Alphavirus Evasion of Zinc Finger Antiviral Protein (ZAP) Correlates with CpG Suppression in a Specific Viral nsP2 Gene Sequence

Figures

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  Figure 1. Alphavirus replication shows differential sensitivity to endogenous ZAP in 293T cells. (A,C) ZAP KO and WT 293T cells untreated (A) or treated with 5 U/mL IFN-β for 24 h (C) were infected with GFP-expressing RRV, SINV, ONNV, or CHIKV vaccine strain 181/clone 25 (MOI = 1 PFU/cell). Cells were harvested and fixed at 24 h post-infection (h.p.i.), and their percentage of infection was determined by flow cytometry. Data are combined from three independent experiments performed with biological replicates in triplicate wells. Error bars represent the standard deviations (SD). Asterisks indicate statistically significant differences (unpaired T test with Holm–Šídák’s multiple comparisons test: ***, p < 0.001; ****, p < 0.0001). (B,D) Fold inhibition of alphavirus replication by ZAP. Data from panels A (B) or C (D) are represented. Fold inhibition relative to ZAP KO was calculated by dividing the percentage of infection of each virus in ZAP KO 293T cells by the percentage of infection in WT 293T cells. Error bars represent the standard deviations (SD). Asterisks indicate statistically significant differences (one-way ANOVA and Tukey’s multiple comparisons test: ***, p < 0.001; ****, p <…

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  Figure 2. Alphavirus production shows differential sensitivity to IFN-induced endogenous ZAP in 293T cells. Following treatment with 5 U/mL IFN-β for 24 h, ZAP KO and WT 293T were infected with RRV (A), SINV (B), ONNV (C), and the CHIKV vaccine strain (181/clone 25) (D) at MOI = 0.1 PFU/cell. Media overlaying the cells were harvested at 0, 8, 24, and 48 h.p.i., and the viral titer was determined by infection of BHK-21 cells in standard plaque assays. Mean values from four biological replicates across two independent experiments are plotted, and error bars represent the SD. Asterisks indicate statistically significant differences (two-way ANOVA and Bonferroni posttest: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

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  Figure 3. ZAPS and ZAPL binding to alphavirus genomic RNA do not correlate with alphavirus replication sensitivity to ZAP. (A,C) Lysates of ZAP KO 293T cells with induced expression of RFP-ZAPS (A) or RFP-ZAPL (C) were incubated with biotinylated Fluc RNA or gRNA of RRV, SINV, ONNV, or CHIKV. Following streptavidin-mediated pull-down of biotinylated RNA, ZAP associated with each RNA and in whole cell lysate (input) was assayed by immunoblot (IB). Expected sizes: RFP-ZAPS = 106 kDa, RFP-ZAPL = 130 kDa. Data are representative of results from three independent experiments. (B,D) ImageJ quantifications of panel A (B) or C (D). Data are combined from three independent experiments. Error bars represent the SD. Asterisks indicate statistically significant differences (one-way ANOVA and Tukey’s multiple comparisons test: *, p < 0.05; **, p < 0.01; ****, p < 0.0001).

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  Figure 4. The alphavirus nsP gene region contains the ZAP sensitivity determinant. (A) Schematics of chimeric viruses generated. The GFP construct is the same across all viruses. (B) ZAP KO 293T cells with doxycycline-inducible expression of ZAPS or ZAPL were infected with GFP-expressing SINV, ONNV, or ONNV nsP/SINV sP chimeric virus at MOI = 0.1 PFU/cell for 18 h before their percentage of infection was determined by flow cytometry. Fold inhibition by ZAP relative to doxycycline-untreated (-dox) cells is shown here. Error bars represent the SD. Data representative of two independent experiments performed with biological replicates in triplicate wells. Asterisks indicate statistically significant differences (two-way ANOVA and Tukey’s multiple comparisons test: **, p < 0.01; ****, p < 0.0001). ns—not significant.

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  Figure 5. ZAPS and ZAPL binding to alphavirus nsP RNA do not correlate with alphavirus replication sensitivity to ZAP. (A,C) Lysates of ZAP KO 293T cells with induced expression of RFP-ZAPS (A) or RFP-ZAPL (C) were incubated with biotinylated Fluc RNA or nsP RNA of RRV, SINV, ONNV, or CHIKV. Following streptavidin-mediated pull-down of biotinylated RNA, ZAP associated with each RNA and in whole cell lysate (input) was assayed by immunoblot. Expected sizes: RFP-ZAPS = 106 kDa, RFP-ZAPL = 130 kDa. Data are representative of results from three independent experiments. (B,D) ImageJ quantifications of panel A (B) or C (D). Data are combined from three independent experiments. Error bars represent the SD. Asterisks indicate statistically significant differences (one-way ANOVA and Tukey’s multiple comparisons test: *, p < 0.05; **, p < 0.01).

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  Figure 6. Sliding window analysis of alphavirus CpG content. The ratio of observed CpG dinucleotide frequency to expected frequency based on mononucleotide content (observed/expected) was calculated across alphavirus genomes using a window size of 500-bp and a step size of 250-bp. The genomes were aligned from the start of their 5′ untranslated regions. Windows with CpG contents that correlate with alphavirus replication sensitivity to ZAP are annotated (windows 1–3).

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  Figure 7. ZAPS and ZAPL binding to one but not all CpG-correlated windows correlates with alphavirus replication sensitivity to ZAP. Lysates of ZAP KO 293T cells with induced expression of RFP-ZAPS (A,C,E) or RFP-ZAPL (B,D,F) were incubated with biotinylated RRV, SINV, ONNV, or CHIKV CpG-correlated window 1 (A,B), window 2 (C,D), or window 3 (E,F) RNA. Following streptavidin-mediated pull-down of biotinylated RNA, ZAP associated with each RNA and in whole cell lysate (input) was assayed by immunoblot. Expected sizes: RFP-ZAPS = 106 kDa, RFP-ZAPL = 130 kDa. Data are representative of results from three independent experiments for window 1 and two independent experiments for windows 2 and 3.

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  Figure 8. ZAPS and ZAPL binding to CpG-correlated window 1 is CpG-dependent. Lysates of ZAP KO 293T cells with induced expression of RFP-ZAPS (A) or RFP-ZAPL (B) were incubated with biotinylated RRV CGlo, SINV CGlo, ONNV CGhi, or CHIKV CGhi window 1 mutant RNA. Following streptavidin-mediated pull-down of biotinylated RNA, ZAP associated with each RNA and in whole cell lysate (input) was assayed by immunoblot. Expected sizes: RFP-ZAPS = 106 kDa, RFP-ZAPL = 130 kDa. Data are representative of results from three independent experiments.