ZIKV Nonstructural Protein Expression in Mammalian Cells

pMOI-GFP and pMOI-PARP11 (Homo sapiens) expression plasmids were purchased from GeneCopoeia and described previously [34 (link)]. The viral RNA of the GZ01/2016 strain was isolated and used in reverse transcription PCR experiments to obtain the complementary DNA (cDNA) sequence of ZIKV nonstructural NS1 and NS3 proteins. ZIKV NS1 and NS3 genes were cloned into the pcDNA6/V5-His expression vector (Invitrogen) using standard molecular techniques and verified by sequencing. DsRed-PARP11, EGFP-PARP12, Flag-PARP11, HA-PARP12, HA-PARP12 ZnF, HA-PARP12 WWE, HA-PARP12 PARP, EGFP-PARP11 WWE domain, EGFP-PARP11 PARP domain, YFP-PARP11, Flag-PARP13 and GFP-PARP11 mutants were cloned using standard molecular cloning and oligonucleotide mutagenesis methods. To create a stable cell line for PARP11 expression, PARP11 was cloned into the pMXsIG-IgkFLAG vector and co-transfected into HEK293T cells with VSV glycoprotein and pCpG helper plasmids. 48 h after transfection, the culture supernatant was collected and added into WT or PARP12^−/− A549 cells for infection. The cells were collected 72 h after infection, and the PARP11-overexpressing cells were then sorted by fluorescence-activated cell sorting (FACS).