hACE2-independent internalization of pseudo-typed SARS-CoV-2.

A, Upper: Schematic drawing of a pseudo-typed SARS-CoV-2 virion, VSVEGFP-S-A647, in which the EGFP-coding sequence was inserted into the genomic RNA of VSV, the G protein of VSV was replaced with S of SARS-CoV-2, and A647 was conjugated with primary amines of superficial envelope proteins. Lower: Confocal images of A647 of purified VSVEGFP-S-A647 (V-A647, red) on coverslips and immunofluorescent staining with antibody against S2 subunit of S-protein (green). B, Bright-field and confocal images of VSVEGFP-S-A647’s A647 (V-A647) and EGFP expression (V-EGFP) in BHK21 cells stably expressing hACE2 (BHKhACE2), BHK21 cells without ACE2 expression (BHK), and BHK cells transiently overexpressed with hACE2-BFP plasmid (BHK+hACE2). Top: Incubation protocol – cells were incubated with VSVEGFP-S-A647 for 1 h at 37°C, followed by washout for 24 h (applies to panels B-G). C, The white dotted box in panel B enlarged to show V-A647 spots in EGFP-labelled cytosol. D, Similar to panel B, except in 293ThACE2 and 293T cells. E, Similar to panel B, except in Vero (containing ACE2), Hela (no ACE2) and MRC5 (no ACE2) cells. F, Similar to panel B, except VSVEGFP-S-A647 was replaced with LentiEGFP-SOmi-A647. G, Fluorescence intensity of VSVEGFP-S-A647’s V-A647 (F647) and V-EGFP (FEGFP) in BHKhACE2 (471 cells, 3 independent experiments), BHK (370 cells, 3 independent experiments), BHK+hACE2 (515 cells, 3 independent experiments). 293ThACE2 (404 cells, 3 independent experiments), 293T (234 cells, 3 independent experiments), Vero (295 cells, 3 independent experiments), Hela (305 cells, 3 independent experiments), and MRC5 (237 cells, 3 independent experiments) cells. LentiEGFP-SOmi-A647’s F647 and FEGFP in BHKhACE2 (364 cells, 3 independent experiments) and BHK (335 cells, 3 independent experiments) cells are also plotted. Virus incubation protocol is shown in panel B. F647: mean ± s.e.m., reflecting viral uptake; FEGFP: mean ± s.e.m., reflecting viral infection. F647 and FEGFP were measured per cell identified in the bright field and were normalized to cells expressing ACE2 in each subgroup (see gaps between subgroups). ***: p < 0.001, t-test.

Endocytosis is the primary route for pseudo-typed SARS-CoV-2 internalization and gene expression.

A, Left upper: incubation protocol for viral attachment and early endocytosis – incubating cells with VSVEGFP-S-A647 for 1 h at 4°C, followed by 3 min washout with 500-750 μM A490 at 37°C (applies to panel A-D). Left lower: strategy for labeling endocytic vesicles taking up PM-attached virions (VSVEGFP-S-A647, red) with a fluorescent dye in the bath (A490, cyan). Right: STED images showing V-A647 spots (red dots) colocalization with A490 spots (cyan) in the cytoplasm (cell boundary beyond the plot). Representative images are from BHK cells. B, STED XY-plane images of V-A647 and A490 at different Z sections as labeled showing a cytosolic A490 spot (endocytic vesicle) containing a single V-A647 spot that reflects a single VSV-S virion. Representative images are from BHK cells. C, STED XY-plane images of V-A647 and A490 showing a cytosolic A490 spot may contain 1-4 V-A647 spots, each reflecting a single virion. Top: the V-A647 spot number (NA647) is labeled. Representative images from BHK cells. D, Left: WH distribution of A490 spots (SpotA490) containing V-A647 spots (55 SpotA490, 3 independent experiments from BHK cells). Middle: NA647 plotted versus the corresponding SpotA490 WH (55 SpotA490, 3 independent experiments, from BHK cells). A linear regression fit (correlation coefficient: 0.53) showed that larger SpotA490 contains more V-A647 spots. Right: V-A647-containing SpotA490 WH in BHKhACE2 (70 SpotA490, 3 independent experiments) and BHK cells (55 SpotA490, 3 independent experiments). E, Left: EM images showing that endosomes may contain one or more VSVEGFP-S-A647 virions (arrows). Representative images are from BHKhACE2 cells. Incubation protocol: VSVEGFP-S-A647 for 1 h at 4°C, 15 min washout at 37°C. Right: WH (mean ± s.e.m.) of EM-observed virus (VSVEGFP-S-A647)-containing endosome (38 endosomes, 2 independent experiments) and STED-observed virus (VSVEGFP-S-A647)-containing SpotA490 (70 SpotA490, 3 independent experiments). F, Bright field and confocal images of V-A647 (uptake) and V-EGFP (viral gene expression) in BHKhACE2 cells in control (Ctrl) or treated with dynasore (DnS, 80 μM, bath). Top: VSVEGFP-S-A647 incubation protocol – 1 h incubation, 24 h washout, all at 37°C. G, VSVEGFP-S-A647’s F647 (uptake) and FEGFP (gene expression) in cells in Ctrl (789 cells, 3 independent experiments), treated with DnS (653 cells, 3 independent experiments), or overexpressed with dynamin 2 K44A (19 cells, 2 independent experiments). Virus incubation protocol shown in panel F. **: p < 0.01; ***: p < 0.001, t-test. H, Left: bright field and confocal images of V-A647 (uptake) in BHKhACE2 cells in Ctrl or treated with cathepsin inhibitor 1 (CPS Inh, 10 μM). Top: VSVEGFP-S-A647 incubation protocol – 1 h incubation at 37°C. Right: bright field and confocal images of V-A647 (uptake) and V-EGFP (gene expression) in BHKhACE2 cells in Ctrl or treated with CPS Inh. Top: VSVEGFP-S-A647 incubation protocol – 1 h incubation, 24 h washout, all at 37°C. I, F647 (uptake) or FEGFP (gene expression) in cells in Ctrl (540 cells, 3 independent experiments) or treated with CPS Inh (742 cells, 3 independent experiments). F647 and FEGFP were measured after VSVEGFP-S-A647 incubation in panel H-left and H-right, respectively. ***: p < 0.001, t-test.

Heparan sulfate is the receptor for pseudo-typed SARS-CoV-2 attachment at the cell surface.

A, Immunolabelling (A647) of hACE2 in a BHKhACE2 (left) and a BHK (right) cell. B, ACE2-independent viral cell-surface attachment. Images: STED images showing virus cell-surface attachment in a BHKhACE2 (left) or BHK (right) cell. Middle: images in green (left) and yellow (right) boxes replotted on a larger scale. Top: incubation protocol – virus (VSVEGFP-S-A490) 1 h at 4°C for viral attachment at the cell surface (applies to panels B, C, E, and H). Bar graph: Cell-surface virus fluorescence intensity (FVirus) per BHKhACE2 (92 cells, 3 independent experiments) or BHK cell (98 cells, 3 independent experiments). Data normalized to the mean of the BHKhACE2 group. C, Cell-surface-attached viruses colocalized with HS puncta. Images: STED images of viral spots (VSVEGFP-S-A490’s V-A490) and immunolabelled HS (Alexa 488-conjugated antibody) at BHKhACE2 or BHK cell surface. Top: incubation protocol – VSVEGFP-S-A647 1 h at 4°C for cell-surface attachment. Bar graph: the percentage of viral spots overlapping with HS puncta in BHKhACE2 (8 cells, 4 independent experiments) or BHK cells (8 cells, 4 independent experiments). D, Bright field and confocal images of immunolabelled HS (left) and HS fluorescence intensity (FHS, right) in BHKhACE2 cells in Ctrl (188 cells, 3 independent experiments) or treated with Heparinases I/II/III (HPRase, 264 cells, 3 independent experiments). ***: p < 0.001, t-test. E, Images: bright field and confocal image of V-A647 in BHKhACE2 cells in Ctrl or treated with HPRase. Top: incubation protocol – VSVEGFP-S-A647 1 h at 4°C for cell-surface attachment. Bar graph: cell-surface F647 per cell for BHKhACE2 cells (left) in Ctrl (125 cells, 3 independent experiments) or treated with HPRase (150 cells, 3 independent experiments), and for BHK cells (right) in Ctrl (147 cells, 3 independent experiments) or treated with HPRase (127 cells, 3 independent experiments). ***: p < 0.001, t-test. F, Images: bright field and confocal image of V-A647 (uptake) in BHKhACE2 cells in Ctrl or treated with HPRase. Top: incubation protocol – VSVEGFP-S-A647 1 h at 4°C (for viral attachment), 1 h washout at 37°C (for endocytosis of cell-surface-attached viruses). ***: p < 0.001, t-test. Bar graph: F647 (uptake) for BHKhACE2 cells in Ctrl (201 cells, 3 independent experiments) or treated with HPRase (197 cells, 3 independent experiments), and for BHK cells in Ctrl (202 cells, 3 independent experiments) or treated with HPRase (206 cells, 3 independent experiments). G, Images: bright field and confocal image of V-EGFP expression in BHKhACE2 cells in control or treated with HPRase. Top: incubation protocol – VSVEGFP-S-A647 1 h at 4°C, 24 h washout at 37°C. ***: p < 0.001, t-test. Bar graph: FEGFP (viral gene expression) per BHKhACE2 cell in Ctrl (482 cells, 3 independent experiments) or treated with HPRase (391 cells, 3 independent experiments). H, STED images of viral spots (VSVEGFP-S-A490’s A490) and immunolabelled hACE2 puncta (A647) at a BHKhACE2 cell surface. I, STED images of viral spots (VSVEGFP-S-A490’s A490) and immunolabelled hACE2 puncta (A647) inside BHKhACE2 cells – hACE2 is sometimes taken up with viruses in endosomes.

SARS-CoV-2 cell entry path is analogous to pseudo-typed SARS-CoV-2

A, Images (bright field and confocal images) of VSVEGFP-Somi-A647’s V-A647 (left) and F647 (right, viral uptake) in BHKhACE2 (146 cells, 3 independent experiments) or BHK (149 cells, 3 independent experiments) cells. Top: incubation protocol – VSVEGFP-Somi-A647 1 h at 37°C (for virus endocytosis). B, Images (bright field and confocal images) of VSVEGFP-Somi-A647’s V-EGFP (left) and FEGFP (right, viral gene expression) in BHKhACE2 (273 cells, 3 independent experiments) or BHK (200 cells, 3 independent experiments) cells. Top: incubation protocol – VSVEGFP-Somi-A647 1 h, 24 h washout, all at 37°C (for viral gene expression). C, Images (bright field and confocal images) of CoV2mCh-Somi-A647’s A647 fluorescence (V- A647, left) and F647 (right, uptake) in BHKhACE2 (120 cells, 3 independent experiments) or BHK (120 cells, 3 independent experiments) cells. Top: incubation protocol – CoV2mCh-Somi-A647 1 h at 37°C (for virus endocytosis). D, Images (bright field and confocal images) of CoV2mCh-Somi-A647’s mCherry expression (V-mCherry, pseudo color, left) and FmCherry (right, uptake) in BHKhACE2 (120 cells, 3 independent experiments) or BHK (120 cells, 3 independent experiments) cells. Top: incubation protocol – CoV2mCh-Somi-A647 1 h, 6 h washout, all at 37°C (for viral gene expression). E, STED XY-plane images of CoV2mCh-Somi-A647’s V-A647 and A490 in cytosol showing that the number (NA647) of V-A647 spots (virions) in a A490 spot (endocytic vesicle) may vary as labelled. Top: incubation protocol – CoV2mCh-Somi-A647 1 h at 4°C, 3 min washout with 500-750 μM A490 at 37°C. F, Images (bright field and confocal images) of CoV2mCh-Somi-A647 ’s V-A647 (left) and surface F647 per cell (right) for BHKhACE2 cells in Ctrl (120 cells, 3 independent experiments) or treated with HPRase (120 cells, 3 independent experiments). Top: incubation protocol – CoV2mCh-Somi-A647 1 h at 4°C for cell-surface attachment.

Structural arrangement of HS as the viral receptor at the cell surface

A, Confocal (CONF) XY-plane images of immunolabelled HS (A647-conjugated secondary antibody) at various Z-axis locations as labeled (16 cells, 4 independent experiments). B, MINFLUX (2D) pixel-based rendering of immunolabelled HS (HS-Ab-F647, see Methods), the corresponding confocal (CONF) image of immunolabelled HS, and confocal PHmNG (labeling PM) image at a BHKhACE2 cell surface [extracellular (Extra) compartment is above the PM]. C, MINFLUX image of immunolabelled HS and STED image of CoV2mCh-Somi-A490 (Virus) at a BHKhACE2 cell surface (merged at the bottom). Cells were incubated with CoV2mCh-Somi-A490 for 1 h at 4°C for viral cell-surface attachment. Top is the extracellular side. D, MINFLUX coordinate-based rendering of HS clusters at the cell surface with each trace ID (TID) labeled in different colors (four different regions are shown). Extra: extracellular side; PM: plasma membrane position. E, Left: MINFLUX pixel-based rendering (upper) and confocal image (lower) of immunolabelled (HS-Ab-F647) single HS molecules in vitro (on the coverslip). Right: MINFLUX coordinate-based rendering of single HS molecules color-coded by trace IDs (TIDs) (data I, II, III, IV from left panel). F, Distribution of HS-molecule number per HS-cluster (NHS/cluster, 51 clusters, 8 cells, 3 independent experiments). G, MINFLUX pixel-based rendering (upper) and confocal image (lower) of CoV2-Somi (CoV2mCh-Somi-A647’s A647) at the cell surface (extracellular side: above the confocal spots). Cells were incubated with CoV2mCh-Somi-A647 for 1 h at 4°C for viral cell-surface attachment. H, MINFLUX coordinate-based rendering of CoV2mCh-Somi-A647 showing three individual virions from panel G (I, II, III in panel G). I, SARS-CoV-2 cell-entry model: heparan sulfate molecules in clusters protruding tens to hundreds of nanometers from the plasma membrane serve as the receptor for viral cell-surface attachment; endocytosis is the primary route to internalize surface-attached virions. If endocytosis co-internalizes local hACE2 in the close vicinity to the heparan sulfate cluster at the cell surface, viral RNA genome can be released from endosomes for viral genome expression to infect cells.

Attachment of SARS-CoV-2 JN.1 variant at the cell surface depends on heparan sulfate.

A, CoV2-SJN.1-A647 attachment at the cell surface: sampled bright field and confocal images of CoV2-SJN.1-A647’s V-A647 in wild-type CHO cells (CHO-K1), GAGs-deficient CHO-pgsA-745 cells lacking HS, and GAGs-deficient CHO-pgsB-618 cells lacking HS (images merged in the right) after incubation of CoV2-SJN.1 for 1 h at 4°C for labeling cell-surface attachment (incubation protocol shown on the top). B, Quantification of CoV2-SJN.1-A647 attachment at the cell surface: cell-surface fluorescence of CoV2-SJN.1-A647 (F647) per cell (mean + s.e.m.) in CHO-K1 (117 cells, 2 repeats), CHO-pgsA-745 (93 cells, 2 repeats), and CHO-pgsB-618 cells (90 cells, 2 repeats). ***: p < 0.001 (ANOVA test).

SARS-CoV-2 JN.1 variant attachment and infection in primary human airway cells depend on HS and are inhibited by a clinically used HS-binding agent.

A, Images for virus attachment: bright field, confocal, and merged images of CoV2-SJN.1-A647 in normal human bronchial/tracheal epithelial cells (NHBE) pre-treated with control (Ctrl), pixantrone (PIX), or anti-ACE2 antibody (ACE2 Ab). Top: incubation protocol – CoV2-SJN.1-A647 1 h at 4°C for cell-surface attachment. Bar graph for virus attachment: cell-surface fluorescence of CoV2-SJN.1-A647 (F647) per cell for normal human bronchial/tracheal epithelial (NHBE) cells pre-treated with control (Ctrl, 16 cells, 2 independent experiments), pixantrone (PIX, 18 cells, 2 independent experiments), or anti-ACE2 antibody (ACE2 Ab, 24 cells, 2 independent experiments). ***: p < 0.001 (ANOVA test). B, Cell-surface-attached viruses colocalized with HS puncta: confocal images of viral spots (CoV2-SJN.1-A647’s V-647, red) and immunolabelled HS (Alexa 488-conjugated antibody, green) at NHBE cell surface (images merged in the bottom). Top: incubation protocol – CoV2-SJN.1-A647 1 h at 4°C for cell-surface attachment. C, PIX, but not ACE2 antibody, inhibits CoV2-SJN.1 attachment in human primary small airway epithelial cells in air-liquid interface (ALI) format. Images: sampled confocal images of Hoechst (gray, labeling cell nucleus), antibody-labeled Nucleocapsid protein (N Protein of CoV2-SJN.1, green), and Spike protein (anti-S2P6 of CoV2-SJN.1, red) in ALI cells were incubated with CoV2-SJN.1 for 1 h at 4°C (for viral cell-surface attachment, protocol shown at the top). Before virus incubation, cells were pretreated with 1) control solution (Ctrl), 2) Pixantrone (PIX), or 3) anti-ACE2 antibody (ACE2 Ab). Images were projection images. Bar graphs: quantification of the fluorescence of immunolabelled nucleocapsid protein (FN protein, upper) and spike (FSpike, lower) for ALI cells incubated with CoV2-SJN.1 for 1 h at 4°C in three pre-treated conditions stated above (Ctrl: 2 independent experiments; PIX: 2 independent experiments; ACE2 Ab: 2 independent experiments). ***: p < 0.001 (ANOVA test). D, Both PIX and ACE2 antibody inhibit CoV2-SJN.1 infection of ALI cells. Images: Immunolabelled N-protein images from ALI cells being incubated with viruses with a protocol for viral genome expression in three conditions, including 1) control (Ctrl), 2) pretreated with PIX (before virus incubation), and 3) pretreated with ACE2 Ab. The images are from the entire ALI culture using 20x objective (scale bar: 1 mm). Incubation protocol (shown at the top): CoV2-SJN.1 incubation for 1 h at 4°C, followed by incubation at 37°C for 24 h. Bar graph: quantification of the fluorescence of immunolabelled N protein (anti-Nucleocapsid protein antibody) for ALI cells being incubated with CoV2-SJN.1 with a protocol for viral genome expression (protocol shown in the left) in three conditions stated above (Ctrl: 3 repeats; PIX: 3 repeats; ACE2 Ab: 3 repeats). ***: p < 0.001 (ANOVA test).

SARS-CoV-2 pseudovirus gene expression, but not internalization, depends on hACE2

The percentage (%) of cells containing VSVEGFP-S-A647’s V-A647 (%, mean ± s.e.m., upper) and V-EGFP (FEGFP, mean ± s.e.m., lower) in BHKhACE2 (3 experiments, 471 cells), BHK (3 experiments, 370 cells), BHK+hACE2 (3 experiments, 515 cells). 293ThACE2 3 experiments, 404 cells), 293T (3 experiments, 234 cells), Vero (3 experiments, 295 cells), Hela (3 experiments, 305 cells), and MRC5 (3 experiments, 237 cells) cells. LentiEGFP-SOmi-A647 virus’ V-A647 and V-EGFP in BHKhACE2 (3 experiments, 364 cells) and BHK (3 experiments, 335 cells) cells are also plotted. The virus incubation protocol is shown in Fig. 1B (1 h viral incubation, 24 h washout). The percentage of cells containing V-A647 or V-EGFP refers to cells identified in the bright field. ***: p < 0.001, t-test.

hACE2-dependent V-EGFP expression detected with flow cytometry

A, Cell counts (normalized to mode) plotted versus the EGFP fluorescence intensity (FEGFP) in BHK (left) and BHKhACE2 (right) cells incubated with VSVEGFP-S-A647 (green) or without VSVEGFP-S-A647 (black, control) at 37°C for 18 h. Ice-cold DPBS supplemented with EDTA solution was used instead of trypsinization, to avoid potential digestion of heparan sulfate proteoglycans. B, Percentage of cells expressing EGFP (V-EGFP) as detected by flow cytometry in BHK cells (biological triplicates for each experiment, 2 experiments) and in BHKhACE2 cells (biological triplicates for each experiment, 2 experiments) incubated with VSVEGFP-S-A647 (virus +) or without VSVEGFP-S-A647 (virus –, control) at 37°C for 18 h. ***: p < 0.001 (ANOVA test); n.s.: p > 0.05.

Uptake of both VSVEGFP-S-A647 and the bath A490 into the cytosol reflects viral endocytosis

A, Top: incubation protocol – VSVEGFP-S-A647 plus 50 μM A490 for 1 h at 37°C, followed by 24 h washout at 37°C. Bottom: strategy for labeling endocytic vesicles taking up PM-attached virions (VSVEGFP-S-A647, red) with a fluorescent dye in the bath (A490, cyan). B, Confocal images showing V-A647 spot (red dots) colocalization with A490 spots (green) in the cytosol. White dash line: cell outline from bright field images. C, The percentage of cytosolic V-A647 spots colocalized with A490 spots in BHKhACE2 (27 cells, 3 experiments) and BHK cells (21 cells, 3 experiments) after the incubation protocol shown in panel A.

Virus cell-surface attachment labels the cell outline

STED images showing most V-A647 spots (red dots) decorating the outline of the cell plasma membrane and A490 spots (cyan) within the outline. Incubation protocol is shown on the top: 1 h VSVEGFP-S-A647 at 4°C, 3 min washout with 500 μM A490.

Dynasore reduces VSVEGFP-S viral uptake and gene expression.

A, VSVEGFP-S-A647’s F647 in cells in control (Ctrl_1h, 256 cells) and treated with dynasore (DnS_1h, 248 cells) for 1 h, reflecting viral uptake. BHKhACE2 cells were treated with control (Ctrl) or dynasore (DnS, 80 μM, bath) with VSVEGFP-S-A647 at 37°C for 1 h for confocal images. Data are normalized to control group. ***: p < 0.001, t-test. B, VSVEGFP-S-A647’s FEGFP in cells in control (Ctrl_8h, 316 cells) and treated with dynasore (DnS_8h, 227 cells) for 8 h, reflecting viral gene expression. BHKhACE2 cells were treated with control (Ctrl) or dynasore (DnS, 80 μM, bath) with VSVEGFP-S-A647 at 37°C for 1 h, washout and imaged at 8 h. Data are normalized to control groups. ***: p < 0.001, t-test.

hACE2-independent viral attachment detected with flow cytometry

A, Cell counts (normalized to mode) plotted versus fluorescence intensity of VSVEGFP-S-A647’s V-A647 (F647) in BHK (left) and BHKhACE2 (right) cells incubated with VSVEGFP-S-A647 (red) or without VSVEGFP-S-A647 (black, control) at 4°C for 1 h. Ice-cold DPBS supplemented with EDTA solution was used instead of trypsinization, to avoid potential digestion of heparan sulfate proteoglycans. B, Percentage of cells associated with VSVEGFP-S-A647’ A647 (V-A647) detected with flow cytometry in BHK cells (biological triplicates for each experiment, 2 experiments) and in BHKhACE2 cells (biological triplicates for each experiment, 2 experiments) incubated with VSVEGFP-S-A647 (virus +) or without VSVEGFP-S-A647 (virus –, control) at 4°C for 1 h. **: p < 0.01; ***: p < 0.001 (ANOVA test); n.s.: p > 0.05.

hACE2-independent viral attachment detected across a virus dilution series.

Percentage of cells associated with VSVEGFP-S-A647’ A647 (V-A647) detected with flow cytometry in BHK and in BHKhACE2 cells incubated with VSVEGFP-S-A647 at 4°C for 1 h across different virus concentrations generated by serial dilution. Similar attachment was observed in BHK and BHKhACE2 cells across the tested concentration range, indicating that VSV-S cell-surface attachment is largely independent of hACE2.

Sample images of the cell plasma membrane labelled with PHSNAP

Confocal XY-plane images of PHSNAP at various Z-axis locations as labelled. A series of XY-plane images along the Z-axis every 700 nm were used to estimate the cell-surface area.

MINFLUX images of HS clusters at cell surface and single HS molecules in vitro

A, Left: MINFLUX image of a cluster of immunolabelled HS at the plasma membrane (PM). Height and width were measured by fitting the localizations in the cluster with an ellipse (Image J). Right: Same data as in the left, but plotted with localization trace IDs (TID) labeled in different colors. B, Height plotted versus width of HS clusters at the plasma membrane (53 clusters, 9 cells). The line is a linear regression fit (correlation coefficient: 0.8). C, Distribution of the number of TIDs per HS cluster (NTID/cluster, 53 clusters, 9 cells). D, Left: MINFLUX localization image of a single HS molecule in vitro. Purified HS was plated on the coverslip and labeled with antibodies. Height and width were measured by fitting the localizations with an ellipse (Image J). Right: Same data as in the left, but plotted with localization TIDs labeled in different colors. E, Height plotted versus width of single HS-molecule localizations in vitro (67 HS-molecules). The dotted box is enlarged in the inset. F, Distribution of the number of TIDs per single HS-molecule (NTID/HS-molecule). The dotted box is enlarged in the inset.

PIX, but not ACE2 antibody, inhibits CoV2-SJN.1 attachment in human primary small airway epithelial cells in air-liquid interface (ALI) format.

Sampled confocal images of Hoechst (gray, labeling cell nucleus), antibody-labeled Nucleocapsid protein (N Protein of CoV2-SJN.1, green) and Spike protein (anti-S2P6 of CoV2-SJN.1, red) in human primary small airway epithelial cells in air-liquid interface (ALI) format; ALI cells in control (Ctrl), pre-treated with Pixantrone (PIX), or pre-treated with Anti-ACE2 antibody (ACE2 Ab) were incubated with CoV2-SJN.1 for 1 h at 4°C (for viral cell-surface attachment, protocol shown at the top). Images were projection images. XZ- and YZ-plane images are also shown on the bottom and right, respectively.