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. 2019 Sep 4;5(9):eaax3567.
doi: 10.1126/sciadv.aax3567. eCollection 2019 Sep.

Human Adenovirus Type 26 Uses Sialic Acid-Bearing Glycans as a Primary Cell Entry Receptor

Free PMC article

Human Adenovirus Type 26 Uses Sialic Acid-Bearing Glycans as a Primary Cell Entry Receptor

Alexander T Baker et al. Sci Adv. .
Free PMC article


Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis. As non-enveloped, double-stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus type 26 (HAdV-D26) is both a cause of EKC and other diseases and a promising vaccine vector. HAdV-D26-derived vaccines are under investigation as protective platforms against HIV, Zika, and respiratory syncytial virus infections and are in phase 3 clinical trials for Ebola. We recently demonstrated that HAdV-D26 does not use CD46 or Desmoglein-2 as entry receptors, while the putative interaction with coxsackie and adenovirus receptor is low affinity and unlikely to represent the primary cell receptor. Here, we establish sialic acid as a primary entry receptor used by HAdV-D26. We demonstrate that removal of cell surface sialic acid inhibits HAdV-D26 infection, and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.


Fig. 1
Fig. 1. HAdV-D26K forms a local basic area in the apical depression to facilitate sialic acid binding despite an overall acidic predicted isoelectric point.
(A) HAdV-D26K has low (56.76%) sequence identity with fiber knobs known to bind sialic acid by a similar mechanism and an acidic isoelectric point. The electrostatic potential surfaces of HAdV-D8K (B), HAdV-D64/37 (C), and HAdV-D19p (D) fiber knobs are highly basic, especially about the central depression about the threefold axis. (E) HAdV-D26 fiber knob is less basic overall but maintains positive potential in the central depression. Surfaces are displayed at ±10 mV, and the two residues that differ between HAdV-D19p and HAdV-D37/64 are shown as green sticks.
Fig. 2
Fig. 2. HAdV-D26K shares key binding residues with sialic acid–using adenoviruses and exploits sialic acid to infect cells.
(A) Sequence alignment of HAdV-D26K shows conservation of key binding residues with known sialic acid–binding adenoviruses; top numbering is according to HAdV-D26K. Residues boxed in red form polar contacts with sialic acid, those boxed in black denote contact sialic acid via water bridge, and those boxed in orange indicate hydrophobic contacts; all HAdV-D26K polar contacts also form water bridges. Neuraminidase treatment does not reduce the ability of HAdV-D5/B35K (B) or HAdV-C5 (C) to infect SKOV-3 (ovarian adenocarcinoma), BT-20 (breast carcinoma), or MDA-231 (metastatic breast adenocarcinoma) cells, while HAdV-D5/D26K (D) is significantly inhibited. n = 3 biological replicates; error bars indicate ±SD.
Fig. 3
Fig. 3. Sialic acid binds in the apical depression of HAdV-D26 fiber knob protein.
The map shows clear density for a ligand (A), which is best described by a double conformer of sialic acid (B). (C) Sialic acid (orange) is seen to bind in three locations in the apical depression of the HAdV-D26 fiber knob, bridging between monomers (shades of blue) of the trimeric assembly. Crystallization statistics are provided in table S1; 2FoFc map (blue mesh, σ = 1.5) and FoFc (green mesh, σ = 3.0).
Fig. 4
Fig. 4. HAdV-D26K forms a complex interaction network of hydrophobic and electrostatic interactions with sialic acid.
Sialic acid (orange) is seen to bind HAdV-D26 (A) and HAdV-D37 (B) through a network of polar contacts (red dashes) and hydrogen bonds (blue dashes). The interaction is stabilized by hydrophobic interactions (red regions on white surface) with the N-acetyl CH3 group, but different residues in HAdV-D26 (C) and HAdV-D37 (D). Waters are shown as cyan spheres, residues forming comparable contacts in HAdV-D26 and HAdV-D37 are shown as blue sticks, and other residues are shown as green sticks. Oxygen and nitrogen are seen in red and blue, respectively.
Fig. 5
Fig. 5. HAdV-D26K uses an induced-fit mechanism in sialic acid binding.
HAdV-D26K residue Gln348 can occupy multiple conformations, with a greater preference for conformation A (capable of forming a polar contact with the glycerol arm of sialic acid) at pH 8.0 (A) than at pH 4.0 (B). (C) Ile324 has two conformations when HAdV-D26K is unliganded (PDB 6FJO). (D) However, upon sialic acid binding, the Ile324 adopts a single confirmation, creating a hydrophobic indentation around the N-acetyl methyl group bounded by Ile324, Ile310, and the ring of Tyr312.

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