were amplified from the full-length template and cloned into the pcDNA3.1 vector. The pIFN-β-luc reporter plasmid was constructed by cloning a 125 bp fragment of the IFN-β promoter into the pGL3-Basic vector using the NheI and HindIII sites upstream of the luciferase reporter gene [8 (link), 22 (link)]. All constructs were verified by DNA sequencing. All primers used for plasmid construction are listed in Additional file 3: Table S3.

Protein interactions were detected by mammalian two-hybrid system as described previously [22 (link)]. pFN10A(ACT) and pFN11A(BIND) were purchased from Promega. pFN10A(ACT)-human-IRF3 and pFN11A(BIND)-APPV-N^pro were transfected into HEK293T cells using Lipofectamine 3000. The empty vector pBIND was served as controls. Cells were incubated for 24 h at 37°C in the presence of 5% CO₂, and then the luciferase activity was detected as described above.