a549 expressing hace2  (ATCC)

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: rH234A exhibited impaired replication in human lung-derived epithelial cells. ( A and B ) A549-AT cells were infected with rWT or rH234A at 0.1 and 1 MOI. The culture supernatants were collected for extracellular viral titration ( A ). Whole cell lysates were collected for RNA extraction and subsequent quantification of viral N mRNA level ( B ). Data in A and B were shown as mean ± SD ( n = 3), and data in B were analyzed with a two-way analysis of variance Tukey’s multiple comparison test. **, P < 0.01; ***, P < 0.001. ( C ) Western blotting of viral N protein in A549-AT cells (0.1 MOI). β-Actin was served as a loading control. ( D−F ) Growth curves of rWT and rH234A in A549-A (0.1 or 5 MOI) or Calu-3 cells (0.1 MOI). Data in this figure are representative of at least two independent experiments. Data in D−F are shown as mean ± SD ( n = 3).

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Derivative Assay, Infection, Titration, RNA Extraction, Comparison, Western Blot, Control

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: rH234A exhibited impaired replication in primary human lung epithelial cells. ( A ) Schematic diagram of HBEC differentiation. ( B and C ) Differentiated HBECs in Transwell inserts were inoculated with rWT or rH234A at 0.1 MOI. ( B ) The apical mucus layers were collected for viral titration, and ( C ) the cells on the membrane were harvested for viral N mRNA quantification. ( D ) Schematic diagram of alveolar type 2 organoids induction and differentiation. ( E and F ) AT2 organoids grown in Matrigel were inoculated with rWT or rH234A at 0.1 MOI. ( E ) The culture Matrigel content was collected for viral titration. ( F ) The organoids were harvested for viral N mRNA quantification. Data in this figure are representative of two independent experiments and shown as mean ± SD ( n = 3). Data in C and F were analyzed with a two-way analysis of variance Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01. The images in A and D were prepared using Biorender.com .

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Titration, Membrane, Comparison

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: SARS-CoV-2 Nsp15 antagonizes type I and III IFN activation. ( A ) Quantification of IFN- β, IFN- λ 3 , ISG56 mRNA, and viral N gene in A549-A cells infected with 5 MOI of rWT or rH234A by RT-qPCR. ( B ) Immunoblotting phosphorylated Stat1 (p-Stat1), total Stat1 (t-Stat1), viral N protein (N pro), and β-actin of A549-A cells infected with 5 MOI of rWT or rH234A. ( C ) RT-qPCR quantification of IFN- β, IFN- λ 3 , ISG56 mRNA, and viral N gene in Calu-3 cells infected with 1 MOI of rWT or rH234A. ( D ) RT-qPCR quantification of IFN- β, IFN- λ 3 , ISG56 mRNA, and viral N gene in Caco2-AT cells infected with 0.1 MOI of rWT or rH234A. Data in A , C , and D are shown as mean ± SD ( n = 3) and were analyzed with a two-way analysis of variance Tukey’s multiple comparison test. Data in this figure are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Activation Assay, Infection, Quantitative RT-PCR, Western Blot, Comparison

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: The H14 and W332 residues are important for Nsp15-mediated IFN antagonism. ( A ) Growth kinetic curves of recombinant viruses in Vero-AT cells (0.01 MOI). ( B ) Growth curves (left) and titer fold difference (right) of recombinant viruses in A549-A cells (0.1 MOI) (right). ( C ) RT-qPCR quantification of IFN- β, IFN- λ 1 , and ISG56 mRNA in Caco2-AT cells infected with 0.1 MOI of indicated recombinant virus. Data in A and B are representative of at least two independent experiments and are shown as mean ± SD ( n = 3-6). Data in C are the pooled results of two independent experiments, shown as mean ± SD ( n = 4-6), and were analyzed with a two-way analysis of variance Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Recombinant, Quantitative RT-PCR, Infection, Virus, Comparison

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: rH234A infection differentially promotes the nuclear trafficking of PABPC1. ( A ) RNA integrity analysis of A549-A cells infected with rWT or rH234A at 5 MOI. The cells were harvested at indicated times for whole RNA extraction and subsequent TapeStation analysis. ( B ) The RIN of the total RNA collected in A . ( C ) Immunofluorescence assay (IFA) of viral dsRNA and host PABPC1 protein. A549-A cells were infected with rWT or rH234A at 5 MOI and fixed at 24 HPI. Viral dsRNA and host PABPC1 protein were probed with specific antibodies, and the nucleus was stained with Hoechst 33342. Infected cells with or without PABPC1 nuclear translocation are indicated with yellow- and white-color arrows, respectively. Scale bar: 20 µM. ( D ) Quantification of PABPC1 nuclear localization in virus-infected cells from 14 to 15 views of IFA slides. Data are shown as mean ± SEM ( n = 14-15) and analyzed using unpaired t -test with Welch’s correction. Data in the figure are representative of at least two independent experiments.

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Infection, RNA Extraction, Immunofluorescence, Staining, Translocation Assay, Virus

Journal: Journal of Virology

Article Title: SARS-CoV-2 Nsp15 endoribonuclease subverts host defenses to enhance viral fitness in lung cells

doi: 10.1128/jvi.01175-25

Figure Lengend Snippet: rH234A infection does not trigger stress granule formation in human lung cells. ( A ) Immunoblotting p-PKR, t-PKR, p-eIF2α, t-eIF2α, and β-actin of A549-A cells infected with 5 MOI of either rWT or rH234A and harvested at the indicated HPIs. The band intensities were measured with ImageJ. The relative protein levels were calculated as the band intensity relative to β-actin compared to the mock groups. ( B ) Immunofluorescence assay of viral N protein (N pro, red) and host G3BP1 protein (green). A549-A cells were cultured with 2% fetal calf serum media (Starvation) or infected with rWT or rH234A at 5 MOI and fixed at 24 HPI. The N protein and G3BP1 were detected with specific antibodies, and the nucleus was stained with Hoechst 33342. Scale bar: 20 µM. ( C ) Some cells in B show colocalization of the N protein and G3BP1. The RGB profile shows the fluorescent intensity signals of the N protein and G3BP1. Scale bar: 20 µM. Data in this figure are representative of at least two independent experiments.

Article Snippet: A Caco2 cell line expressing hACE2 and hTMPRSS2 (Caco2-AT) and an A549 cell line expressing hACE2 and hTMPRSS2 (A549-AT), gifts from Dr. Mohsan Saeed (Boston University), were cultured in DMEM with 10% FBS, 1× Pen/Strep, 1 μg/mL puromycin, and 1 μg/mL blasticidin (InvivoGen, ant-bl-05).

Techniques: Infection, Western Blot, Immunofluorescence, Cell Culture, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Diabetes exacerbates SARS-CoV-2 replication through ineffective pulmonary interferon responses, delayed cell-mediated immunity, and disruption of leptin signaling

doi: 10.3389/fcimb.2025.1513687

Figure Lengend Snippet: Silencing of the leptin receptor in A459-hACE2 cells enhances SARS-CoV-2 replication in the presence of high leptin levels. (A) Experiment design for evaluating the effect of LEPR-deficiency and high concentration of leptin on SARS-CoV-2 replication in vitro . A549-hACE2 were seeded on 24-well plates and treated with anti-LEPR siRNA (A549-hACE2 LEPR- ) or a control siRNA (A549-hACE2 CTL ), followed by the addition of recombinant leptin (rLeptin). Cells were infected with a recombinant SARS-CoV-2 USA-WA1/2020 expressing enhanced GFP (ic-SARS-CoV-2-eGFP) following the addition of recombinant leptin. Cells were harvested and the percentage of eGFP + cells were counted by flow cytometry. (B) Relative gene expression of LEPR in A549-hACE2, A549-hACE2 LEPR- and A549-hACE2 CTL cells at 48h post-transfection. Bars represent the mean ± standard deviation. (C) Representative multiplex immunofluorescence staining of LEPR (green), human ACE2 (red), and nucleus (grey) in A549, A549-hACE2, A549-hACE2 LEPR- and A549-hACE2 CTL cells at 48h post-transfection (Scale bar: 20 μm). Percentage of eGFP + A549-hACE2 LEPR- and A549-hACE2 CTL cells at 12 hpi (D) , 24 hpi (E) , and 48 hpi (F) in the presence of 0, 10, and 100 ng/ml of rLeptin. The insets illustrate the fold change of eGFP + cells between A549-hACE2 LEPR- and A549-hACE2 CTL , both treated with 100 ng/mL of rLeptin, at 12 and 24 hpi. Bars represent the mean ± standard deviation of four independent experiments performed in triplicate. Each dot represents the average of the triplicate. Relative gene expression of IFNb1 in A549-hACE2 LEPR- and A549-hACE2 CTL at 12 hpi (G) and 24 hpi (H) demonstrated an increase of IFNb1 expression in A549-hACE2 LEPR- and with concurrent administration of rLeptin. Bars represent the mean ± standard deviation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: Human lung carcinoma cells (A549; NR-52268) and A549 expressing human angiotensin-converting enzyme 2 (A549-hACE2; NR-53821) were obtained from BEI Resources (Manassas, VA) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% FBS, 1% 1× non-essential amino acids (Gibco).

Techniques: Concentration Assay, In Vitro, Control, Recombinant, Infection, Expressing, Flow Cytometry, Gene Expression, Transfection, Standard Deviation, Multiplex Assay, Immunofluorescence, Staining

Journal: iScience

Article Title: Characterizing fitness and immune escape of SARS-CoV-2 EG.5 sublineage using elderly serum and nasal organoid

doi: 10.1016/j.isci.2024.109706

Figure Lengend Snippet: Comparative fitness of EG.5.1 and XBB.1.9.1 (A) Beta-galactosidase staining on differentiated nasal organoid monolayers from a younger adult (28 years old) and an older adult (68 years old). Upper panel: light microscopy; Lower panel: Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm (left), 50 μm (right). (B) Viral replication in VeroE6/TMPRSS2 cell line, A549/TMPRSS2/ACE2 cell line or nasal organoids derived from the younger adult or the older adult. The viral load was determined using RT-qPCR. Data represent mean, and error bar represents one standard deviation. The viral load at each time point was compared using Student’s t test. ∗, p < 0.05. (C) Competition assay between EG.5.1 and XBB.1.9.1. Human nasal organoid derived from the young adult was infected with EG.5.1 and XBB.1.9.1 at a EG.5.1:XBB.1.9.1 TCID 50 ratio of 1:1. Next generation sequencing was performed on the cell culture supernatants that were collected at 24, 48 and 72 hpi. The percentage of reads corresponding to the EG.5.1 was determined using the spike amino acid residues 52 and 456. The data at 0 h time point represents the sequencing result of the 1:1 mixture. Data represent mean, and error bar represents one standard deviation.

Article Snippet: TMPRSS2 and ACE2 over-expressing A549 cells (A549/TMPRSS2/ACE2) were obtained from InvivoGen (Catalog code: a549-hace2tpsa).

Techniques: Staining, Light Microscopy, Derivative Assay, Quantitative RT-PCR, Standard Deviation, Competitive Binding Assay, Infection, Next-Generation Sequencing, Cell Culture, Sequencing

Journal: iScience

Article Title: Characterizing fitness and immune escape of SARS-CoV-2 EG.5 sublineage using elderly serum and nasal organoid

doi: 10.1016/j.isci.2024.109706

Figure Lengend Snippet:

Article Snippet: TMPRSS2 and ACE2 over-expressing A549 cells (A549/TMPRSS2/ACE2) were obtained from InvivoGen (Catalog code: a549-hace2tpsa).

Techniques: Virus, Recombinant, Modification, Saline, Plasmid Preparation, Staining, Sequencing, Software