Journal: Nagoya Journal of Medical Science

Article Title: Endosomal maturation leads to nucleocapsid conformation change in seasonal coronaviruses

doi: 10.18999/nagjms.88.1.109

Figure Lengend Snippet: High-titer coronavirus purification Fig. 1A: Schematics of virus purification steps. HCoV-229E and/or HCoV-OC43 were infected in Huh7 cells at 35 °C for more than one week. Supernatant of cell culture was harvested by low-speed centrifugation and followed to ultracentrifugation. Viral pellets were suspended in HEPES buffer, and the viral fraction was fractionated by sucrose density gradient ultracentrifugation. Fig. 1B: SDS-PAGE and CBB staining of viral fractions. Nucleoprotein was highly detected in fractions no. 9–11, and these fractions were pooled and concentrated as final purified virus fraction. Fig. 1C: Purified viruses infect cultured cells. The titer of purified viral samples was determined by TCID50, and the expression of nucleoprotein (N) when infected with Huh7 cells at MOI=1 was confirmed by Western blot. Fig. 1D: HCoV-229E is taken up into cells by endocytosis. Results of RT-qPCR quantification of viral infection after 24 hours. Viral uptake measured by RT-qPCR was completely inhibited by Bafilomycin A1 treatment. Unpaired student’s t-test; P=0.0001. Fig. 1E: Subcellular distribution of nucleoproteins 0.5 or 2 hours post infection at MOI=5 or 10. Viral nucleoproteins were stained with anti-Rab5A and/or EEA1 antibodies (shown in magenta). Co-localisation signals were shown in the arrowhead. The green signal shows nucleoproteins. Scale bar: 10 µm. SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis CBB: coomassie brilliant blue TCID50: tissue culture infectious dose 50 MOI: multiplicity of infection RT-qPCR: real-time quantitative polymerase chain reaction

Article Snippet: HCoV-229E or HCoV-OC43 were purchased from ATCC and amplified by Huh7 cells.

Techniques: Purification, Virus, Infection, Cell Culture, Centrifugation, SDS Page, Staining, Expressing, Western Blot, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Nagoya Journal of Medical Science

Article Title: Endosomal maturation leads to nucleocapsid conformation change in seasonal coronaviruses

doi: 10.18999/nagjms.88.1.109

Figure Lengend Snippet: Unanchored ubiquitin is not detected within purified seasonal coronavirus particles Fig. 2A: TEM images of various viruses. Left, influenza A virus (IAV); center, HCoV-229E; right, HCoV-OC43. Scale bar shows 100 nm. Fig. 2B: Detection of ubiquitin chains within viral extracts, 16.8 µg, 12.7 µg, and 27.8 µg of IAV, HCoV-229E, and HCoV-OC43 viral extract were loaded, respectively. Ubiquitin signals were detected with various anti-ubiquitin and anti-diglycyl lysine antibodies. Asterisks indicate non-specific signals. TEM: transmission electron microscopy HCoV: human coronavirus

Article Snippet: HCoV-229E or HCoV-OC43 were purchased from ATCC and amplified by Huh7 cells.

Techniques: Ubiquitin Proteomics, Purification, Virus, Transmission Assay, Electron Microscopy

Journal: European Journal of Microbiology & Immunology

Article Title: Phytochemicals and micronutrients in suppressing infectivity caused by SARS-CoV-2 virions and seasonal coronavirus HCoV-229E in vivo

doi: 10.1556/1886.2023.00010

Figure Lengend Snippet: Effect of test composition on weight change, viral load and spike protein in lung tissue infected and re-infected with human coronavirus 229E (HCoV-229E) . A. Effect of four-days of oral administration of MixV in K18-hACE2 mice with no significant weight change after 3 dpi and 5 dpi and oral administration of MixV with mid-term re-infection. B. The viral load in lung tissue after 3 dpi with oral administration of MixV and 5 dpi with oral administration and mid-term re-infection, determined by RT-qPCR as described in Materials and Methods section. C. Spike protein detection by WB in representative lung tissue samples. D. Quantification of spike protein performed as described in Materials and Methods section. Data are presented as mean ± SD; infected untreated animal n = 8, infected treated animal n = 8, re-infected untreated animal n = 8, re-infected treated animal n = 8; # P ≤ 0.05, ∆ P ≤ 0.01, dpi - days post-infection

Article Snippet: HCoV-229E anti-spike antibody was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Infection, Quantitative RT-PCR

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: Cell Host & Microbe

Article Title: Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response

doi: 10.1016/j.chom.2021.12.005

Figure Lengend Snippet:

Article Snippet: hCoV-229E spike protein , Sino Biological , 40605-V08B.

Techniques: Recombinant, Blocking Assay, Neutralization, Expressing, Transfection, Plasmid Preparation, Binding Assay, Software

Journal: PLOS ONE

Article Title: Evaluation of the Kaira COVID-19/Flu/RSV Detection Kit for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus: A comparative study with the PowerChek SARS-CoV-2, influenza A&B, RSV Multiplex Real-time PCR Kit

doi: 10.1371/journal.pone.0278530

Figure Lengend Snippet: Analytical specificity evaluation results of the Kaira assay.

Article Snippet: Human coronavirus 229E , ATCC (VR-740D) , Negative.

Techniques: Virus

Journal: Scientific Reports

Article Title: Commercially available SARS-CoV-2 RT-qPCR diagnostic tests need obligatory internal validation

doi: 10.1038/s41598-023-34220-w

Figure Lengend Snippet: List of commercially available molecular standards (nucleic acid solutions) used in analysis of the specificity of the three studied RT-qPCR assays.

Article Snippet: Quantitative Genomic RNA from Human coronavirus 229E , ATCC-VR-740DQ.

Techniques: Virus

Journal: Scientific Reports

Article Title: Commercially available SARS-CoV-2 RT-qPCR diagnostic tests need obligatory internal validation

doi: 10.1038/s41598-023-34220-w

Figure Lengend Snippet: RT-qPCR amplification parameters for Novel Coronavirus (2019-nCoV) Real Time Multiplex RT-PCR Kit (Liferiver), Vitassay qPCR SARS-CoV-2 (Vitassay) and TaqPath COVID‑19 CE-IVD RT-PCR Kit (Thermo Fisher Scientific).

Article Snippet: Quantitative Genomic RNA from Human coronavirus 229E , ATCC-VR-740DQ.

Techniques: Amplification, Multiplex Assay

Journal: Virology Journal

Article Title: Mycophenolate mofetil exerts broad-spectrum antiviral activity against coronaviruses including SARS-CoV-2

doi: 10.1186/s12985-025-02673-2

Figure Lengend Snippet: Primers used in qRT-PCR assay

Article Snippet: The primary antibodies used were antibodies against glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Boston, MA, USA, 1:1000), HCoV-229E NP (Sino Biological, Beijing, China, 1:8000), HCoV-OC43 nucleocapsid protein (NP; Millipore, Billerica, MA, USA,1:1000), and IMPDH2 (Abcam, Cambridge, UK, 1:1000).

Techniques: Sequencing

Journal: Virology Journal

Article Title: Mycophenolate mofetil exerts broad-spectrum antiviral activity against coronaviruses including SARS-CoV-2

doi: 10.1186/s12985-025-02673-2

Figure Lengend Snippet: The antiviral activity of MMF is achieved by non-competitive inhibition of the IMPDH enzyme. a Schematic diagram of time-of-addition assay. b , c Time-of-addition assay of MMF. Cells were seeded onto 96-well plates and infected with HCoV-229E or HCoV-OC43 (MOI = 3.5) for 1 h on ice. 5 μM MMF was added at different time points and cells were fixed at 9 h. Intracellular viral NP protein was detected with immunofluorescence (IF) assay. Green particles represent NP and the blue ones represent cell nucleus. The quantitative study on immunofluorescence was comparison of the total fluorescence values of each experimental group with the control group and analyzed by Image J software. d , e CETSA assay to detect the binding between IMPDH and MMF in the cell lysis of Huh7.5 and H460. Each lane represents the protein level of IMPDH at various temperatures after incubation with MMF or DMSO control, and relative density of IMPDH was normalized by the protein gray value at 47 °C ( n = 3). f , g Cells were infected with HCoV-229E (MOI = 0.35) or HCoV-OC43 (MOI = 0.35), and treated with different concentrations of guanosine and MMF for 24 h. The cells were harvested for western blotting assay ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. virus control (Con) or # P < 0.05, ## P < 0.01, ### P < 0.001 vs. MMF treated group (The third group), one-way ANOVA with Holm-Sidak multiple comparisons test

Article Snippet: The primary antibodies used were antibodies against glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Boston, MA, USA, 1:1000), HCoV-229E NP (Sino Biological, Beijing, China, 1:8000), HCoV-OC43 nucleocapsid protein (NP; Millipore, Billerica, MA, USA,1:1000), and IMPDH2 (Abcam, Cambridge, UK, 1:1000).

Techniques: Activity Assay, Inhibition, Infection, Immunofluorescence, Comparison, Fluorescence, Control, Software, Binding Assay, Lysis, Incubation, Western Blot, Virus

Journal: Virology Journal

Article Title: Mycophenolate mofetil exerts broad-spectrum antiviral activity against coronaviruses including SARS-CoV-2

doi: 10.1186/s12985-025-02673-2

Figure Lengend Snippet: Anti-coronavirus activities of MPA and MMF

Article Snippet: The primary antibodies used were antibodies against glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Boston, MA, USA, 1:1000), HCoV-229E NP (Sino Biological, Beijing, China, 1:8000), HCoV-OC43 nucleocapsid protein (NP; Millipore, Billerica, MA, USA,1:1000), and IMPDH2 (Abcam, Cambridge, UK, 1:1000).

Techniques:

Journal: Virology Journal

Article Title: Mycophenolate mofetil exerts broad-spectrum antiviral activity against coronaviruses including SARS-CoV-2

doi: 10.1186/s12985-025-02673-2

Figure Lengend Snippet: MMF and MPA inhibited viral protein levels and viral yield of HCoV-229E and HCoV-OC43. Cells were infected with HCoV-229E (MOI = 0.35) or HCoV-OC43 (MOI = 0.35), and treated with different concentrations of MMF or MPA for 24 h. a–f The cells were harvested for western blotting assay ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. virus control (Con), one-way ANOVA with Holm-Sidak multiple comparisons test. g–j The cells were fixed and NP was detected with immunofluorescence (IF) assay. Green particles represent NP and the blue ones represent cell nucleus. The quantitative study on immunofluorescence was comparison of the total fluorescence values of each experimental group with the control group and analyzed by Image J software. k–l Viral titers were detected by the 50% tissue culture infectious dose assay (TCID 50 ) ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. virus control (Con), one-way ANOVA with Holm-Sidak multiple comparisons test

Article Snippet: The primary antibodies used were antibodies against glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Boston, MA, USA, 1:1000), HCoV-229E NP (Sino Biological, Beijing, China, 1:8000), HCoV-OC43 nucleocapsid protein (NP; Millipore, Billerica, MA, USA,1:1000), and IMPDH2 (Abcam, Cambridge, UK, 1:1000).

Techniques: Infection, Western Blot, Virus, Control, Immunofluorescence, Comparison, Fluorescence, Software

Journal: Virology Journal

Article Title: Mycophenolate mofetil exerts broad-spectrum antiviral activity against coronaviruses including SARS-CoV-2

doi: 10.1186/s12985-025-02673-2

Figure Lengend Snippet: Combinatorial effects of MMF and other antiviral compounds. a , b Dose–response matrixs and dose–response curves of combination of MMF and molnupiravir. c , d Dose–response matrixs and dose–response curves of combination of MMF and GC376. e , f Dose–response matrixs and dose–response curves of combination of MMF and E64d. In detail, Huh7.5 cells were infected with HCoV-229E (MOI = 0.35) for 24 h (a, c, e, n = 3, mean ± SD) and H460 cells were infected with HCoV-OC43 for 24 h (MOI = 0.35) (b, d, f, n = 3, mean ± SD). The horizontal and vertical coordinates of the dose–response matrix are the concentrations of the combined drugs. NP mRNA levels was determined by qRT-PCR assay and relative quantification. The inhibition of virus replication of non-administrated group was regarded as zero to calculate the inhibition of each administration group

Article Snippet: The primary antibodies used were antibodies against glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Boston, MA, USA, 1:1000), HCoV-229E NP (Sino Biological, Beijing, China, 1:8000), HCoV-OC43 nucleocapsid protein (NP; Millipore, Billerica, MA, USA,1:1000), and IMPDH2 (Abcam, Cambridge, UK, 1:1000).

Techniques: Infection, Quantitative RT-PCR, Inhibition, Virus