Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: The antiviral effect of Tg against HCoV-229E infection does not require IRE1, ATF6, or PERK expression. (A) Stable UPR knockdown A549 cell lines were generated via lentiviral transduction. Silencing of protein expression was confirmed via western blot. (B–F) A549 shCTRL, shIRE1, shATF6, and shPERK cells were primed with DMSO or Tg (0.05 µM) for 30 minutes, then washed prior to infection with HCoV-229E (MOI 0.05). Cell lysates (B–E) or supernatants (F) were collected at 24 hpi. (B–D) Protein expression of IRE1, ATF6, PERK and HCoV-229E N were assessed by western blot. (E) Changes in gene expression were quantified by RT-qPCR. Data are normalized to actin and set relative to shCTRL DMSO. (F) Supernatants were used to determine viral titers by plaque assay on Huh7 cells. Due to variability between replicates, titration data is expressed as a percentage relative to the shCTRL DMSO in each experiment. Graphs show means ± SD from 3 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Infection, Expressing, Knockdown, Generated, Transduction, Western Blot, Gene Expression, Quantitative RT-PCR, Plaque Assay, Titration

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological inhibition of IRE1 or ATF6 does not affect the antiviral activity of Tg against HCoV-229E infection. (A and B) A549 cells were primed with DMSO or Tg (0.05 µM) in the absence or presence of KIRA6 (10 µM) or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hours post-treatment. Xbp1s (A) or HERPUD1 (B) protein expression was assessed by western blot. (C) A549 cells were treated with DMSO, KIRA6 (10 µM), or Ceapin-A7 (6 µM) for 24 hours. Alamar blue assay was used to assess cell viability, which is expressed as a percentage relative to DMSO. (D and F) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or KIRA6 (10 µM). Cell lysates were collected at 24 hpi to assess XBP1s and N gene expression by RT-qPCR (D and F), or Xbp1s and HCoV-229E N protein expression by western blot (E). (G and I) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of Ceapin-A7 (6 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hpi to assess HERPUD1 and N expression by RT-qPCR (G and I), or HERPUD1 and HCoV-229E N protein expression by western blot (H). RT-qPCR data are normalized to actin and set relative to DMSO. (J) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM) or Ceapin-A7 (6 µM), alone or in combination, prior to infection with HCoV-229E (MOI 0.05). Inoculum was removed 2 hours later and replaced with media containing the respective inhibitor(s). Viral supernatants were collected 24 hpi and titrated by plaque assay on Huh7 cells. Graphs show mean ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate (* p < 0.05, *** p < 0.001; **** p < 0.0001). Representative western blots are shown.

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Inhibition, Activity Assay, Infection, Expressing, Western Blot, Alamar Blue Assay, Gene Expression, Quantitative RT-PCR, Plaque Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological activation of PERK inhibits HCoV-229E replication. (A–C) A549 cells were treated with DMSO or one of the selective UPR activators for 24 hours. IXA4 (10 µM) activates IRE1, AA147 (10 µM) activates ATF6, and CCT020312 (5 µM) activates PERK. RNA lysates were collected 24 hours post-treatment and the expression of genes downstream of each UPR pathway was assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (D) A549 cells were primed with Tg (0.5 µM) for 30 minutes, then washed and infected with HCoV-229E (MOI 0.5). Alternatively, cells were treated with AA147 (10 µM) or CCT020312 (5 µM) for 24 hpi. Cell lysates were collected to assess the expression of HERPUD1, PERK and 229E N protein by western blot. (F and G) A549 cells were primed with DMSO or Tg (0.05 µM) for 30 minutes prior to infection with HCoV-229E (MOI 0.05). Alternatively, cells were infected and then treated with media containing DMSO or the selective UPR activators alone or in combination. Cell lysates and supernatants were collected at 24 hpi. (F) Changes in HCoV-229E N gene expression were assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (F) Viral titer was assessed by plaque assay on Huh7 cells. (H–J) A549 cells were infected with HCoV-229E (MOI 0.05), then incubated with increasing concentrations of CCT020312 for 24 h. Cell lysates were collected to assess changes in HCoV-229E N gene (H) and CHOP (I) expression by RT-qPCR. (J) Cell viability was assessed by Alamar blue assay. Graphs show means ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way ANOVA or t -test (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Infection, Western Blot, Gene Expression, Plaque Assay, Incubation, Alamar Blue Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: UPR activation by tunicamycin does not recapitulate the antiviral effect of Tg. (A–C) A549 cells were primed with Tg (0.5 µM) for 30 minutes or with Tm (1 µg mL −1 ) for 4 hours. Cells were then washed and either mock-infected or infected with HCoV-229E (MOI 0.05). At 24 hpi, cell lysates were collected to evaluate expression of UPR target genes ( CHOP , HERPUD1 and Xbp1s ) or HCoV-229E N gene by qPCR. Graphs show means ± SD from 2 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way or two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Infection, Expressing

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Enzyme-linked Immunospot

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Expressing