IRES Mutation Confers a Replicative Advantage to SVA by Enhancing Translation Efficiency

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  Figure 1. Conditioned medium enhances cellular antiviral innate immune responses. (A) The transcriptional levels in 3D4/21(CRL‐2843) cells after infection with SVA. (B) The procedure for preparing conditioned medium. (C) The transcriptional levels in 3D4/21(CRL‐2843) cells after incubation with CCM for 18 h. (D) The expression levels and nuclear translocation of phosphorylated IRF3 (P‐IRF3) and phosphorylated STAT1 (P‐STAT1) in 3D4/21 (CRL‐2843) cells following 18 h incubation with CCM were assessed by immunofluorescence assay (IFA). Cells were incubated with CCM for 18 h. Following fixation, the cells were incubated overnight (12 h) with primary antibodies against P‐IRF3 and P‐STAT1, then incubated with appropriate secondary antibodies for 1 h. Fluorescence signals were subsequently visualized using a Laser Scanning Confocal Microscope (Zeiss LSM980, Carl Zeiss), and fluorescence intensity was quantitatively analyzed using Image J. (E) The immune activation status of cells after 18 h of incubation with CCM was assessed using dual‐luciferase reporter assay. Cells (CRL‐2843, left column; HEK‐293T, right column) were seeded in 96‐well plates and cotransfected with dual‐luciferase reporter…

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  Figure 2. Serial passaging of viruses and whole‐genome next‐generation sequencing analysis. (A) Procedure for serial passaging of SVA in CRL‐2843 cells with antiviral innate immunity preactivated by CCM. (B) Sequencing coverage and depth. (C) Comparison of stably inherited nucleotide mutations in the P60–P80 genomic regions of CRL‐FJ and IB‐FJ. In the figure, distinct symbols represent different types of nucleotide substitutions, and the frequency of each symbol corresponds to the number of occurrences of that specific mutation type. (D) Two distinct mutations occurred in the 5′‐UTR of CRL‐FJ: 108U109 and G265A.

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  Figure 3. IRES‐dependent translation initiation assay. (A) Key components of the pCMV‐hR‐Luc‐IRES‐F‐Luc plasmid, including the positions and types of introduced mutations. (B, C) The pCMV‐hR‐Luc‐IRES‐F‐Luc plasmid was transfected into 3D4/21 cells (B) and HEK293T cells (C), Following 24 h of incubation, cells were lysed completely on ice. R‐Luc and F‐Luc activities were measured using a Dual‐Luciferase Reporter Assay System. (D) The secondary structures of both the wild‐type and mutant SVA IRES were predicted and modeled using the RNAfold web server (http://rna.tbi.univie.ac.at). In the structural diagrams, the blue circle highlights the region affected by the mutation, the red nucleotides indicate the specific sites of the mutated bases, and the yellow box marks the regions corresponding to the key structural domains of the IRES reported in prior literature. All experiments were repeated three times with consistent results. Student’s t‐test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

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  Figure 4. Recovery and growth kinetics assessment of SVA. (A) 5′ UTR mutations in the reverse genetics system for rescuing SVA were identified through sequencing. (B) 5′ UTR mutations in the rescued and recovered SVA strains were identified through sequencing. (C) CPE induced by the wild‐type and mutant viruses in 3D4/21 cells. (D, E) Viral RNA copy numbers of wild‐type and mutant SVA in culture supernatants from 3D4/21 cells (D) and IBRS‐2 cells (E) were quantified via RT‐qPCR at 12, 24, and 36 h postinfection. (F, G) Viral titers of wild‐type and mutant SVA in cell culture supernatants from 3D4/21 cells (F) and IBRS‐2 cells (G) were determined via the TCID50 assay at 12, 24, and 36 h postinfection. All experiments were repeated three times with consistent results. Student’s t‐test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

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  Figure 5. Detection of Viral Infection‐Induced Transcription of Antiviral Innate Immune Factors. 3D4/21 cells were infected with either the wild‐type virus (SVA‐IRES‐WT) or a single‐point mutation virus (IRES‐108U109, IRES‐ G265A), or a two‐point mutant virus (SVA‐IRES‐Mut), and cocultured for 24 h. (A, B) RT‐qPCR analysis of immune factor mRNA transcription levels in IRES‐108U109, IRES‐ G265A, and IRES‐WT infected cells. Results were compared with a mock control without virus infection. Special note: for graphical representation, the fold changes of the mock group were not log‐transformed, whereas those of the IRES‐WT, IRES‐108U109, and IRES‐G265A groups were expressed as log10 values. (C) Volcano plots based on transcriptome sequencing data showed upregulated and downregulated genes in 3D4/21 cells 24 h after infection with the wild‐type virus (SVA‐IRES‐WT). (D) Volcano plots based on transcriptome sequencing data showed upregulated and downregulated genes in 3D4/21 cells 24 h after infection with the two‐point mutant virus (SVA‐IRES‐Mut). (E) The clustering heatmap of transcriptome sequencing data revealed distinct gene expression profiles in 3D4/21 cells at 24 h postinfection with…