Fig 2: Binding of L-SIGN to the SARS-CoV-2 spike protein mediates viral entry.(A) Protein domain structures of SARS-CoV-2 spike protein and human L-SIGN (numbers represent amino acids). TM, transmembrane domain; CP, cytoplasmic domain; EPN, Glu-Pro-Asn motif; SS, signal sequence. (B) Protein expression of ACE2-myc3 and L-SIGN-flag in transfected HEK293T cells by immunoblotting. GAPDH was used as a loading control. (C and D) Representative flow cytometry plots of VSVG-type (histogram) and SARS-CoV-2–type (density plot) pseudo-typed virus infection in response to mock-transfected, ACE2-myc3–transfected, and L-SIGN-flag–transfected HEK293T cells defined by ZsGreen expression. Uninfected or mock-transfected cells were used as negative control. Dashed boxes indicate infected cells. (E) Quantification of pseudo-typed virus infection in C and D. Each dot represents an individual experiment. (F) Representative overlaid flow cytometry plot of exogenous L-SIGN expression and CoV-2–type infection on L-SIGN-flag–transfected HEK293T cells (top) and quantification of the percentage of CoV-2 infection based on different L-SIGN expression levels (bottom). CoV-2 infectivity positively correlates with L-SIGN expression level. Percentages of infection were normalized with percentages of expression of L-SIGN in HEK293T cells. N.C., negative control; uninfected, L-SIGN–stained HEK293T cells; infected, L-SIGN–stained CoV-2–type–infected HEK293T cells; Inter, intermediate. Bars indicate the mean; error bars represent mean ± SEM. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01. All experiments were repeated at least 3 times.

Fig 3: L-SIGN binding to spike protein depends on glycan recognition and calcium.(A) Representative flow cytometry histograms of surface expression of L-SIGN mutants, spike/Fc binding, and VSVG-type or SARS-CoV-2–type pseudo-typed viral infection in response to mock-transfected HEK293T cells (mock) or transfected HEK293T cells expressing the indicated L-SIGN construct. N.C. represents either isotype-matched, IgG-stained, Fc control or no viral infection. Arrows indicate positive staining or infection. (B) Quantification of the percentage of spike/Fc binding (left) and the percentage of CoV-2 infection (right) in A. (C) Cartoon of spike/Fc binding and CoV-2–type infection on L-SIGN expressing cells. QPD and AAA represent loss-of-function mutations of the carbohydrate recognition domain of L-SIGN. N343, Asn343. (D) DNA sequences of surrounding N-glycosylation sites (Asn-X-Thr), which have been mutated to (Gln-X-Thr) in each spike mutant. (E) Representative flow cytometry plot of N-glycosylation at Asn343-deficient CoV-2 infection in response to ACE2-myc3–transfected HEK293T cells (left) and quantification of CoV-2 infection (right). (F) Representative flow cytometry plots of each N-glycosylation–deficient CoV-2 infection in response to L-SIGN-flag–transfected HEK293T cells (left) and quantification of CoV-2 infection (right). Uninfected cells were used as negative control. Dashed boxes indicate infected cells. For all analyses, n = 3. Bars indicate the mean; error bars represent mean ± SEM. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: **P < 0.01.

Fig 4: L-SIGN is a receptor for SARS-CoV-2 virus.(A) Representative confocal microscopy images of SARS-CoV-2 nucleocapsid protein in LSECs from a COVID-19 liver autopsy sample. An uninfected normal human liver autopsy sample was used as a negative control. LSECs were L-SIGN+ (green). Arrows mark SARS-CoV-2 (red). DAPI (gray), nuclear staining. Scale bar: 10 µm. (B) Representative of 3D rendering of Z-stacked microscopy of liver sections from COVID-19 liver autopsy samples. Scale bars: 3 µm. Arrows mark SARS-CoV-2 protein particles (red) inside LSECs. Scale bar: 5 µm. (C) Representative 3D reconstructed confocal microscopy images of sections from a COVID-19 liver autopsy sample (left). Images on the right show orthogonal xy, xz, and yz projections of the dotted area to reveal the virus staining inside the LSEC. Scale bars: 10 µm. The results represent data from biopsy of autopsy samples of 4 independent patients with COVID-19 and 3 uninfected patients. (D) Representative confocal microscopy images of cultured L-SIGN-flag–expressing LSECs infected with authentic SARS-CoV-2 (MOI = 1). Scale bar: 5 µm. Blue (DAPI), nuclear staining; green, L-SIGN; red, nucleocapsid protein. (E) A 3D microscopy view of L-SIGN-flag–expressing LSECs infected with authentic SARS-CoV-2 (MOI = 1). Scale bar: 5 µm. Green, L-SIGN; red, nucleocapsid protein. (F) Percentage of nucleocapsid protein+ cells per total cells in 4–8 ×40 fields in mock- or L-SIGN-flag–transduced LSECs. (G) Median tissue culture infectious dose (TCID50) virus titer assay of supernatants 2 days after infection in mock-, L-SIGN-flag–, or ACE2-myc3–transduced LSECs. Infected Vero-E6 cells were positive control in terms of their permissiveness for high viral productivity. (H) Percentage of nucleocapsid protein+ cells per total L-SIGN-flag–transduced LSECs in the presence of mannan (100 and 500 µg/ml) in 4–8 ×40 fields in each group. *P < 0.01; **P < 0.0001. Bars indicate the mean; error bars represent mean ± SD. All experiments were at least repeated 3 times.

Fig 5: LSECs from liver autopsy samples from patients with COVID-19 express increased levels of L-SIGN, vWF, and FVIII.(A) Representative 3D rendering of Z-stacked confocal microscopy images of liver sections from normal human and COVID-19 liver autopsy samples. Scale bars: 20 µm. Green, L-SIGN; red, vWF; blue (DAPI), nuclear staining. (B) Representative single-color or merged-color confocal images of immune-stained human liver sections. Arrow indicates central vein. L-SIGN marks LSECs. vWF (green), L-SIGN (blue), FVIII (red), DAPI (white). Scale bar: 20 µm. (C) Quantification of relative mean fluorescence intensity of vWF or FVIII associated with L-SIGN+ LSECs. **P < 0.01. For all analyses, n = 4. Bars indicate the mean; error bars represent mean ± SEM. (D) Representative confocal microscopy images of Lyve1 (red) in a COVID-19–infected liver autopsy sample. An uninfected normal human liver autopsy sample was used as a positive control. Scale bar: 10 µm.