Antiviral potential of endogenous ZAP against JEV infection.

<p>(A) Western blot analysis for the expression of ZAP in A549 cells transduced with lentiviruses carrying shRNA targeting LacZ and ZAP-L/S. (B and C) A549-shLacZ and -shZAP-L/S cells were infected with JEV (MOI = 5) for 24 h. Total RNA and culture supernatants were harvested for RT-qPCR (B) and viral titration (C), respectively. Relative JEV RNA level normalized by GAPDH was determined using RT-qPCR. Viral titer was measured using plaque assay. Representative data from two independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s <i>t</i> test. ** <i>P</i>≤0.01. (D) Confocal microscopy of mock and JEV (MOI = 1) infected A549 cells at 16 hpi stained with anti-dsRNA and anti-ZAP antibodies. Cell nuclei were counterstained with DAPI. Scale bar = 20 μm. (E) Co-localization of dsRNA with ZAP was estimated by Pearson’s correlation coefficient (PCC). Mean ± SD was calculated from 30 cells each group and the statistical significance was analyzed by two-tailed Student’s <i>t</i> test. *** <i>P</i>≤0.001. (F) Cell lysates from mock and JEV (MOI = 1) infected A549 cells after 16 h of infection were incubated with anti-ZC3HAV1 (ZAP) antibody or control IgG. Western blot analysis of the immunoprecipitated ZAP isoforms (upper panel). The ZAP-binding viral RNA pulled down by antibodies was amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input JEV viral RNA (lower panel).