Small Interfering RNAs Targeting VP4, VP3, 2B, or 3A Coding Regions of Enterovirus A71 Inhibit Viral Replication In Vitro

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  Figure 1. Schematic representation of the enterovirus A71 (EV-A71) genome and siRNA target sites. The EV-A71 RNA genome consists of a single open reading frame with untranslated regions (UTRs) at the 5′ and 3′ ends. At the 5′ UTR end, the genome is covalently connected to viral protein genome-linked (VPg), a small peptide that plays a crucial role in protein priming and replication. The 3′ UTR incorporates a poly-A tail. The open reading frame encodes a polyprotein that includes four structural proteins (VP4, VP2, VP3, and VP1) in the P1 region, as well as seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) in the P2 and P3 regions. Fifteen target sites are indicated on the EV-A71 genome, corresponding to specific siRNAs listed in Table 1. Each target site is labeled with the name of the corresponding siRNA.

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  Figure 2. Monitoring of cells transfected with diverse concentrations of siRNA. HeLa cells were transiently transfected with different concentrations (0, 5, 10, 25, 50, 100, 200, and 300 nM) of FAM (fluorescein amidite)-labeled scrambled control siRNAs. The control group (0 nM) was not treated with siRNA and received only Lipofectamine. (A,B) Evaluation of siRNA effects using fluorescence detection of FAM. Confocal microscopy images showing green fluorescence indicating the expression of siRNA at 48 h (A) and 72 h post-transfection (B). The nucleus was stained with Hoechst 33342. Scale bar represents 100 μm. (C) The proportions of FAM-labeled HeLa cells transfected different concentrations of FAM-labeled scrambled control siRNAs at 72 h post-transfection were shown using flow cytometry. (D,E) Cytotoxicity assessment of siRNA-transfected HeLa cells. Cellular viability was quantified using a water-soluble tetrazolium salt assay at 48 h (D) and 72 h post-transfection (E). The results are presented as the percentage of viable cells compared to that of the siRNA-untreated group (0 nM) and are represented as the means ± SDs from three independent experiments. Statistical significance is…

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  Figure 3. Evaluation of the antiviral activity of individual siRNAs against enterovirus A71 (EV-A71) infection. HeLa cells were transiently transfected with 25 nM siRNA for 24 h prior to virus infection. Subsequently, the cells were infected with EV-A71 at a multiplicity of infection of 0.001. (A,B) Extracellular viral titers were measured by plaque assays in the cell supernatants at 24 hpi (A) and 48 hpi (B). As controls, HeLa cells treated with Lipofectamine alone (Lipo only) and cells treated with scrambled siRNA (scr siRNA) alone were infected with the virus. The viral titers are presented as plaque-forming units per milliliter (PFU/mL), quantified from the supernatants of infected cells. The data represent the mean ± SD of three independent experiments. (C) Immunofluorescence analysis was performed at 36 hpi to visualize EV-A71 replication and siRNA-mediated inhibition. Cells were stained with EV-A71 monoclonal antibody (red) and Hoechst 33342 (blue) and visualized using a fluorescence microscope. The images show Lipofectamine-treated HeLa cells without siRNA (a), scr-siRNA-treated HeLa cells (b), 2B-114 siRNA-treated HeLa cells (c), and 3A-150 siRNA-treated HeLa cells (d). The scale…

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  Figure 4. Effect of siRNAs on immune response and viral protein synthesis in enterovirus A71 (EV-A71)-infected HeLa cells. HeLa cells were transfected with each siRNA at a concentration of 25 nM. Twenty-four hours post-transfection, the cells were infected with EV-A71 at a multiplicity of infection of 0.001. Western blot analysis was performed on cell lysates collected at 24 h post-infection to assess the levels of PKR, phospho-PKR (p-PKR), and EV-A71 VP1. The relative protein quantities of the cells with siRNAs targeting structural proteins are shown in (A), while those with siRNAs targeting nonstructural proteins are shown in (B), compared to scrambled (scr)-siRNA-treated cell lysates. β-Actin was detected as the internal control. (C–F) The intensity of each band was quantified using the Image J software (version 1.53). The VP1 protein expression levels were normalized by β-actin (C,E). The p-PKR protein expression levels were normalized by PKR (D,F). All data are presented as means ± SD from three independent experiments, compared with the scr siRNA group. Statistical significance is indicated by asterisks, denoting t test p values (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not…

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  Figure 5. Evaluation of the antiviral activity of enterovirus A71 (EV-A71)-specific siRNAs against coxsackievirus B3 (CVB3) infection. HeLa cells were transiently transfected with 25 nM siRNA for 24 h prior to virus infection and then infected with CVB3 at a multiplicity of infection of 0.00001. (A) Cell supernatants were harvested at 48 hpi, and extracellular viral titers were measured by plaque assays. As controls, HeLa cells treated with Lipofectamine alone (Lipo only) and cells treated with scr siRNA alone were infected with the virus. The viral titers are presented as plaque-forming units per milliliter (PFU/mL), quantified from the supernatants of infected cells. The data represent the mean ± SD of three independent experiments. Statistical significance is indicated by asterisks, denoting t test p values (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant). (B) After 48 h of infection, cell survival was measured using a water-soluble tetrazolium salt assay. The mock-infected group represents cells without virus infection. The results are shown as the percentage of viable cells compared to scr-siRNA-transfected cells for each siRNA-transfected cell. Data represent the mean…