Heat Shock Protein A6 Is Especially Involved in Enterovirus 71 Infection

Figures

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  Figure 1. EV71 infection specially upregulates HSPA6 gene expression. (A) Heat map of RNA-Seq transcriptome analysis for genes changed in RD cells infected with EV71 for 8 h. (B) RT-PCR measurement of HSPA6 and HSPA1, HSPA8, and HSPB1 expression in RD cells infected with EV71 for 8 h. (C) RT-PCR measurement of EV71 VP1 gene and positive (POS) strand in RD cells infected with EV71 for 8 h. (D) RT-PCR measurement of HSPA6 expression in RD cells infected with EV71 for 8 h and 24 h. (E) RT-PCR measurement of HSPA6 expression in U251 cells infected with EV71 for 24 h and 48 h. Control presented cell control, and PDL presented treating the cells with 10 μM of pendulectin at the same time of EV71 infection (**p < 0.01).

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  Figure 2. EV71 infection specially upregulates HSPA6 protein expression. (A) Western blotting analysis of HSPA1A, HSPA8, HSPB1, and viral VP1 expression in RD cells infected with EV71 for 8 or 24 h. (B) Western blotting analysis of HSPA6 expression in RD cells infected with EV71 for 8 or 24 h. The relative expression of HSPA6 was quantitatively analyzed based on the bands’ intensities from the Western blotting analysis. PDL (10 μM) here was used as anti-EV71 control (*p < 0.05, **p < 0.01).

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  Figure 3. HSPA6 knockdown inhibits EV71 replication and protein expression. (A) RT-PCR measurement of HSPA6 as well as viral VP1, positive (POS) strand, and negative (NEG) strand expression in RD cells transfected with siRNAs targeting HSPA6 (si-HSPA6) and infected with EV71. (B) Western blotting analysis of protein level of HSPA6 protein and viral VP1 in RD cells transfected with siRNAs and infected with EV71. Mock is the infected but untreated control, si-NC is the negative control siRNA targeting no known genes. Control in B is the cell control without viral infection and siRNA transfection. si-SCARB2 is siRNA targeting the receptor of EV71, using it as the positive control (vs. si-NC, **p < 0.01).

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  Figure 4. HSPA6 knockdown inhibits EV71 CPE formation and daughter virus production. (A) siRNA targeting HSPA6 protected RD cells from EV71-induced CPE. (B) siRNA targeting HSPA6 inhibited the production of the EV71 daughter virus. RD cells were transfected with siRNAs for 24 h and then infected with 0.2 MOI EV71 for 72 h, followed by cytopathic analysis. At the same time, the culture supernatant was subjected to plaque reduction assay.

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  Figure 5. EV71 infection enhances the cytoplasmic aggregation of HSPA6. (A) Confocal microscopy analysis of RD cells infected with EV71 at 8 hpi. (B) Confocal microscopy analysis of RD cells infected with EV71 at 24 hpi. Cells were stained with VP1/ Alexa Fluor 488, HSPA6/Alexa Fluor 594, and nuclei/DAPI.

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  Figure 6. HSPA6 is involved in the infection of EV71 but not other HFMD-associated viruses. (A) RT-PCR and Western blotting analysis of HSPA6 expression in RD cells infected with various HFMD viruses. RD cells were infected with 0.2 MOI of EV71 C4 strain, EV71 BrCr strain, CVA6, and CVA10, while Vero cells were infected with 1MOI of CVA16 and CVB1-3. After 8 h infection, the cells were subjected to RT-PCR analysis. Western blotting was also performed to analyze the expression of HSPA6 protein in RD cells infected with 0.2 MOI of EV71 or CVA6 at 36 hpi. (B) Survival analysis of RD cells transfected with siRNA targeting HSPA6 and infected with various viruses. RD cells or Vero cells were transfected with 100 nM of si-HSPA6 or si-NC for 24 h and then infected with the viruses for 72 h, followed by the cell survival detection with Alamar blue reagent.