Fig. 1.

Human coronaviruses induce HSF1 phosphorylation and DNA-binding activity. A The human coronavirus lipid bilayer comprising the spike protein (blue), the membrane protein (orange) and the envelope protein (green), and the viral RNA (purple) associated with the nucleocapsid protein (pink) are shown. B Schematic representation of genome structure, classification and receptors of the human coronaviruses HCoV-229E, HCoV-NL63 and HCoV-OC43. ORF1a and ORF1b are represented as light blue boxes; genes encoding structural proteins spike (S), nucleocapsid (N), envelope (E), membrane (M), and hemagglutinin-esterase (HE) and genes encoding accessory proteins are shown. hAPN, human aminopeptidase N; 9-O-Ac-Sia, N-acetyl-9-O-acetylneuraminic acid; ACE2, angiotensin-converting enzyme 2. C Immunoblot (IB) analysis of pHSF1-Ser326, HSF1, viral nucleocapsid (N) and β-actin protein levels in MRC-5 and Caco-2 hACE2 cells mock-infected (−) or infected (+) with HCoV-229E or HCoV-OC43 (MRC-5) for 24 h, or HCoV-NL63 (Caco-2 hACE2) for 72 h at a m.o.i. of 0.1 TCID₅₀/cell. D, E Whole-cell extracts (WCE) from samples mock-infected (Mock) or infected with HCoV-229E (1 TCID₅₀/cell) were analyzed for pHSF1-Ser326, HSF1, N and α-tubulin protein levels at early (D) or late (E) times post infection (p.i.) by IB. F Schematic representation of HSF1 domain organization: DBD, DNA Binding Domain; HR-A/HR-B, Heptad Repeats A and B; RD, Regulatory Domain; HR-C, Heptad Repeat C; AD, Activation Domain. Phosphorylation sites Ser121, Ser303, and Ser326 are shown. G MRC-5 cells were treated with bortezomib (BTZ, 20 nM) for 16 h, exposed to heat stress (HS, 43 °C, 40 min), mock-infected (Mock) or infected with HCoV-229E (0.1 TCID₅₀/cell) for 40 h. WCE were analyzed for levels of HSF1-Ser326, -Ser303 and -Ser121 phosphorylation, HSF1, viral N and α-tubulin proteins by IB (top panels). In the same samples, HSF1 DNA-binding activity was analyzed by EMSA (bottom panel). Positions of the HSF DNA-binding complex (HSF), constitutive HSE-binding activity (CHBA) and nonspecific protein-DNA interaction (NS) are shown. H MRC-5 cells were mock-infected or infected with HCoV-229E (1 TCID₅₀/cell). At different times p.i., HSF1 DNA-binding activity was analyzed by EMSA. I WCE from samples infected with HCoV-229E (0.1 TCID₅₀/cell, 40 h p.i.) were preincubated with different dilutions of anti-HSF1 or anti-HSF2 antibodies and analyzed by gel mobility supershift assay. The position of the nonsupershifted virus-induced HSF1 complex is indicated at the left (No Ab)