ZIKV cell binding and entry experiments were performed on the basis of the protocol described previously (34). Briefly, for the virus-binding assay, 2 × 105 cells were seeded in a 12-well plate and cultured for 24 hours, followed by infection with ZIKV (MOI = 2) and incubation at 4°C for 1 hour. Unbound virus was removed, cells were washed with 1× PBS twice, and then, cell lysates were harvested to determine the amount of viral RNA by qRT-PCR. For the entry assay, after binding at 4°C for 1 hour, the infected cells were washed with 1× PBS twice, followed by incubation with prewarmed DMEM for 10 min at 37°C. Subsequently, cells were rinsed three times with 1× PBS, then treated with 0.25% trypsin for 10 min, and again washed three times with 1× PBS. Total cellular RNA was extracted to quantify viral RNA by qRT-PCR.

A SZ01 ZIKV replicon carrying the seven ZIKV nonstructural proteins and RLuc gene was developed as described (49). A replicon assay was performed as previously published (49, 50), with minor modifications. Briefly, 2 × 105 BHK-21 cells in a 24-well plate were transfected with 200 ng of the in vitro–transcribed replicon containing the seven ZIKV nonstructural proteins and the RLuc reporter and with 200 ng of pMOI-PARP12 or control plasmid using a Lipofectamine 3000 reagent (Thermo Fisher Scientific). The cell lysates were collected after 48 hours, and the RLuc activity was measured using the Renilla Luciferase Assay system (Promega) in a GloMax 96 microplate luminometer.

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