The Eukaryotic Translation Initiation Factor 4F Complex Restricts Rotavirus Infection via Regulating the Expression of IRF1 and IRF7

Figures

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  Figure 1. Knockdown of eIF4A supports rotavirus infection. (A) Western blot assay detected eIF4A in Caco2 cells transduced with lentiviral RNAi vectors against eIF4A. (B) Successful knockdown by shRNA vectors measured by RT-qPCR (n = 6, ** P < 0.01, Mann–Whitney test). (C) Silence of eIF4A increased rotavirus genomic RNA level (n = 6, * P < 0.05, ** P < 0.01, Mann–Whitney test). (D) Silence of eIF4A increased viral VP4 protein level in SA11 rotavirus infected Caco2 cells. (E) Western blot detected the eIF4A protein level in Caco2 cells transduced with CRIPSRs against eIF4A, suggesting a successful knockout. (F) Silence of eIF4A by CRISPR/Cas9 increased the rotavirus genomic RNA level (n = 6, * P < 0.05, Mann–Whitney test). (G) Silence of eIF4A by CRISPR/Cas9 increased the viral VP4 protein level in SA11 rotavirus infected Caco2 cells.

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  Figure 2. The eIF4E suppresses rotavirus infection. (A) Western blot assay measured the eIF4E protein in Caco2 cells transduced lentiviral RNAi vectors against eIF4E, indicating that one out of three shRNA vectors exerted a potent knockdown. (B) The knockdown of eIF4E significantly promoted rotavirus genomic RNA (n = 6, ** P < 0.01, Mann–Whitney test). (C) Western blot assay confirmed that eIF4E knockdown increased rotavirus protein level. (D) The eIF4E was knocked out by CRISPR/Cas9 assay. (E) Silence of eIF4E by CRISPR/Cas9 increased the rotavirus RNA level (n = 6, * P < 0.05, Mann–Whitney test). (F) Silence of eIF4E by CRISPR/Cas9 increased the rotavirus VP4 protein level.

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  Figure 3. Silence of eIF4G promotes rotavirus infection. (A) Western blot assay detected eIF4G in Caco2 cells transduced with lentiviral RNAi vectors against eIF4G. (B) Successful knockdown by shRNA vectors detected by RT-qPCR assay (n = 6, *** P < 0.001, Mann–Whitney test). (C) Silence of eIF4G showed an increased rotavirus genomic RNA level (n = 6, * P < 0.05, ** P < 0.01, Mann–Whitney test). (D) Silence of eIF4G promoted the viral VP4 protein level in SA11 rotavirus infected Caco2 cells. (E) Western blot assay detected eIF4G in Caco2 cells transduced with CRIPSRs against eIF4G. (F) Silence of eIF4A by CRISPR/Cas9 increased the rotavirus genomic RNA level (n = 6, * P < 0.05, Mann–Whitney test). (G) Silence of eIF4G by CRISPR/Cas9 increased the viral VP4 protein level in SA11 rotavirus infected Caco2 cells.

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  Figure 4. Silence of eIF4A/E/G promotes rotavirus infection. (A) Western blot assay detected eIF4 A/E/G in Caco2 cells transduced CRIPRs against eIF4A, eIF4E, and eIF4G. (B) Silence of eIF4A/E/G promoted the rotavirus genomic RNA level detected by qPCR assay (n = 6, * P < 0.05, Mann–Whitney test). (C) Silence of eIF4A/E/G showed increased rotavirus protein VP4.

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  Figure 5. Programmed cell death protein 4 (PDCD4) sustains rotavirus infection. (A) Western blot assay detected PDCD4 in Caco2 cells transduced lentiviral RNAi vectors against PDCD4. (B) One out of three shRNA vectors showed successful knockdown detected by RT-qPCR (n = 6, *** P < 0.001, Mann–Whitney test). (C) Silence of PDCD4 showed inhibition of rotavirus genomic RNA levels (n = 8, * P < 0.05, Mann–Whitney test). (D) Silence of PDCD4 inhibited viral VP4 protein level in SA11 rotavirus infected Caco2 cells.

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  Figure 6. The eIF4F complex is required for IRF7 that exerts anti-rotavirus effect. (A) Western blot assay detected the protein level of antiviral factors including IRF1, IRF7, and RIG-I in eIF4A knockdown Caco2 cells. (B) Western blot assay detected the protein level of antiviral factors including IRF1, IRF7, and RIG-I in eIF4E knockdown Caco2 cells. (C) Western blot assay detected the protein level of antiviral factors including IRF1, IRF7, and RIG-I in eIF4G knockdown Caco2 cells. (D) Western blot assay detected the protein level of IRF7 in eIF4G knockout Caco2 cells, and two out of four shRNA vectors showed successful knockdown of IRF7 detected by western blot. (E) Silence of IRF7 increased rotavirus genomic RNA level detected by qPCR assay (n = 6, ** P < 0.01, *** P < 0.001, Mann–Whitney test). (F) Western blot assay detected the protein level of IRF7 in eIF4G knockout Caco2 cells, indicating the successful knockdown of IRF1 detected by western blot. (G) Silence of IRF1 increased the rotavirus genomic RNA level detected by qPCR assay (n = 6, ** P < 0.01, Mann–Whitney test).