african green monkey kidney vero e6 cells  (ATCC)

Journal: iScience

Article Title: FGFR signaling and neddylation facilitate SARS-CoV-2 infection by modulating interferon induction and viral entry, respectively

doi: 10.1016/j.isci.2025.114566

Figure Lengend Snippet: Inhibiting FGFR signaling enhances the interferon response (A) HEK293T-ACE2 cells were transfected with a SARS-CoV-2 nanoluciferase reporter replicon. Then, the cells were treated with MLN4924 (100 nM), BGJ398 (10 μM), remdesivir (10 μM), IFNα (100 unit/mL), or DMSO alone. Luminescence (expressed as arbitrary units) was measured at 24 and 48 h post-treatment. (B) Schematic of FGFR and downstream signaling pathways and inhibitors used to block them (indicated in red). (C) HEK293T-ACE2 cells were pretreated with the indicated inhibitors of signaling pathways downstream of FGFR for 24 h and then infected with SARS-CoV-2 (72B/CA/CALG) using an MOI of 0.1. Twenty-four hours later, media was collected and subjected to the plaque assay to determine viral titers. (D) Normal human bronchial epithelial (NHBE) cells were treated with DMSO alone, MLN4924, or BGJ398 at 1 μM for 24 h and then infected with 400 HAU/mL of Sendai virus. Total cellular RNA was harvested 4 or 8 h post infection (hpi) and then subjected to qRT-PCR to determine relative levels of mRNA encoding type I IFNβ. (E) NHBE cells were treated as described in (D). Total cellular RNA was harvested 8 hpi and then subjected to qRT-PCR to determine relative levels of mRNA encoding ISGs (OAS1, MX1, and MX2). (F) Vero cells were pretreated with MLN4924 (10 μM), BGJ398 (100 μM), remdesivir (10 μM), or DMSO for 24 h and then infected with SARS-CoV-2 (72B/CA/CALG), using an MOI of 0.01. Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. Data shown are averaged from three independent experiments. Error bars represent standard error of the mean. One-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance between the control (DMSO) and drug-treated samples. p value < 0.01 ∗∗, <0.001 ∗∗∗, <0.0001 ∗∗∗∗, ns = not significant (A, C, D, E, and F).

Article Snippet: Vero CCL81, HEK 293T, HEK293FT, Vero E6, Huh 7.5, A549 and Calu-3 cells were sourced from the American Type Culture Collection (Manassas, VA), Vero-TMPRSS2 from JCRB cell bank 1819, and HEK 293T-ACE2 and NCI-H23-ACE2 were developed in-house by stable transduction of ACE2 expressing lentivirus constructs.

Techniques: Transfection, Protein-Protein interactions, Blocking Assay, Infection, Plaque Assay, Virus, Quantitative RT-PCR, Comparison, Control

Journal: iScience

Article Title: FGFR signaling and neddylation facilitate SARS-CoV-2 infection by modulating interferon induction and viral entry, respectively

doi: 10.1016/j.isci.2025.114566

Figure Lengend Snippet: An internal deletion within the spike protein confers resistance to the neddylation inhibitor MLN4924 (A) SARS-CoV-2 (72B/CA/CALG) was passaged in Vero cells in the presence of MLN4924 or DMSO alone. For passage 1, an MOI of 0.01 was used for infection. Twenty microliters of media from infected cells were then used to infect a new batch of cells after 72 h or when 40% of the cells showed CPE. (B) Vero cells were pretreated with DMSO or MLN4924 (10 μM) for 24 h and then infected with aliquots of serially passaged SARS-CoV-2 (72B/CA/CALG) (MOI of 0.01) as explained in (A) for each passage. Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. (C) HEK293T-ACE2 cells were pretreated with DMSO or MLN4924 or TAS4464 both at 100 nM for 24 h and then infected with SARS-CoV-2-WT or a SpikeΔ9 strain (MOI = 0.1). Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. Data shown are averaged from three independent experiments. Error bars represent standard error of the mean. One-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance between the control (DMSO) and drug-treated samples. p value < 0.05 ∗, <0.01 ∗∗, <0.0001 ∗∗∗∗, ns = not significant (B and C). (D) Titers from plaque assays conducted on media from Vero cells electroporated with infectious DNA clones for WT and SpikeΔ9. Error bars represent standard error of the mean. (E) Structural analysis of the spike protein deletion mutant NTD Δ68–76 associated with MLN4924 resistance. Left: mutant spike trimer model with one monomer highlighted (NTD in slate color, RBD in green). Right: NTD domains of wild type (slate) and the deletion mutant (wheat). In the wild-type spike, AlphaFold2 predicts that residues 68–76 (cyan sticks) form a disordered loop-like structure that is absent in cryo-EM structures. In NTDΔ68–76, the missing loop is indicated by an arrow. Residues A67 and K68 (corresponding to K77 in the NTD-WT) are shown in green. Despite the deletion, the overall NTD structure is preserved in the mutant. (F) Analysis of the spike NTDΔ68–76 bound to N12-9 Fab antibody (PDB: 7V23 ). The left image shows the spike trimer with one monomer (deep salmon) bound to the antibody. The antibody’s heavy and light chains bound to the NTD are colored in limon and deep olive, respectively. The right inset overlays the NTDΔ68–76 modeled using AlphaFold2 (wheat), with the NTDΔ68–76 bound to the antibody (PDB: 7V23 , deep salmon). Antibody binding to the NTD induces movement of the Tyr248–Ser256 loop, as indicated by the dotted arrow. In the absence of the disordered loop (Δ68–76), the bordering residues His66, Ala67, Lys68, and Arg69 connect to form a more ordered structure as shown in green. This conformation is consistent between the cryo-EM and AlphaFold2 models. The green ball-and-stick representation in the bottom image highlights the network of interactions formed by these residues. Atoms involved in these interactions are labeled in black, with hydrogen bonds (black) and stacking interactions (pink) shown as dotted lines.

Article Snippet: Vero CCL81, HEK 293T, HEK293FT, Vero E6, Huh 7.5, A549 and Calu-3 cells were sourced from the American Type Culture Collection (Manassas, VA), Vero-TMPRSS2 from JCRB cell bank 1819, and HEK 293T-ACE2 and NCI-H23-ACE2 were developed in-house by stable transduction of ACE2 expressing lentivirus constructs.

Techniques: Infection, Plaque Assay, Comparison, Control, Clone Assay, Mutagenesis, Cryo-EM Sample Prep, Binding Assay, Labeling