Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

FIG 1. (A) Schematic representation of genome organization of E7 virus (top) and the replicon with noncoding synthetic region R1 (ncR1) inserted into the 3′ UTR. (B) Replication of the WT, permuted control CDLR, and replicons in which the ncR1 region was modified to increase frequencies of CpG, UpA, the other pyrimidine/purine dinucleotides (UpG and CpA), and reversed dinucleotides (GpG and ApU). Replication was measured by luciferase expression at 6 h p.t. and expressed as the ratio of replicon luminescence to that of a replicon with the E7 WT ncR1 sequence (normalized to 1.0). Bar heights represent the means for two biological replicates (each the mean of three technical replicates). Error bars show standard deviations (SDs). The significance of differences from the WT construct was determined by ordinary one-way ANOVA with Bonferroni's correction. **, P < 0.0003; ns, no statistically significant difference from WT.

FIG 2. Replication of replicons with R1 sequences containing WT or increased numbers of CpG dinucleotides (n = 51 and n = 102, respectively) with the specified 5′ and 3′ bases (contexts): A, C, G, T, or n (randomized). Replication at 6 h was normalized to WT replication (WT = 1.0). CpG-H and UpA-h mutants that previously showed attenuated replication were included as controls. Bars represent the means for two biological replicates, each with three technical replicates; error bars show SDs. (A) Replication in A549 cells. The significance of differences from WT was calculated as described for Fig. 1. ****, P < 0.0001; *, P < 0.04; ns, no statistically significant difference. (B) The equivalent experiment performed in ZAP and OAS3 KO cells.

FIG 3. (A) Uninfected A549 cells costained for ZAP by specific antibody and Alexa Fluor 488-coupled secondary antibody and for E7 RNA by J2 (assay specificity control; not detected). WT- or CpG-H (assay positive control)-transfected cells costained for ZAP (green) and for dsRNA detected (red) by J2 antibodies in cells at 4 h posttransfection. Nuclear DNA was stained by DAPI (blue). (B and C) 3′ UTR mutant sequences with either (B) WT level (n = 51) or (C) elevated (n = 102) CpG frequencies. Bars, 10 μm. (D) Quantitation of fluorescence intensity of infected cells by Airyscan post-acquisition analysis in representative fields of cell monolayers transfected with E7 WT and compositionally modified mutants of E7. Bar heights show the means for two biological replicates; error bars show SDs. Significances of differences from WT are indicated as follows: **, P = 0.0015; ***, P = 0.0002; ****, P < 0.0001; ns, not significant (P > 0.05).

FIG 4. Binding of ZAP to WT, UpA-H, and CpG-H controls or 3′ UTR mutant RNA transcripts. (A) WB of input long (L) and short (S) isoforms of ZAP (~100 kDa and ~70 kDa, respectively) and GAPDH in uninfected cell lysates detected by immunostaining with anti-ZAP polyclonal antibody or GAPDH polyclonal antibody. Cell lysates from replicon-transfected cells were incubated with anti-ZAP antibody or control antibody (IgG) and immobilized to columns with anti-rabbit IgG. The nitrocellulose membrane was damaged at positions indicated by the black arrowhead. The lower panel shows a WB for immunoprecipitated ZAP. (B) Quantitation of E7 viral RNA transcripts with WT, CpG-H, and UpA-H or 3′ UTR mutant sequences by qPCR in immunoprecipitated ZAP (right column in each pair) or mock-precipitated control (left column). RNA was quantified by qPCR using conserved primers and probe from the 5′ UTR (Table S2). Bar heights and error bars represent means and SDs for two biological replicates. The significance of differences from the WT construct is indicated as follows: ***, P = 0.0002; **, P = 0.0015; *, P < 0.015; ns, not significant (P > 0.05).

FIG 5. Binding of ZAP in IFN-β-prestimulated cell lysates to RNA of different compositions ex vivo. +, ZAP immunoprecipitated with anti-ZAP antibody; −, mock-precipitated control (IP WB). RNA was quantified by qPCR using R1 sequence-specific primers for the different mutants (UpA-H, CpG-H, UpA-L, and combined WT, CpG-L, and CpG/UpA-L; Table S2). ns, no statistically significant difference from the WT.

FIG 6. Coincubation of replicon RNA with fixed concentrations (100 μg/μl) of either (A) CpG-H or (B) UpA-H replicons in the presence of various concentration of the heterologous replicon. RNA binding was quantified by CpG- or UpA-specific PCR to enable transcripts to be differentiated (IP WB) (Fig. S4 and Table S2). Bar heights show the means for two biological repeats; error bars show SDs.

FIG 7. Replication of mutants of E7 replicons with minimized and maximized CpG and UpA dinucleotides. The totals of each dinucleotide in each 3′ UTR sequence are indicated under the graph; red numbers indicate totals that were deliberately maximized and minimized (for these mutants while retaining protein coding). Bar heights show the means for two biological repeats; error bars show SDs. ns, no statistically significant difference from the WT construct; ****, P < 0.0001.

FIG 8. (A) Pulldown of different input quantities of E7 R1 insert RNA sequences by OAS3. Binding was quantified by qPCR. (B and C) Pulldown of 100 ng input WT, CpG-H, and UpA-H RNA transcripts in ZAP KO cells and in OAS3 KO cells, respectively. The significance of differences from WT construct is as follows: **, P = 0.0015; ***, P = 0.0002; ns, no statistically significant difference from WT. (D) Western blot detection of ZAP and OAS3 by specific antibodies in cell lysates with and without IFN-β prestimulation (left) and after immunoprecipitation by ZAP and OAS3 antibodies (center and right). In both IPs, ZAP and OAS3 (after IFN-β induction) coprecipitated, indicating their physical interaction. (E) Western blot detection of ZAP and OAS3 by specific antibodies in IFN-β prestimulation cell lysates in the presence of RNases A/T1 (left) and after immunoprecipitation by ZAP and OAS3 antibodies (center and right). In both IPs, ZAP and OAS3 coprecipitated, indicating their physical interaction.

FIG 9. A549 cells were transfected with the (A) WT or (B) CpG-H replicon transcripts, and at 6 h p.t., cells were fixed with 4% PFA and costained with ZAP (16820-1-AP; Proteintech), OAS3 (ab188111; Abcam), and dsRNA (J2; SCICONS). Nuclear DNA was stained with DAPI. Bars, 10 μm (A) and 1 μm (B). (C) Visualization of E7 dsRNA and ZAP localization with stress granules stained by G3BP (ab220524; Abcam). (D) Quantitation of colocalization of E7 replicon (dsRNA) with ZAP, OAS3, and G3BP in WT and CpG-H replicon-transfected A549 cells using Pearson’s correlation coefficient. Bar heights represent the means for three separate cell fields; error bars show SDs.