HSP90AB1 Is a Host Factor Required for Transmissible Gastroenteritis Virus Infection

Figures

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  Figure 1. qRT-PCR analysis of viral replication kinetics of TGEV in ST cells. ST cells were infected with TGEV, and viral RNA copies were detected at 6, 12, 18, 24, 36, 42, and 48 hpi using qRT-PCR.

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  Figure 2. Identification of HSP90AA1 and HSP90AB1 knockdown cells. (A) qRT-PCR and Western blot of HSP90AA1 gene mRNA and (B) protein levels in WT and HSP90AA1 KD cells. (C) The band density of the protein was quantified using Image J software, and the HSP90AA1 to ACTB ratios were normalized to the control. (D) qRT-PCR and Western blot of HSP90AB1 gene mRNA and (E) protein levels in WT and HSP90AB1 KD cells. (F) The band density of the protein was quantified using Image J software, and the HSP90AB1 to ACTB ratios were normalized to the control. (G,H) Cell viability of WT and (G) HSP90AA1 or (H) HSP90AB1 KD cells were evaluated by CCK-8 assay. * means p ≤ 0.05, ** means p ≤ 0.01, *** means p ≤ 0.001, ns means p > 0.05.

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  Figure 3. Effect of HSP90AA1 and HSP90AB1 knockdown on TGEV infection. (A) TGEV M gene mRNA levels, (B) viral titers in the cell supernatants, and (C) N protein levels in WT and HSP90AA1 KD cells. (D) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control. (E) TGEV M gene mRNA levels, (F) viral titers in the cell supernatants, and (G) N protein levels in WT and HSP90AB1 KD cells. (H) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control. ** means p ≤ 0.01, ns means p > 0.05.

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  Figure 4. Effect of HSP90AB1 overexpression on TGEV infection. (A) qRT-PCR and Western blot of HSP90AB1 gene mRNA and (B) protein levels in WT and OVER cells. (C) The band density of the protein was quantified using Image J software, and the HSP90AB1 to ACTB ratios were normalized to the control. (D) Cell viability of WT and OVER cells was evaluated by CCK-8 assay. (E) TGEV M gene mRNA levels, (F) viral titers in the cell supernatants, and (G) N protein levels in WT and OVER cells. (H) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control. ** means p ≤ 0.01, **** means p ≤ 0.0001, ns means p > 0.05.

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  Figure 5. VER-82576 could inhibit TGEV infection. (A) Schematic diagram of ST cells treated with the HSP90 inhibitors VER-82576 and KW-2478. Blue double-headed arrows indicate KW-2478 or VER-82576 treatment, and red double-headed arrows indicate TGEV incubation. (B,F) The relative mRNA level of the TGEV M gene was determined by qRT-PCR. (C,G) Viral titer in the supernatant was analyzed by TCID50 and (D,H) N protein level was determined by Western blot. (E,I) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control. * means p ≤ 0.05, ** means p ≤ 0.01, *** means p ≤ 0.001, ns means p > 0.05.

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  Figure 6. TGEV infection does not affect HSP90AA1 and HSP90AB1 expression. (A) HSP90AA1 or (B) HSP90AB1 gene mRNA level from ST cells infected with TGEV for 0, 3, 6, 12, 18, and 24 hpi were analyzed by qRT-PCR. (C) HSP90AA1 or (D) HSP90AB1 protein level from ST cells infected with TGEV for 0, 3, 6, 12, 18, and 24 hpi was analyzed by Western blot. (E,F) The band density of the protein was quantified using Image J software, and the (E) HSP90AA1 to ACTB and (F) HSP90AB1 to ACTB ratios were normalized to the control.

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  Figure 7. VER-82576 had no influence on the entry stage of TGEV infection. (A) Schematic diagram of ST cells treated with VER-82576 before (−1–0 h) or during (0–2 h) TGEV incubation. Blue double-headed arrows indicate VER-82576 treatment, and red double-headed arrows indicate TGEV incubation. (B,F) The relative mRNA level of TGEV M gene was determined by qRT-PCR. (C,G) Viral titer in the supernatant was analyzed by TCID50, and (D,H) N protein level was determined by Western blot. (E,I) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control.

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  Figure 8. VER-82576 inhibits TGEV infection at the early stage of viral replication. (A) Schematic diagram of ST cells treated with VER-82576 at different time points after viral entry. Blue arrows indicate the time point at which VER-82576 treatment began, and the blue line indicates the presence of VER-82576. Red down arrows and the red line indicate TGEV incubation. (B) TGEV M gene mRNA levels, (C) viral titers in the cell supernatants, and (D) N protein levels in VER-82576 treated ST cells. (E) The band density of the protein was quantified using Image J software, and the TGEV-N to ACTB ratios were normalized to the control. * means p ≤ 0.05, ** means p ≤ 0.01, ns means p > 0.05.

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  Figure 9. VER-82576 inhibited the production of proinflammatory cytokines induced by TGEV infection. (A–E) qRT-PCR detection of the mRNA levels of proinflammatory cytokines: (A) IL-6, (B) IL-12, (C) TNF-α, (D) CXCL10, and (E) CXCL11 induced by TGEV infection. (F–J) qRT-PCR detection of the inhibitory effect of VER-82576 on the mRNA level of proinflammatory cytokines: (F) IL-6, (G) IL-12, (H) TNF-α, (I) CXCL10, and (J) CXCL11 induced by TGEV infection. * means p ≤ 0.05, ** means p ≤ 0.01, ns means p > 0.05.