Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation

Figures

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  FIG 1. Protein profile in EV-A71-infected and noninfected RD cells. (A and B) Representative silver-stained 2-DE maps of proteins from EV-A71-mock-infected (noninfected) (A) and EV-A71-infected (B) RD cells (n = 3). Spots that were successfully identified by MALDI-TOF-MS and MS/MS scanning are marked with numbers. (C) Genes with significant differences in expression between infected and uninfected RD cells were subjected to GO analyses. The log10 values of the P values are indicated by bar plots. MAPK, mitogen-activated protein kinase. (D) Enlarged sections of the 2-DE maps showing different expressions of spot 10 (GRp78), spot 49 (Hsp27), and spot 7 (PHB) between EV-A71-infected and noninfected (mock-infected) RD cells. (E and F) RD cells were either mock infected or infected with EV-A71 at an MOI of 10 for 6 h. A Western blot assay was used to detect the expression of GRp78, PHB, and Hsp27 (the relative fold change of protein expression is indicated) (E) and other stress response proteins, ERp57, Hsp70, and Hsp90 (F).

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  FIG 2. Upregulation of Hsp27 expression in EV-A71-infected RD cells. (A) RD cells were infected with or without EV-A71 at an MOI of 10. The expression level of Hsp27 was determined by RT-qPCR. (B) RD cells were infected with or without EV-A71 at an MOI of 10 for 3 h, 6 h, 9 h, and 12 h. The Hsp27 expression level was confirmed by Western blot assays. The density of each band was scanned and first normalized to that of actin. The value for the target protein band was set at 1 in the mock-infected control. (C) HeLa cells were infected with EV-A71 at an MOI of 10 for 9 h or 12 h. The expression levels of Hsp27 and viral protein VP1 were determined by a Western blot assay. (D) RD cells were infected with infectious EV-A71 or UV-inactivated EV-A71 at an MOI of 1 or 10 for 12 h. The expression level of Hsp27 was determined by RT-qPCR. GAPDH was used as the internal control. The experiments were conducted in triplicate and repeated at least three times. Statistical analyses were carried out using Student's t test. *, P < 0.05; **, P < 0.01. Data are expressed as the mean ± SD from three experiments.

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  FIG 3. Inhibition of EV-A71 replication by knockdown of Hsp27 in HeLa and RD cells. (A and B) HeLa cells were transfected with negative-control siRNA (NC) or Hsp27 siRNA (siHsp27) for 48 h, and then the cells were infected with EV-A71 and the intracellular viral protein level of VP1 (A) and the viral RNA level (B) were determined by Western blotting and RT-qPCR, respectively. (C to E) RD cells were transfected with negative-control or Hsp27 siRNA for 48 h, and then the cells were infected with EV-A71. (C and D) The EV-A71 mRNA levels in both the cytoplasm (C) and the supernatant (D) were evaluated by RT-qPCR. (E) The Hsp27 level was confirmed by Western blotting at 48 h posttransfection. GAPDH was used as the internal control. Statistical analyses were carried out using Student's t test. *, P < 0.05; **, P < 0.01. Data are expressed as the mean ± SD.

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  FIG 4. Inhibition of EV-A71 replication by knockout of Hsp27. (A) The designed target site in exon 1 of Hsp27 by CRISPR/Cas9. (B) Hsp27 was knocked out in RD cells, and the cells were infected with EV-A71 at an MOI of 1 for 6 h. Viral protein and viral RNA levels were determined by Western blot assay and RT-qPCR, respectively. (C) RD wild-type (WT), Hsp27-KO clone 1 (KO-1), and Hsp27-KO clone 2 (KO-2) cells were infected with EV-A71 at an MOI of 0.01. Photos were taken at 48 h postinfection (p.i.). The wild-type and Hsp27-KO cells were infected with EV-A71 at an MOI of 1 for 6, 9, and 12 h. (D) The viral protein level was determined by Western blot assay. (E) The intracellular viral RNA level was examined by RT-qPCR. (F) The virus titer in the supernatant was determined by TCID50 assay. GAPDH was used as the internal control. Statistical analyses were carried out using Student's t test. *, P < 0.05; **, P < 0.01. Data are expressed as the mean ± SD.

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  FIG 5. Promotion of EV-A71 replication by restored Hsp27 expression in Hsp27-KO cells. Hsp27-KO cells were transfected with the empty vector and plasmid pHsp27, and at 48 h posttransfection, the cells were infected with EV-A71. The intracellular (A) and extracellular (B) viral RNA levels, the viral protein level (C), and the viral titer in the supernatant (D) were determined by RT-qPCR, Western blot assay, and TCID50 assay, respectively. GAPDH was used as the internal control. Statistical analyses were carried out using Student's t test. *, P < 0.05. Data are expressed as the mean ± SD.

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  FIG 6. Promotion of EV-A71 infection by ectopic expression of Hsp27. (A to D) HEK 293T cells were transfected with an Hsp27 expression plasmid for 48 h. (A) The cells were then infected with EV-A71 at an MOI of 1 for 12 h. The expression levels of Hsp27 and viral protein VP1 were detected by Western blot assay. (B and C) The cells were infected with EV-A71 at an MOI of 1 for 6 h, 9 h, and 12 h, and the intracellular (B) and extracellular (C) viral RNA levels were examined by RT-qPCR. (D) The virus titer in the supernatant was determined by TCID50 assay. (E and F) HeLa cells were transfected with the empty vector or plasmid pHsp27. At 48 h posttransfection, the cells were infected with EV-A71 at an MOI of 1 for 9 h and 12 h, and the level of viral protein VP1 and the viral RNA level were detected by Western blot assay and RT-qPCR, respectively. GAPDH was used as the internal control. Statistical analyses were carried out using Student's t test. *, P < 0.05. Data are expressed as the mean ± SD.

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  FIG 7. Upregulation of viral IRES activity by Hsp27. (A) Wild-type and Hsp27-KO RD cells were transfected with the pIRES reporter plasmid. The luciferase activity was determined at 48 h posttransfection as described in the Materials and Methods section. (B) HEK 293T cells were cotransfected with pHsp27 and the pIRES reporter plasmid for 48 h, and luciferase activity was then measured. (C) Wild-type and Hsp27-KO RD cells were cotransfected with the control or 2Apro-expressing plasmid and the pIRES reporter plasmid for 48 h, and then the luciferase activity was measured. (D) HEK 293T cells were cotransfected with pHsp27, the pIRES reporter plasmid and a 2Apro-expressing plasmid, or the empty vector for 48 h, and then the luciferase activity was measured. (E) HEK 293T cells were transfected with pHsp27 for 48 h, and the expression levels of eIF4G and Hsp27 were determined by Western blot assay. (F) HEK 293T cells were cotransfected with pHsp27 and the 2Apro-expressing plasmid or the empty vector for 48 h, and the cleaved eIF4G was determined by Western blot assay. (G) Wild-type and Hsp27-KO RD cells were infected with EV-A71 at an MOI of 1 or 5 for 6 h, and the cleaved eIF4G was determined…

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  FIG 8. Inhibition of EV-A71-induced hnRNP A1 translocation by knockout of Hsp27. (A) Wild-type and Hsp27-KO cells RD were infected with EV-A71 at an MOI of 20 or 40 for 6 h. The cells were fixed, and the subcellular localization of hnRNP A1 was investigated by indirect immunofluorescence assay. (B) Wild-type and Hsp27-KO RD cells were infected with EV-A71 at an MOI of 10 for 6 h. Cell extracts were used for RNA immunoprecipitation assay with anti-hnRNP A1 antibody or normal anti-IgG. After washing and dissociation, the RNA was extracted using the TRIzol reagent and subjected to RT-PCR using primers that were specific to either the EV-A71 5′ UTR or ribosomal protein S16 (RPS16). Statistical analyses were carried out using Student's t test. **, P < 0.01. Data are expressed as the mean ± SD.

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  FIG 9. No effects on FBP1 translocation by knockout of Hsp27. Wild-type and Hsp27-KO RD cells were infected with EV-A71 at an MOI of 20 for 6 h. The cells were fixed, and the subcellular localization of FBP1 was investigated by indirect immunofluorescence assay.

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  FIG 10. Suppression of EV-A71 infection by a small-molecule inhibitor of Hsp27. (A) RD cells were pretreated with TDP for 2 h at the indicated concentrations and then infected with EV-A71 at an MOI of 0.01. Photos were taken at 48 h postinfection (p.i.). RD cells were pretreated with TDP for 2 h at the indicated concentrations and then infected with EV-A71 at an MOI of 1 for 12 h. (B) The intracellular viral RNA level was detected by RT-qPCR. (C) The expression level of eIF4G and the level of viral protein VP1 were determined by Western blot assay. (D) The viral titer in the supernatant was determined by TCID50 assay. (E) The pIRES reporter plasmid was transfected into cells which had been pretreated with TDP for 2 h, and IRES activity was measured at 48 h posttransfection. Statistical analyses were carried out using Student's t test. *, P < 0.05; **, P < 0.01. Data are expressed as the mean ± SD.