Fig 1.
Inhibition of JEV but not DENV infection by human ZAP isoforms.
A549 cells transduced with lentiviruses expressing EGFP (A549-EGFP), V5-tagged ZAP-L (A549-ZAP-L) and V5-tagged ZAP-S (A549-ZAP-S) were infected with JEV or DENV (MOI = 5). Culture supernatants, cell lysates and total RNA were harvested at the indicated time points. (A and D) Viral titration determined by plaque assay. Representative data from three independent experiments are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA. *** P≤0.001; NS, not significant. (B and E) Protein levels of viral NS3, V5-tagged ZAP, EGFP and actin were analyzed by western blot. (C and F) RNA levels of viral genome and actin were performed by RT-PCR. hpi, hours post-infection.
Fig 2.
Antiviral potential of endogenous ZAP against JEV infection.
(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 t test. ** P≤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 t test. *** P≤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).
Fig 3.
Zinc-finger motifs of ZAP are required for JEV RNA binding and its antiviral activity.
(A) Schematic representation of human ZAP isoforms (ZAP-L, 902 a.a. and ZAP-S, 699 a.a.). The four tandem CCCH-type zinc-finger (ZF) motifs within the N-terminus of ZAP are shown as solid black boxes. Deletion of the four ZFs (a.a. 73–193) are indicated with a dashed line. (B) 293T/17 cells infected with JEV (MOI = 5) for 16 h were transfected with plasmids expressing EGFP, WT or ZF-deleted ZAP-L-V5 and ZAP-S-V5 for additional 24 h. The viral RNA bound with V5-tagged proteins was pulled down by anti-V5 agarose affinity gel and amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input viral RNA in JEV-infected cells (lower panel). Western blot analysis of the immunoprecipitated ZAP-L and ZAP-S (WT and del4ZFs) (upper panel). (C-E) The indicated cells were infected with JEV (MOI = 5) for 10 h. Cell lysates, total RNA, and culture supernatants were determined for the indicated proteins by western blot (C), viral RNA by RT-PCR (D), and viral titer by plaque assay (E). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. *** P≤0.001.
Fig 4.
Inhibition of JEV translation and replication by ZAP.
(A and C) A549-EGFP, ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P≤0.05; ** P≤0.01; *** P≤0.001.
Fig 5.
The 3′-5′ RNA exosome-mediated, but not the 5′-3′ XRN1-mediated, RNA degradation is required for the anti-JEV activity of ZAP.
A549 cells with shRNA targeting LacZ, XRN1, and EXOSC5 were transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5. After 10 h of JEV infection (MOI = 5), cells lysates were analyzed by western blot for the indicated proteins (A and D, upper panel). The relative quantification of NS3 normalized by actin was quantified by ImageJ software (A and D, lower panel). Data are mean ± SD (from four independent experiments). Total RNA and culture supernatants were harvested for the measurement of viral RNA by RT-PCR (B and E) and viral titer by plaque assay (C and F). Data are representative results from repeated experiments and shown as mean ± SD (n = 3). The statistical significance was estimated by two-tailed Student’s t test. * P≤0.05; ** P≤0.01; *** P≤0.001; NS, not significant.
Fig 6.
ZAP-enhanced innate immune responses in JEV-infected cells.
(A-C) A549 cells overexpressing EGFP, ZAP-L and ZAP-S were uninfected (Mock) or infected with JEV (MOI = 5) for 16 h. Total RNA was collected for IFN-β, TNF-α, and IL-6 mRNA detection by RT-qPCR. RT-qPCR results are presented relative to the expression of GAPDH. Representative data from repeated experiments with triplicate samples (mean ± SD, n = 3) were analyzed by two-tailed Student’s t test. * P≤0.05; ** P≤0.01; *** P≤0.001. (D) Western blot analysis for the knockdown effects of RIG-I and MDA5 in A549-shLacZ, -shRIG-I and -shMDA5 cells established by transduction with lentiviruses expressing the indicated shRNA. (E and F) A549-shLacZ, -shRIG-I and -shMDA5 cells transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5 were infected with JEV (MOI = 5) for 16 h. Western blots analysis was performed for the indicated proteins.
Fig 7.
ZAP mainly targets JEV 3′-UTR.
(A) Map of ZAP-S binding sites in full-length JEV genome by CLIP-seq of RNA isolated from JEV-infected ZAP-S overexpressing A549 cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (B) Schematic diagram of the reporter RNAs (left panel). 293T/17-EGFP and -ZAP-S cells were cotransfected with 5′-capped firefly luciferase (Fluc) flanked by JEV 5′-UTR197 and/or 3′-UTR RNA and control Renilla luciferase (Rluc) RNA for 18 h. Relative luciferase activity was measured by dual-luciferase reporter assay (right panel). Representative data from two independent experiments are mean ± SD (n = 3) and analyzed by two-tailed Student’s t test. *** P≤0.001; NS, not significant. (C) Biotin-labeled JEV 3′-UTR RNA (at the indicated amounts) was incubated with ZAP-S or ZAP-S-del4ZF overexpressing A549 cell extracts and then pulled down by using streptavidin beads. The co-precipitated ZAP-S-V5 (WT and del4ZFs) was assayed by western blot.
Fig 8.
Mapping of ZAP-responsive element (ZRE) in JEV 3′-UTR.
(A) The CLIP-seq diagram of ZAP-S binding RNA mapped to JEV 3′-UTR. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. The JEV 3′-UTR is divided into domain I, II and III as indicated. The positions of CG dinucleotide are shown by dots. (B) Biotin-labeled JEV 3′-UTR RNA probes (full-length and individual domain I, II, III, and I+II) incubated with A549-ZAP-S cell extracts were pulled down with streptavidin beads and determined for ZAP-S-V5 protein by western blot (left panel). The intensity of ZAP-S was quantified by ImageJ software. Mean ± SD was calculated from 3 independent experiments and analyzed by two-tailed Student’s t test (right panel). (C) 5′-capped full-length and deleted Fluc/5′+3′-UTR RNA (left panel) and control Rluc RNA were cotransfected into 293T/17-EGFP and -ZAP-S cells. At 18 h post-transfection, cell lysates were collected to perform dual-luciferase assay (right panel). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P≤0.01; *** P≤0.001; NS, not significant.