SEC11A identified as a critical host factor for HMPV through virus-induced alternative splicing

Figures

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  Fig. 1. Selection of infection models and transcriptomic analysis. A) Under identical infection conditions (MOI=0.05), total nucleic acids were extracted from the supernatants of two infection models (LLC-MK2 and BEAS-2B cells) at 2 days post-infection, followed by RT-qPCR quantification of HMPV N gene copy numbers (Unpaired t test, ****P < 0.0001). B) A significant increase in HMPV N gene copy number was observed in supernatants from BEAS-2B cells at 2 and 3 dpi (two-way ANOVA). C) Volcano plots of differentially expressed genes at different time points post-infection (|Log2(Fold Change)| ≥ 1, Q value ≤ 0.05). KEGG (D) and GO (E) enrichment analysis results of DEGs at 2 and 3 dpi. F) Venn analysis was performed to compare DEGs at 2 and 3 dpi, identifying a core subset of genes with significant changes common to both time points. This core gene set was subjected to KEGG pathway enrichment analysis (G) and GO biological process enrichment analysis (H). Abbreviations: MOI, multiplicity of infection; RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction; HMPV, human metapneumovirus; KEGG, Kyoto encyclopedia of genes and genomes; GO, gene ontology; DEGs,…

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  Fig. 2. HMPV infection regulates the intracellular mRNA alternative splicing. A) RNA-Seq data were further analyzed for alternative splicing changes, and the splicing changes were categorized (FDR ≤ 0.05). B) GO analysis was performed on differentially spliced genes (ΔPSI ≥ 0.6) to assess functional enrichment. C) Heatmap depicting expression fold changes of splicing-related genes following HMPV infection. Key splicing factors are shown at specified time points post-infection (dpi). Each tile’s color intensity represents the fold change in gene expression (infected versus time-matched uninfected controls, whose FPKM values are normalized to 1). D) Splicing changes in selected transcripts (FASTKD1, TATDN1, SEC11A, GNG2, NASP, PCGF6, and REV1) were validated by reverse transcription PCR and agarose gel electrophoresis in BEAS-2B cells infected with HMPV at an MOI of 0.05 for 3 days. GAPDH mRNA served as a loading control. Abbreviations: MOI, multiplicity of infection; PCR, polymerase chain reaction; HMPV, human metapneumovirus; GO, gene ontology; FDR, false discovery rate; dpi, days post-infection; FPKM, fragments per kilobase of exon model per million mapped fragments; mRNA, messenger…

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  Fig. 3. Polyclonal knockout cells were cultured following transfection with the RNP complex to evaluate the knockout efficiency. Cell lysates were prepared and analyzed by western blot using polyclonal antibodies specific to each target: A) SEC11A knockout cells, B) FASTKD1 knockout cells, C) PCGF6 knockout cells, and D) TATDN1 knockout cells. β-actin was used as an internal loading control. Total RNA was extracted from the polyclonal knockout cells, reverse-transcribed, and subjected to SYBR green-based quantitative PCR to determine the RNA expression levels of the target genes. GAPDH mRNA served as a loading control, as determined by unpaired t test. * P < 0.05, ** P < 0.01, **** P < 0.001, ns indicates no significant difference (P > 0.05). Abbreviations: RNP complex, sgRNA:Cas9 molar ratio ranging from 5:1 to 10:1; mRNA, messenger ribonucleic acid; PCR, polymerase chain reaction; WT, wild type.

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  Fig. 4. HMPV-driven alterations in SEC11A mRNA splicing facilitate viral replication. A) Different polyclonal knockout cells were infected at an MOI of 0.05, and the copy number of the HMPV N gene in the cell culture supernatant was measured at 2 days post-infection (triplicate replicates). A significant reduction in HMPV infection was observed in SEC11A-KO cell lines, as determined by ordinary one-way ANOVA. B) Infected cells were subjected to immunofluorescence staining using anti-HMPV-F protein antibody, with nuclei counterstained by DAPI. C) The relative fluorescence intensity was quantified using ImageJ software, as determined by ordinary one-way ANOVA. D) Cell pellets collected before and after infection were lysed for western blot analysis to detect the expression of HMPV-F protein and host target proteins, as determined by two-way ANOVA. At 2 and 3 dpi with HMPV, both the copy number of the HMPV N gene (E) and the number of infectious viral particles (F) in the supernatant of SEC11A-KO cells showed a significant reduction compared to wild-type cells, as determined by two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant difference (P >…

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  Fig. 5. The specificity of SEC11A in HMPV-host interaction. A) Infection models were established as follows: A549 cells infected with IAV (TCID50 = 100) or HAdV (MOI = 0.01), HEp-2 cells infected with RSV (MOI = 0.01), BEAS-2B cells infected with HPIV3 (MOI = 0.01), and H1-HeLa cells infected with HRV-A81 (MOI = 0.01). Total RNA was extracted from all cell models at 2 dpi. Subsequently, SYBR green-based quantitative PCR was performed using virus-specific primers to detect viral mRNA expression levels (one-way ANOVA, ****P < 0.0001). B) Alternative splicing of the host target SEC11A mRNA in different infection models was analyzed by RT-PCR and agarose gel electrophoresis using the aforementioned primers. GAPDH mRNA served as a loading control. Abbreviations: MOI, multiplicity of infection; HMPV, human metapneumovirus; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcription-polymerase chain reaction; dpi, days post-infection; HPIV3, human parainfluenza virus type 3; HRV-A81, human rhinovirus A81.