Research ArticleVIROLOGY

Heteromultivalent topology-matched nanostructures as potent and broad-spectrum influenza A virus inhibitors

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabd3803
DOI: 10.1126/sciadv.abd3803
  • Fig. 1 Design of heteromultivalent topography-matching nanostructures for virus inhibition.

    (A and B) Design and synthesis of a heteromultivalent nanobowl (Hetero-MNB) for IAV inhibition, including the coating of red blood cell membranes (RBCm) onto the NB surface and the further modification with lipidic Zan. (C) Proposed binding patterns between IAV and the Hetero-MNB, where sialic acid and Zan bind to HA and NA, respectively, and the bowl shape with facilitating the capping to the surface of the virus particle. (D) Inhibition curves for RBCm vesicles at different concentrations from a TIRF setup. Values are expressed as means ± SD, n = 4. (E) Typical immunofluorescent staining images of viral nucleoprotein (NP) to show the infected cells with the treatment of RBCm vesicles. Scale bars, 50 μm. Multiplicity of infection, 0.1. DAPI, 4′,6-diamidino-2-phenylindole. (F) Propagation of IAV in the presence of RBCm vesicles. Values are expressed as means ± SD, n = 4. PFU, plaque-forming units.

  • Fig. 2 Heteromultivalent surfaces for the interaction with IAV virions.

    (A) TIRF-based assay for assessing the interaction of IAVs with homo- or heteromultivalent membranes. The corresponding glycostructures (either sialic acids or lipid-conjugated Zan) are incorporated in an SLB, and the IAV-SLB interaction is recorded using TIRF microscopy. Application of single-virus tracking allows for extracting changes of (B) the rate of IAV attachment to the membranes and (C) the motion of SLB-bound IAVs, when using the different homo- and heteromultivalent structures. Each color represents the track of a single virion. Values in (B) are expressed as means ± SEM, n = 10. **P < 0.01 and ***P < 0.001 by Student’s t test. Note the different scale bars used for the immobile and mobile IAVs in (C).

  • Fig. 3 Topography-matching principle for maximized virus interaction.

    (A) High-resolution transmission electron microscopy (HR-TEM) images for NS and NB with RBCm coating. Scale bars, 50 nm. (B) IC50infection inhibition values for the homomultivalent NS-RBCm and NB-RBCm. Values are expressed as means ± SD, n = 4. (C) Geometry analysis for a virion binding to a convex inhibitor and concave inhibitor. The actual contact between them is marked with θ1; the steric shielding by the inhibitor is marked with purple lines with θ2. Steric shielding is estimated by placing inhibitors with the same curvature. (D) Negatively stained HR-TEM image for the IAV particle binding with the Hetero-MNB. Scale bar, 50 nm. Images for other samples are shown in fig. S11.

  • Fig. 4 Inhibition of IAV infection by the heteromultivalent topography-matching nanostructure.

    (A) Flow cytometry for the virus attachment to MDCK-II cells and (B) corresponding mean and median fluorescence intensities (FI) for the samples. The error bars are generated by the CytExpert Acquisition and Analysis Software Version 2.3. Fluorescence intensity is measured in arbitrary units. (C) Projection confocal laser scanning microscopy images for the viral attachment to MDCK-II cells in the presence of Hetero-MNB. Scale bars, 20 μm. (D) Typical immunofluorescence images to show the IAV [A/X31 (H3N2)] infection in the presence of the inhibitors. Other images are shown in figs. S12 and S13. Scale bars, 100 μm. (E) Detection of viral NP in cells infected with four typical human IAV strains, including A/X31 (H3N2), A/PR/8/34 (H1N1), A/Panama/2007/1999 (H3N2), and A/Bayern/63/2009 (H1N1pdm) in the presence or absence of inhibitors. (F) Inhibition of A/X31 (H3N2) of the inhibitors at different dosages revealed by cell viability. (G) Inhibition toward IAV propagation [A/X31 (H3N2)] for the inhibitors. (H) Inhibition toward IAV propagation for the inhibitors against three other IAV strains, including A/PR/8/34 (H1N1), A/Panama/2007/1999 (H3N2), and A/Bayern/63/2009 (H1N1pdm). The inhibitors are used after the first cycle of infection. Inhibitor dosage, 100 μg/ml. Values are expressed as means ± SD, n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test, respectively.

  • Table 1 Inhibition constants of RBCm vesicles of different IAV strains from hemagglutination inhibition assay.

    Values are expressed as means ± SD, n = 4.

    KiHAI (μg/ml)KiHAI (nM sialic acid)
    A/X31 (H3N2)41.6 ± 20.68.4 ± 4.2
    A/PR/8/34 (H1N1)23.4 ± 9.04.8 ± 1.8
    A/Panama/2007/1999
    (H3N2)
    27.3 ± 7.85.5 ± 1.7
  • Table 2 Inhibition constants of samples against A/X31 (H3N2) from hemagglutination inhibition assay.

    Values are expressed as means ± SD, n = 4.

    KiHAI (μg/ml)KiHAI (nM sialic acid)
    RBCm41.6 ± 20.68.4 ± 4.2
    MDCK-II membrane6.8 ± 1.91.6 ± 0.5
    RBCm vesicles and
    free Zan
    11.7 ± 4.52.4 ± 0.9
    RBCm-Zan hybrid
    vesicles
    5.9 ± 2.31.2 ± 0.5
    NB-RBCm51.9 ± 23.83.1 ± 1.4
    NB-RBCm and Zan19.5 ± 7.81.2 ± 0.5
    Hetero-MNB13.7 ± 3.90.8 ± 0.2

Supplementary Materials

  • Supplementary Materials

    Heteromultivalent topology-matched nanostructures as potent and broad-spectrum influenza A virus inhibitors

    Chuanxiong Nie, Marlena Stadtmüller, Badri Parshad, Matthias Wallert, Vahid Ahmadi, Yannic Kerkhoff, Sumati Bhatia, Stephan Block, Chong Cheng, Thorsten Wolff, Rainer Haag

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