Fig. 3.

HCoV infection turns on an HSF1-driven transcriptional program in human lung cells. A–D Expression profile of selected HSF1-target genes affected by HCoV-229E infection (0.1 TCID₅₀/cell) for 24 h in MRC-5 cells relative to mock-infected cells as determined by qRT-PCR array (PAHS-076ZD-2-Qiagen). Heat Map (A) and Volcano plot (B) of 84 human HSPs and chaperones/cochaperones gene expression. In A each row represents a single gene, each column represents a sample [mock-infected or HCoV-229E infected cells (229E); n = 3]. The gradual color ranging from blue to red represents the mRNA expression level (Z-score). In the Volcano plot (B) fold regulation threshold is set to 2 and p-value cut off is 0.05; each dot represents a gene: red dots indicate significantly upregulated genes and blue dots indicate significantly downregulated genes. Selected HSPs and chaperones/cochaperones genes whose expression is highly induced by HCoV infection are shown in C; levels of heat shock factors (HSF1, HSF2 and HSF4) gene expression affected by HCoV infection are shown in D. E Expression of non-canonical HSF1-target genes AIRAP, COX-2 and NKRF in samples treated as in A as determined by qRT-PCR. Error bars indicate means ± S.D. (n = 3). *p < 0.05; Student’s t-test (D, E). F Levels of HSP90, GRP94, GRP78, HSP70, HSPA6, HSP60, AIRAP, viral spike (S) and β-actin proteins were determined by IB in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID₅₀/cell) at different times p.i.. G Schematic representation of the puromycin-labeling experimental protocol. H MRC-5 cells were mock-infected (−) or infected with HCoV-229E (+) (1 TCID₅₀/cell), or treated with vehicle (−) or cycloheximide (CHX, 100 µg/ml, +) for 3 h, as positive control of translation inhibition. At different times p.i., puromycin (2.5 µg/ml, +) was added thirty minutes before harvesting and IB analysis. Blot membranes were stained with Ponceau S solution to assess the steady-state proteomes (top panel) and then hybridized with anti-puromycin antibodies to detect de novo synthesized nascent polypeptides (middle panel). HCoV-N protein is indicated by red arrowheads. Levels of GRP94, GRP78, HSP70, HCoV-N and β-actin proteins detected by IB in the same samples are shown (bottom panels)