Fig 2: SARS-CoV-2 virions bind to DC/L-SIGN receptor primarily through high-mannose glycans(A) Virions from Vero, A549-ACE2, and 16HBE cells were inactivated with or without ß-propiolactone and then bound to L-SIGN receptor by ELISA assay.(B) 16HBE (ACE2) cell origin virions which were inactivated with or without ß-propiolactone were treated with lectin HHA (2 µg mL-1) before binding to L-SIGN receptor in ELISA assay.(C and D) 16HBE (ACE2) cell origin virions were pre-treated with or without lectin HHA and then infected HepG2 (L-SIGN) cells (MOI = 1). Viral loads of the infected cells were determined based on the number of viral envelope gene RNA copies detected by qRT-PCR at the indicated h.p.i. (C); The virus-infected cells were visualized using anti-viral nucleocapsid protein antibody by confocal microscopy at 24 h.p.i. (D).(E) Virions from Vero, A549-ACE2, and 293T cells were pre-treated with or without lectin Con A (0.5 µM) and then bound to DC-SIGN receptor by ELISA assay. The error bars represent the standard deviation from four repeats. *: P<0.05 based on Student's t-test.

Fig 3: DC- and L-SIGN mediated SARS-CoV-2 trans/cis-infections(A and B) MDDCs (A) and MLE-12-L-SIGN cells (B) were first incubated with virions of different cell origins at a MOI = 1 for 1.5 h and then co-cultivated with Vero cells for 48 h after intensive washing. Viral titers of the different co-culture supernatants were determined by the CCID50 assay. MDDCs and MLE-12-L-SIGN cells (without virus incubation) were used as negative controls. For DC- and L-SIGN blockade, antibodies against the two receptors were used.(C and D) HepG-2 cells expressing DC-SIGN (C) and L-SIGN (D) were infected with viruses of different cell origins (MOI = 1), and viral titers were determined at 24 h.p.i. by CCID50 assay. HepG-2 cells transduced with empty lentiviral particles were used as a negative control (NC). For DC- and L-SIGN blockade, antibodies against the two receptors were used.(E–H) HepG-2 cells expressing L-SIGN were infected with viruses of different cell origins at a MOI of 0.01 (E and F) or 0.001 (G and H), viral titers were assessed at 24 –72 h.p.i. by the CCID50 assay (E and G), and virus-infected cells were visualized using anti-viral nucleocapsid protein antibody by confocal microscopy at 24 h.p.i. (F and H). The error bars represent the standard deviation from four repeats. *: P<0.05 based on Student's t-test.

Fig 4: L-SIGN is expressed on LSECs in human biopsy or autopsy tissue samples.(A) Representative images of immunohistochemical staining of L-SIGN (ORIGENE, TA810067) in different normal human autopsy tissues. Arrows indicate liver sinusoids. Arrowheads indicate blood vessels. No L-SIGN was detected in blood vessels. Scale bar: 30 µm. (B) Representative confocal microscopy images of immunofluorescence staining of different normal human autopsy liver samples. Arrows indicate LSECs. Arrowheads indicate blood vessels. LSECs are vWF+Lyve1+L-SIGN+. Blood vessels are vWF++Lyve1–L-SIGN–. DAPI, nuclear staining. Scale bar: 20 µm. The results represent at least 3 experiments from biopsy or autopsy samples of 3 uninfected patients.

Fig 5: DC- and L-SIGN does not support productive infection of SARS-CoV-2(A) Expression of ACE2 receptor in Vero, HepG-2, A549, 16HBE, and MLE-12 cells by western blot detection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference control.(B) SARS-CoV-2 infection, replication, and proliferation through MLE-12 cells that stably express ACE2, DC-SIGN, or L-SIGN receptor (from the top down). Cells transduced with empty lentiviral particles were used as a negative control (NC). Left panels, viral loads of the infected cells were determined based on the number of viral envelope gene RNA copies detected by qRT-PCR at the indicated h.p.i.; middle panels, virus-infected cells were visualized using anti-viral nucleocapsid protein antibody by confocal microscopy at 24 h.p.i.; right panels, viral titers from the culture supernatants of the infected cells were determined using a CCID50 assay at the indicated h.p.i..(C–E) Binding of ACE2 (C), DC-SIGN (D), and L-SIGN (E) receptors to virions of different cell origins by ELISA.(F) Viral titers from the culture supernatants of infected ACE2-, DC-SIGN-, and L-SIGN-expressing MLE-12 cells upon infection with the virions of different cell origins at 24 h.p.i.. The error bars represent the standard deviation from four repeats.