Fig 2: Replication of E7 WT and compositionally altered mutants in different k/o cell lines Merged images (E7 RNA, ZAP and nuclear DNA) of E7 WT and UpA- or CpG-high mutants of E7 in ZAP, RNASeL and OAS3 k/o cell lines.

Fig 3: Replication of replicons with 3′UTRs of different dinucleotide compositions compared to WT virus. (A–C) Effect of pathway knockout on the attenuation of replicons with different CpG and UpA compositions, scored by luciferase expression at 6 h post-transfection. Replicons were modified through insertion of mutated R1 sequences in the non-translated 3′UTR. The UpA-H R2R1 mutant possessed a further insertion of UpA-high sequence in the R2 (coding) region. (A) Attenuation of replicons relative to E7 WT in the parental A549 cell line, a control CRISPR k/o cell line (pLenti) and two ZAP k/o cell lines, B6 and B8. (B) Attenuation in two different RNAseL k/o cell lines and those with k/o of OAS1 and OAS3. (C) Attenuation of UpA-H mutants in A549, RD and E4, an RD-derived cell line with ZAP k/o. Bar heights represent the mean of three biological replicates; error bars show standard deviations. (D) Effect of reconstitution of ZAP and RNAseL on replicon replication in the corresponding k/o cell lines (B8 – ZAP and E10: RNaseL). These were transfected with plasmids expressing ZAP or RNAseL with a fused GFP tag to enable FACS-based cell sorting for expression prior to transfection with WT and CpG- and UpA-high replicons at 15 h. Luciferase expression was subsequently recorded at 6 h post transfection, and normalized to expression with WT virus. Expression in A549 and the corresponding k/o cell lines were determined as controls. Bars represent the mean of two biological replicates, each with three technical replicates, error bars show standard deviations.

Fig 4: Cryptolepine increases JAK1, TYK2, STAT1, STAT2, IRF9, and OAS3 mRNA levels in THP1-derived macrophages. The derived macrophages from the parent THP1 cells were cultured with cryptolepine (CRYPT), fludarabine (FLUD), or IFN-α as indicated. The target genes were assessed after 24 h by RT-qPCR with gene-specific primers and probes using β-actin as an endogenous control. Data are shown as the means, from three varied experiments with each done in triplicate, and error bars represent the standard deviations. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (one-way ANOVA and Bonferroni's test).

Fig 5: RNA-Seq Analysis of ISG mRNA Levels in PC3 Cells Silenced for MAP3K7 and/or CHD1Total-cell RNA extracts were prepared from mock-infected shControl, shMAP3K7, shCHD1, and shMAP3K7/CHD1 cells. Samples were depleted of rRNA, reverse transcribed, sequenced, and aligned to the human genome. Gene counts were log2 transformed and centered about their means for each sample. (A–M) Data shown are mRNAs encoded by the same ISGs as in Figure 4: (A) MX1, (B) OAS3, (C) IFIT1, (D) IFIT5, (E) IFITM3, (F) IRF3, (G) ISG15, (H) EIF2AK2, (I) STAT1, (J) OAS2, (K) IFIT2, (L) IFIT3, and (M) PRKRA. Data are represented as mean ± SD on a log2 scale from three independent experiments. Statistical significance was determined for each ISG protein from log-transformed data using one-way ANOVA with Holm’s correction for multiple comparisons. ∗p < 0.05, for differences between cells.