Microbiology and Infectious Disease

Nanoscopy Reveals Heparan Sulfate Clusters as Docking Sites for SARS-CoV-2 Attachment and Entry

Sue Han
Xin Wang
Tiansheng Li
Ammar Mohseni
Ivan Kosik
Chung Yu Chan
Alberto Domingo López-Muñoz
Jessica Matthias
Reid Suddaby
Zhixiong Wang
Albert J Jin
Christian A Wurm
Jonathan W Yewdell author has email address
Ling-Gang Wu author has email address

National Institute of Neurological Disorders and Stroke, Bethesda, United States
Cellular Biology Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States
Abberior Instruments America LLC, Bethesda, United States
Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States

https://doi.org/10.7554/eLife.108925.1

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Figures and data

hACE2-independent internalization of pseudo-typed SARS-CoV-2.
A, Upper: Schematic drawing of a pseudo-typed SARS-CoV-2 virion, VSV_EGFP-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 envelop proteins. Lower: Confocal images of A647 of purified VSV_EGFP-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 VSV_EGFP-S-A647’s A647 (V-A647) and EGFP expression (V-EGFP) in BHK21 cells stably expressing hACE2 (BHK_hACE2), BHK21 cells without ACE2 expression (BHK), and BHK cells transiently overexpressed with hACE2- BFP plasmid (BHK+hACE2). Top: Incubation protocol – cells were incubated with VSV_EGFP-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 293T_hACE2 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 VSV_EGFP-S-A647 was replaced with Lenti_EGFP-S_Omi-A647. G, Fluorescence intensity of VSV_EGFP-S-A647’s V-A647 (F₆₄₇) and V-EGFP (F_EGFP) in BHK_hACE2 (471 cells, 3 experiments), BHK (370 cells, 3 experiments), BHK+hACE2 (515 cells, 3 experiments). 293T_hACE2 (404 cells, 3 experiments), 293T (234 cells, 3 experiments), Vero (295 cells, 3 experiments), Hela (305 cells, 3 experiments), and MRC5 (237 cells, 3 experiments) cells. Lenti_EGFP-S_Omi-A647’s F₆₄₇ and F_EGFP in BHK_hACE2 (364 cells, 3 experiments) and BHK (335 cells, 3 experiments) cells are also plotted. Virus incubation protocol is shown in panel B. F₆₄₇: mean ± s.e.m., reflecting viral uptake; F_EGFP: mean ± s.e.m., reflecting viral infection. F₆₄₇ and F_EGFP 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 VSV_EGFP-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 (VSV_EGFP-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). 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. 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 (N_A647) is labeled. D, Left: W_H distribution of A490 spots (Spot_A490) containing V-A647 spots (55 Spot_A490, 3 experiments from BHK cells). Middle: N_A647 plotted versus the corresponding Spot_A490 W_H (55 Spot_A490, 3 experiments, from BHK cells). A linear regression fit (correlation coefficient: 0.53) showed that larger Spot_A490 contains more V-A647 spots. Right: V-A647-containing Spot_A490 W_H in BHK_hACE2 (70 Spot_A490, 3 experiments) and BHK cells (55 Spot_A490, 3 experiments). E, Left: EM images showing that endosomes may contain one or more VSV_EGFP-S-A647 virions (arrows). Incubation protocol: VSV_EGFP-S-A647 for 1 h at 4°C, 15 min washout at 37°C. Right: W_H (mean ± s.e.m.) of EM-observed virus (VSV_EGFP-S-A647)-containing endosome (38 endosomes, 2 experiments) and STED-observed virus (VSV_EGFP-S-A647)-containing Spot_A490 (70 Spot_A490, 3 experiments). F, Bright field and confocal images of V-A647 (uptake) and V-EGFP (viral gene expression) in BHK_hACE2 cells in control (Ctrl) or treated with dynasore (DnS, 80 μM, bath). Top: VSV_EGFP-S-A647 incubation protocol – 1 h incubation, 24 h washout, all at 37°C. G, VSV_EGFP-S-A647’s F₆₄₇ (uptake) and F_EGFP (gene expression) in cells in Ctrl (789 cells, 3 experiments), treated with DnS (653 cells, 3 experiments), or overexpressed with dynamin 2 K44A (19 cells, 2 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 BHK_hACE2 cells in Ctrl or treated with cathepsin inhibitor 1 (CPS Inh, 10 μM). Top: VSV_EGFP-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 BHK_hACE2 cells in Ctrl or treated with CPS Inh. Top: VSV_EGFP-S-A647 incubation protocol – 1 h incubation, 24 h washout, all at 37°C. I, F₆₄₇ (uptake) or F_EGFP (gene expression) in cells in Ctrl (540 cells, 3 experiments) or treated with CPS Inh (742 cells, 3 experiments). F₆₄₇ and F_EGFPwere measured after VSV_EGFP-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 BHK_hACE2 (left) and a BHK (right) cell. B, ACE2-independent viral cell-surface attachment. Images: STED images showing virus cell-surface attachment in a BHK_hACE2 (left) or BHK (right) cell. Middle: images in green (left) and yellow (right) boxes replotted on a larger scale. Top: incubation protocol – virus (VSV_EGFP-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 (F_Virus) per BHK_hACE2 (92 cells, 3 experiments) or BHK cell (98 cells, 3 experiments). Data normalized to the mean of the BHK_hACE2 group. C, Cell-surface-attached viruses colocalized with HS puncta. Images: STED images of viral spots (VSV_EGFP-S-A490’s V-A490) and immunolabelled HS (Alexa 488-conjugated antibody) at BHK_hACE2 or BHK cell surface. Top: incubation protocol – VSV_EGFP-S-A647 1 h at 4°C for cell-surface attachment. Bar graph: the percentage of viral spots overlapping with HS puncta in BHK_hACE2 (8 cells, 4 experiments) or BHK cells (8 cells, 4 experiments). D, Bright field and confocal images of immunolabelled HS (left) and HS fluorescence intensity (F_HS, right) in BHK_hACE2 cells in Ctrl (188 cells, 3 experiments) or treated with Heparinases I/II/III (HPRase, 264 cells, 3 experiments). ***: p < 0.001, t-test. E, Images: bright field and confocal image of V-A647 in BHK_hACE2 cells in Ctrl or treated with HPRase. Top: incubation protocol – VSV_EGFP-S-A647 1 h at 4°C for cell-surface attachment. Bar graph: cell-surface F₆₄₇ per cell for BHK_hACE2 cells (left) in Ctrl (125 cells, 3 experiments) or treated with HPRase (150 cells, 3 experiments), and for BHK cells (right) in Ctrl (147 cells, 3 experiments) or treated with HPRase (127 cells, 3 experiments). ***: p < 0.001, t-test. F, Images: bright field and confocal image of V-A647 (uptake) in BHK_hACE2 cells in Ctrl or treated with HPRase. Top: incubation protocol – VSV_EGFP-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 BHK_hACE2 cells in Ctrl (201 cells, 3 experiments) or treated with HPRase (197 cells, 3 experiments), and for BHK cells in Ctrl (202 cells, 3 experiments) or treated with HPRase (206 cells, 3 experiments). G, Images: bright field and confocal image of V-EGFP expression in BHK_hACE2 cells in control or treated with HPRase. Top: incubation protocol – VSV_EGFP-S-A647 1 h at 4°C, 24 h washout at 37°C. ***: p < 0.001, t-test. Bar graph: F_EGFP (viral gene expression) per BHK_hACE2 cell in Ctrl (482 cells, 3 experiments) or treated with HPRase (391 cells, 3 experiments). H, STED images of viral spots (VSV_EGFP-S-A490’s A490) and immunolabeled hACE2 puncta (A647) at a BHK_hACE2 cell surface. I, STED images of viral spots (VSV_EGFP-S-A490’s A490) and immunolabeled hACE2 puncta (A647) inside BHK_hACE2 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 VSV_EGFP-S_omi-A647’s V-A647 (left) and F₆₄₇ (right, viral uptake) in BHK_hACE2 (146 cells, 3 experiments) or BHK (149 cells, 3 experiments) cells. Top: incubation protocol – VSV_EGFP-S_omi-A647 1 h at 37°C (for virus endocytosis). B, Images (bright field and confocal images) of VSV_EGFP-S_omi-A647’s V-EGFP (left) and F_EGFP (right, viral gene expression) in BHK_hACE2 (273 cells, 3 experiments) or BHK (200 cells, 3 experiments) cells. Top: incubation protocol – VSV_EGFP-S_omi-A647 1 h, 24 h washout, all at 37°C (for viral gene expression). C, Images (bright field and confocal images) of CoV2_mCh-S_omi-A647’s A647 fluorescence (V- A647, left) and F₆₄₇ (right, uptake) in BHK_hACE2 (120 cells, 3 experiments) or BHK (120 cells, 3 experiments) cells. Top: incubation protocol – CoV2_mCh-S_omi-A647 1 h at 37°C (for virus endocytosis). D, Images (bright field and confocal images) of CoV2_mCh-S_omi-A647’s mCherry expression (V- mCherry, pseudo color, left) and F_mCherry (right, uptake) in BHK_hACE2 (120 cells, 3 experiments) or BHK (120 cells, 3 experiments) cells. Top: incubation protocol – CoV2_mCh-S_omi-A647 1 h, 6 h washout, all at 37°C (for viral gene expression). E, STED XY-plane images of CoV2_mCh-S_omi-A647’s V-A647 and A490 in cytosol showing that the number (N_A647) of V-A647 spots (virions) in a A490 spot (endocytic vesicle) may vary as labelled. Top: incubation protocol – CoV2_mCh-S_omi-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 CoV2_mCh-S_omi-A647 ’s V-A647 (left) and surface F₆₄₇ per cell (right) for BHK_hACE2 cells in Ctrl (120 cells, 3 experiments) or treated with HPRase (120 cells, 3 experiments). Top: incubation protocol – CoV2_mCh-S_omi-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 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 PH_mNG (labeling PM) image at a BHK_hACE2 cell surface [extracellular (Extra) compartment is above the PM]. C, MINFLUX image of immunolabelled HS and STED image of CoV2_mCh-S_omi-A490 (Virus) at a BHK_hACE2 cell surface (merged at the bottom). Cells were incubated with CoV2_mCh-S_omi- 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 (N_HS/cluster, 51 clusters, 8 cells, 3 experiments). G, MINFLUX pixel-based rendering (upper) and confocal image (lower) of CoV2-S_omi (CoV2_mCh-S_omi-A647’s A647) at the cell surface (extracellular side: above the confocal spots). Cells were incubated with CoV2_mCh-S_omi-A647 for 1 h at 4°C for viral cell-surface attachment. H, MINLFUX coordinate-based rendering of CoV2_mCh-S_omi-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-S_JN.1-A647 attachment at the cell surface: sampled bright field and confocal images of CoV2-S_JN.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-S_JN.1 for 1 h at 4°C for labeling cell-surface attachment (incubation protocol shown on the top). B, Quantification of CoV2-S_JN.1-A647 attachment at the cell surface: cell-surface fluorescence of CoV2-S_JN.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-S_JN.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-S_JN.1-A647 1 h at 4°C for cell-surface attachment. Bar graph for virus attachment: cell-surface fluorescence of CoV2-S_JN.1-A647 (F₆₄₇) per cell for normal human bronchial/tracheal epithelial (NHBE) cells pre-treated with control (Ctrl, 16 cells, 2 experiments), pixantrone (PIX, 18 cells, 2 experiments), or anti-ACE2 antibody (ACE2 Ab, 24 cells, 2 experiments). ***: p < 0.001 (ANOVA test). B, Cell-surface-attached viruses colocalized with HS puncta: confocal images of viral spots (CoV2-S_JN.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-S_JN.1-A647 1 h at 4°C for cell-surface attachment. C, PIX, but not ACE2 antibody, inhibits CoV2-S_JN.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-S_JN.1, green), and Spike protein (anti-S2P6 of CoV2-S_JN.1, red) in ALI cells were incubated with CoV2-S_JN.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 (F_N _protein, upper) and spike (F_Spike, lower) for ALI cells incubated with CoV2-S_JN.1 for 1 h at 4°C in three pre-treated conditions stated above (Ctrl: 2 experiments; PIX: 2 experiments; ACE2 Ab: 2 experiments). ***: p < 0.001 (ANOVA test). D, Both PIX and ACE2 antibody inhibit CoV2-S_JN.1 infection of ALI cells. Images: Immunolabeled 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-S_JN.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-S_JN.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).

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