Journal: iScience

Article Title: Direct interaction of HMGB1 with SARS-CoV-2 facilitates its infection via RAGE-dependent endocytosis

doi: 10.1016/j.isci.2025.113063

Figure Lengend Snippet: HMGB1 induces SARS-CoV-2 infection in an ACE2-independent RAGE-dependent manner (A) Western blot analysis of receptors responsible for SARS-CoV-2 infection and HMGB1 binding. S.E., short exposure; L.E., long exposure. (B) Flow cytometry analysis of ectodomain ACE2 in A549 and NCI-H1975 cells used in this study. Vero E6 cells were used as control. (C) A549 cells were transfected with shRNA-ACE2 for 48 h prior to infection with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h at 37°C, cultured further for 3 h, and subjected to western blotting. (D and E) A549 cells were infected for 1 h with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h, in the presence of 40 μg/mL sRAGE at 37°C as indicated. Cells were harvested at 3 hpi and subjected to western blotting (D) n = 3, and qRT-PCR for viral RNA measurement (E) n = 3. (F) NCI-H1975 cells were infected with SARS-CoV-2 preincubated with 20 μg/mL HMGB1 in the presence of azeliragon. Cells were harvested at 3 hpi and subjected to western blotting. (G) A549 cells were infected with 5 MOI SARS-CoV-2, which was treated as above to observe NP. Representative confocal images are shown. The percentage of infected cells and NP intensity were measured by counting at least 700 visible cells. n = 4. (H) Cycloheximide pretreated NCI-H1975 cells were infected with 5 MOI SARS-CoV-2 (preincubated with HMGB1). Cells were stained for NP before permeabilization (green; external) and post-permeabilization (red; external and internal). Representative images and their magnifications are shown. The percentage of intracellular spots was measured by counting at least 200 visible cells. n = 4. (I and J) SARS2pp was preincubated with or without HMGB1 in the presence of sRAGE before transduction in NCI-H1975 cells. NanoLuc luciferase activity was measured 72 h post-transduction. n = 3 Scale bars represent 5 μm. Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant, using one-way ANOVA with Tukey’s multiple comparison test and Student’s unpaired t-test. HMGB1, high-mobility group box 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription polymerase chain reaction; NP, nucleocapsid protein; SEM, standard error of the mean; ANOVA, analysis of variance; hpi, hours post-infection; RAGE, receptor for advanced glycation end-products; ACE2, angiotensin-converting enzyme 2; SARS2pp, SARS-CoV-2 spike protein (S)-pseudotyped retrovirus.

Article Snippet: 40 μg/mL sRAGE (11629-HCCH, Sino Biological, Oklahoma City, OK, USA), azeliragon (S6415, Selleckchem, Houston, TX, USA), dynasore (D7693, Sigma-Aldrich, St. Louis, MO, USA) and chloroquine (C6628, Sigma-Aldrich) were pretreated for 2 h at 37°C, prior to the infection procedure.

Techniques: Infection, Western Blot, Binding Assay, Flow Cytometry, Control, Transfection, shRNA, Cell Culture, Quantitative RT-PCR, Staining, Transduction, Luciferase, Activity Assay, Comparison, Reverse Transcription, Polymerase Chain Reaction