Extended receptor repertoire of an adenovirus associated with human obesity

Figures

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  Fig 1. HAdV-D36 uses CAR and sialic-acid-containing glycotopes to attach to A549 cells via the fiber knob. (A) Binding of 35S-labelled HAdV-D36 virions to A549 cells pretreated with different concentrations of the protease ficin. (B) Binding of 35S-labelled HAdV-D36 virions to A549 cells cultured in the presence and absence of the metabolic inhibitor BenzylGalNAc (inhibitor of O-linked glycan synthesis). 35S-labelled virion binding to cell was shown as CPM (counts per minute) in both A and B. (C) Binding of 35S-labelled HAdV-D36 FK, HAdV-D37 FK, and HAdV-C5 FK to CHO cell lines expressing different human cell surface proteins (CAR, CD46, CD21; see the legend on the right), or overexpressing SA (Pro5). The CHO-K1 and CHO mock cells represent- control cell lines. Y-axis shows knob binding to cells, represented as Geo Mean (Geometrical mean). All experiments were performed three times with duplicate samples. Error bars represent mean ± SD. The statistical significance was determined by unpaired two-tailed t test; ns = not significant, * P of < 0.05 and ** P of < 0.01.

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  Fig 2. CAR and Neu5Ac engagement have a synergistic effect on virion attachment. (A) 35S-labelled HAdV-D36 and -D37 virion binding to A549 cells, treated with (black bars) or without (white bars) neuraminidase. (B) Binding of 35S-labelled HAdV-D36 and -D37 virion, pre-incubated with increasing concentration of Neu5Ac, to A549 cells. (C) 35S-labelled HAdV-D36 and -C5 virion binding to A549 cells. Virions were preincubated with increasing concentrations of soluble CAR-D1 before binding. (D) 35S-labelled HAdV-D36 virion binding to A549 cells in absence (control) or presence of CAR-D1 (50 μg/mL), Neu5Ac (50 mM), or both. All data are presented as % of control, where control for (A) refers to binding of virions to cells not treated with Neuraminidase and for (B-D) refers to binding of virions to cells where virions were not incubated with Neu5Ac or CAR-D1 or both before binding. All experiments were performed three times with duplicate samples. Error bars represent mean ± SD. The statistical significance was determined by unpaired two-tailed t test. * P of < 0.05, ** P of < 0.01 and *** P of < 0.001.

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  Fig 3. Structural features of the HAdV-D36 FK. (A) Poisson-Boltzmann electrostatic potential surface representation of the HAdV-D36 FK (left) and HAdV-D37 FK (right) calculated at ±3 kT/e and shown from a top view along the threefold axis. (B) Comparison of the quaternary arrangement of the FKs of HAdV-D36 (gray) and HAdV-D37 (green, PDB-ID 1UXE) displayed as surfaces and as ribbon representation of the beta sheet cores. The superposition was calculated using all atoms of both trimers. (C) The HAdV-D36 FK possesses long DG loops (red) that are partially ordered and contact the counterclockwise neighboring monomer when viewed from the top. (D) Close-up view of the interactions between the DG loop and the counterclockwise neighboring monomer. Polar and hydrophobic interactions are displayed as black and yellow dashes, respectively.

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  Fig 4. Glycan array screening of the HAdV-D36 FK and comparison with the HAdV-D37 FK. (A) A broad-spectrum glycan screening array containing 492 glycans (see legend on the upper right and S1 Table) showed a low overall signal with one outstanding signal elicited by 4-O-Ac-3’SL (7,279 fluorescence units). Numerical scores for the binding intensity are shown as means of fluorescence intensities of duplicate spots at 5 fmol/spot, with error bars representing half of the difference between the two values. (B) A small, focused array designed to test for HAdV-D36 FK binding to 3’SL variants. Lipid-linked glycan probes with their corresponding sequences, including lipid tags are listed on the right panel (more information on lipid tag in S4 Table). The doses of the glycan probes arrayed per spot are indicated. Notably, HAdV-D36 FK demonstrated selective binding exclusively to 4-O-Ac-3’SL, contrasting with the preference of HAdV-D37 FK for 3’SL. Linker (or tag) effects in glycan binding are commonly observed in glycan array studies and are likely associated with glycan presentation [117]. Error bars represent half of the difference between the two values.

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  Fig 5. Structure of the HAdV-D36 FK in complex with 4-O-Ac-3’SL and comparison of Neu5Ac and Neu4,5Ac2 engagement as well as functional relevance. (A) Simulated annealing omit map (green: 3σ; yellow: 2.5σ) and 2Fo-Fc map after refinement (1σ, blue) of 4-O-Ac-3´SL in complex with the HAdV-D36 FK. The lactose stem is generally poorly ordered and only partially visible in one of three cases. (B) 4-O-Ac-3’SL colored by -factors ranging from 10Å2 (blue) to 60 Å2 (red). (C) Superposition of the Neu5Ac (yellow) and Neu4,5Ac2 (green) complex structures. Neu4,5Ac2 is forced towards the threefold axis by the steric influence of N313, Y315, and P323, while the polar key contacts are only slightly altered. (D) This movement causes the three neighboring Neu4,5Ac2 moieties to form a triangular hydrophobic contact (green dashes) that is not possible for Neu5Ac. (E) Steric influence of Y315 and P320 on Neu4,5Ac2. Short-ranged van-der-Waals contacts are formed with both acetyl groups of the sugar. As a result, even slight alterations in the Y315 conformation would interfere with the binding. (F) Binding of preincubated HAdV-D36 virions with Neu5NAc and Neu4,5NAc2 to A549 cells. Data are presented in %…