Fig 1.
Schematic structures of sialylglyco-polyglutamic acid (PGA) and sialylglycopeptide (SGP).
Structures of compounds investigated are shown in a line-column formation. First row: a series of SGP; Second Raw: γ-PGA bearing glycans; Third Row: α-PGA bearing glycans. First column: glycan carrying α-2-3-linked glycosides of sialic acid; Second column: glycan carrying α-2-6-linked glycosides of sialic acid; Third column: compounds without sialic acid. Cartoon presentations of glycans are shown as suggested by the Consortium for Functional Glycomics. Average molecular weights for α- and γ-PGA together with degree of substitution (DS) provide overall structures of polymeric compounds. Polymers carrying “lipo”-functions form self-aggregates.
Fig 2.
α2-6SLN-lipo-PGA inhibited SARS-CoV-2 infection.
(A, B) SARS-CoV-2 or SARS-CoV infection assays were performed in the presence or absence of the indicated compounds. SARS-CoV-2 or SARS-CoV infection was determined by detecting SARS-CoV-2 RNA in the culture supernatant (A) and viral N protein in the cells (B). Scale bar 50 μm. Lopinavir, 16 μM; remdesivir, 10 μM; α2-3SLN-PGA, 10 mg/ml; α2-6SLN-PGA, 10 mg/ml; α2-6SLN-lipo-PGA, 1 mg/ml. (C) Dose-response curve of α2-6SLN-lipo-PGA upon SARS-CoV-2 (red) or SARS-CoV (blue) infection. Secreted viral RNA was quantified upon treatment with α2-6SLN-lipo-PGA at the concentration as shown. (D, E) Anti-SARS-CoV-2 or anti-SARS-CoV activity of N-acetylneuraminic acid (Neu5Ac, the component of α2-6SLN-lipo-PGA) and lactosylsphingosine-PGA (LS-PGA, the similar structure with α2-6SLN-lipo-PGA without having sialic acid). Remdesivir, 10 μM; Neu5Ac, 80 mM; α2-6SLN-lipo-PGA, 20 μM; LS-PGA, 20 μM. (F) Anti-SARS-CoV-2 activity of indicated compounds in human lung epithelial-derived cell line, Calu-3 cells. Remdesivir, 10 μM; α2-6SLN-lipo-PGA, 10 μM.
Fig 3.
α2-6SLN-lipo-PGA inhibited SARS-CoV-2 attachment.
(A) Schematic representation of the schedule for treating VeroE6/TMPRSS2 cells with compounds and SARS-CoV-2 in time of addition analysis. Black and white boxes indicate the periods with and without treatment, respectively. (B) The antiviral activities of indicated compounds under treatment protocol as shown in (A) are estimated by quantifying the levels of secreted viral RNA at 24 h post-inoculation. Remdesivir, 8 μM; chloroquine, 8 μM; α2-6SLN-lipo-PGA, 20 μM. (C) SARS-CoV-2 attachment/entry was evaluated using SARS-CoV-2 pseudovirus. VeroE6/TMPRSS2 cells were inoculated with SARS-CoV-2 pseudovirus in the presence or absence of compounds, and at 24 h post-inoculation, cells were lysed and assessed for luciferase activity generated by SARS-CoV-2 pseudovirus infection. Heparin, 50 U/ml; α2-6SLN-lipo-PGA, 10 μM. (D) Virus-cell attachment assay. VeroE6/TMPRSS2 cells were incubated with viruses in the presence or absence of the indicated compounds for 5 min at 4°C to allow virus-cell attachment. After extensive washing, cells were lysed and cell-attached viral RNA was quantified. Heparin, 50 U/ml; chroloquine, 100 μM; α2-6SLN-lipo-PGA, 100 μM.
Fig 4.
α2-6-linked sialosides interacted with S1 subunit of SARS-CoV-2 spike protein.
(A) The attachment of compound-pretreated virus to cells. SARS-CoV-2 particles were pretreated with the compounds at 37°C for 60 min, then the free compounds were removed by ultrafiltration. The prepared viruses were treated to VeroE6/TMPRSS2 cells in the absence of compounds at 4°C for 30 min to examine virus-cell attachment. (B) The viral attachment to compound-pretreated cells. The indicated compounds were treated to VeroE6/TMPRSS2 cells at 37°C for 30 min and were then washed out extensively. The prepared cells were incubated with SARS-CoV-2 in the absence of compounds at 4°C for 30 min to examine virus-cell attachment. (A, B) Anti-ACE2 antibody, 100 μg/ml; heparin, 10 U/ml; α2-6SLN-lipo-PGA, 10 μM. (C) Schematic representation of glycan array. Glycan tip immobilized with sialylglycopeptides on a slide glass was incubated with recombinant SARS-CoV-2 S1 at room temperature for 1 h, and then the fluorescence signal was detected. Anti-SARS-CoV-2 S1 antibody was used as primary antibody, and Cy3-conjugated anti-rabbit IgG was used as secondary antibody. (D) The fluorescent response of the glycan array due to the interaction between recombinant SARS-CoV-2 S1 (aa 16–685, Sino Biological) and indicated protein or glycans. The amount of glycan on the neoglycoproteins were 10.8 mol (α2-6-SGP), 10.5 mol (α2-3-SGP) and 10.8 mol (asialo-SGP) of glycan per 1 mol of BSA. The dashed line indicates the nonspecific response level derived from negative control asialo-SGP. Asialo-SGP, 0.7 mg/ml; α2-3-SGP, 0.1 and 1 mg/ml; α2-6-SGP, 0.1 and 1 mg/ml; recombinant ACE2, 0.1 mg/ml.
Fig 5.
Host sialic acid contributed to the efficient spread of SARS-CoV-2 infection.
(A) SARS-CoV-2 or SARS-CoV pseudovirus infection to neuraminidase-treated cells. VeroE6/TMPRSS2 cells were pretreated with or without indicated neuraminidases for 2 h at 37°C, followed by inoculation with SARS-CoV-2 or SARS-CoV pseudovirus. At 24 h post-inoculation, cells were lysed and luciferase activity was quantified. (B) SARS-CoV-2 attachment on neuraminidase-treated cells. VeroE6/TMPRSS2 cells were treated with or without neuraminidase for 2 h at 37°C, then the cells were inoculated with SARS-CoV-2 for 1 h at 4°C. After washing unbound viruses, the cells were lysed and attached viral RNA was detected. The attached viral spike protein and endogenous protein expression of ACE2 and actin in VeroE6/TMPRSS2 cells was determined by immunoblotting. The number under the blot shows the relative intensity (fold) of the band measured using ImageJ program. (C) The interaction between ACE2 and SARS-CoV-2 spike under pretreatment with neuraminidase. VeroE6/TMPRSS2 cells were treated with or without neuraminidase for 2 h at 37°C, then were inoculated with SARS-CoV-2 for 1 h at 4°C. After washing unbound viruses, the cells were harvested to perform immunoprecipitation with anti-ACE2 antibody (IP: ACE2) or normal mouse IgG as a negative control (IP: IgG). Viral spike protein and ACE2 in the precipitates were detected by immunoblotting. (D) The effect of sialic acid on ACE2 for the interaction with SARS-CoV-2 spike S1. The fluorescent response due to the interaction between recombinant SARS-CoV-2 spike S1 and recombinant ACE2 protein was detected. Recombinant ACE2 proteins were incubated with or without 2 U/ml neuraminidase (Arthrobacter ureafaciens) for 2 h at 37°C, and each ACE2 sample were immobilized on a glass slide to analyze the interaction with SARS-CoV-2 spike S1 protein. The dashed line indicates the nonspecific response level of protein-free glass slide. The schematic representation of the assay is shown under the data. The removal of sialic acid on ACE2 by treatment with neuraminidase was confirmed by immunoblotting. (E) Combination treatment with anti-ACE2 antibody and neuraminidase was performed using SARS-CoV-2 and SARS-CoV pseudovirus. (F) The spread of SARS-CoV-2 and SARS-CoV infection was examined by (a) immunofluorescent observation of infected cells (scale bar 50 μm), (b) quantification of infected area and (c) detection of viral RNA in culture supernatant. (A-F) Neuraminidase from Vibrio cholerae, 40 mU/ml (A); from Arthrobacter ureafaciens, 100 mU/ml (A, E), 200 mU/ml (B, C) and 2 U/ml (D, F); anti-ACE2 antibody, 40 μg/ml (E, left) and 7 μg/ml (E, right).