Journal: bioRxiv
Article Title: Coronavirus protein interaction mapping in bat and human cells identifies molecular and genetic switches for immune evasion and replication
doi: 10.1101/2025.07.26.666918
Figure Lengend Snippet: (A) Schematic illustrating the generation of the RFe-AT cell line by introducing human ACE2 and TMPRSS2 into immortalized RFe cells (top). Western blot analysis (bottom) shows expression of human ACE2 and TMPRSS2 (full-length and cleaved forms) in the indicated cell lines. β-actin serves as a loading control. (B) Functional assessment of SARS-CoV-2 entry using pseudotyped HIV based particles. Luciferase-based entry assays were performed using HIV-based particles pseudotyped with SARS-CoV-2 WA/01 Spike (top) or VSV-G (bottom) in RFe, RFe-AT, HEK293T, and HEK293T-AT cells. Firefly luciferase activity is shown as relative luminescence units (RLU). (C) Assessment of SARS-CoV-2 entry mediated by ACE2 orthologs from human (hACE2) or bat (bACE2). HEK293T cells were transfected with vector control, hACE2, or bACE2, followed by infection with SARS- CoV-2 WA/01 at MOI 1. Top panel depicts the percentage of ACE2-positive cells determined by flow cytometry. Percentage of infected (N-positive) cells in ACE2-positive population was measured by intracellular staining for viral nucleocapsid (N) protein (bottom). Data represent ± SD from one of three independent experiments. (D) Replication kinetics of SARS-CoV-2 WA/01 in RFe-AT (red) and MRC5-hACE2 (blue) cell lines. Cells were infected with the virus at MOI 0.1. Virus supernatant was collected at 24, 48 and 72 h post-infection and plaque assays were performed in Vero E6 cells. (E) Schematic overview of the SARS-CoV-2 adaptation experiment in bat cells (top). The virus was serially passaged (P1-P3), followed by plaque purification and viral genome sequencing. Bar plots (bottom) show the frequency of specific missense mutations (N_P80T and Nsp4_T269I) in the viral population across passages. (F) Replication kinetics of RFe adapted SARS-CoV-2 WA1 virus in RFe-AT and MRC5-hACE2 cell lines. Cells were infected at MOI 0.1 in triplicates. Supernatant was collected at 24, 48 and 72 h post-infection and plaque assays were performed in Vero E6 cells. (G) Growth kinetics of recombinant SARS-CoV-2 wild-type (WT) and viruses carrying mutations leading to N_P80T or Nsp4_T269I amino acid substitutions in RFe-AT (left) and MRC5-hACE2 (right) cells. Cells were infected at MOI 0.1, and virus titers in supernatants were measured by plaque assay at the indicated time points. Data are presented as mean ± SD from one representative biological replicate (n = 3). *P < 0.05, **P < 0.005, ****P < 0.00005. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.
Article Snippet: To generate lentiviruses, HEK293T cells were transfected with human ACE2 or TMPRSS2 lentiviral plasmids (Addgene #154981 and #170390, respectively), along with packaging plasmids for Gag-Pol (psPAX2) and VSV-G (pMD2.G) in a 15 cm dish.
Techniques: Western Blot, Expressing, Control, Functional Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Infection, Flow Cytometry, Staining, Virus, Purification, Sequencing, Recombinant, Plaque Assay
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