Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses

Abstract

Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base, and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and nonhuman mastadenoviruses. Knowledge of the structure of enteric adenoviruses provides a starting point for the design of vectors suitable for oral delivery or intestinal targeting.

Fig. 1. The cryo-EM structure of HAdV-F41 at 4.0 Å.

(A) Radially colored surface rendering of the HAdV-F41 map viewed down the twofold icosahedral axis. One facet is identified by a white triangle. Icosahedral symmetry axes are indicated with white symbols: fivefold (pentagon), threefold (triangle), and twofold (oval). A Gaussian filter has been applied to the map to accentuate the main features. (B) The molecular models of the major and minor coat proteins traced in the icosahedral HAdV-F41 facet. Top: view from outside the capsid. Bottom: view inside the capsid. Protein colors as indicated by the legend at the left hand side. The four hexon trimers in the asymmetric unit (AU) are numbered H1 to H4. (C) Cartoon depicting the icosahedral AU and surrounding hexons, as seen from outside the capsid. The icosahedral symmetry axes are indicated with red filled symbols. A hollow red triangle indicates the L3 axis between hexons 2, 3, and 4.

Fig. 2. Hexon and penton base structures.

(A) Superposition of the HAdV-C5 [Protein Data Bank (PDB) ID: 6B1T chain A, cyan] and HAdV-F41 hexon monomers, colored by root mean square deviation (RMSD). Residues exceeding the Chimera RMSD cutoff, or where RMSD calculation is not possible because they are not traced in HAdV-C5, are in purple. The orientation with respect to the capsid (outside/inside) is indicated. n.a., not available. (B) Sequence alignment of hexon HVRs from HAdV-C5 and HAdV-F41. Here and in (D), gray bars indicate traced regions. The HAdV-C5 acidic region is highlighted by a black rectangle. Amino acids are colored by polarity. (C) Superposition of the HAdV-C5 penton base monomer (PDB ID: 6B1T chain M, pink) and HAdV-F41 colored by RMSD as in (A). The variable loop and fiber binding rearrangement regions are shown with the map in gray mesh. (D) Sequence alignments focusing on the penton base (PB) N-terminal arm, variable and hypervariable loops, and hydrophobic rim; and on the N-terminal peptides of the HAdV-C5 (PDB ID: 3IZO) and the l (long) and s (short) HAdV-F41 fibers. Black rectangles highlight the RGD/IGDD sequence motifs, the conserved fiber peptide, and the nonconserved Ser⁴²⁶ in the hydrophobic rim. The histogram indicates the mean pairwise identity over all pairs in the column. (E) The penton base pentamer is shown with one monomer in black and the rest in white. Hydrophobic residues conserved with HAdV-C5 in the fiber binding ring are colored cyan (Phe⁴¹², Tyr⁴¹³, Tyr⁴¹⁹, and Leu⁴²²), and those nonconserved (Ser⁴²⁶) are in maroon. (F) HAdV-C5 and HAdV-F41 penton base pentamers colored by surface electrostatic potential.

Fig. 3. Protein IX triskelion structure.

(A) Schematics showing the organization of protein IX in HAdV-C5, BAdV-3, and HAdV-F41, where only the N-terminal domain is ordered. Triskelions located at the I3 symmetry axis (filled triangle) are in cyan, and those located at the L3 axis (empty triangle) are in several shades of pink. Dashed lines: rope domains in HAdV-C5 One IX monomer is depicted in cyan on top of each schematic. (B) Structure-based sequence alignment of protein IX in HAdV-C5, HAdV-D26, HAdV-F41, and BAdV-3. Black text: residues modeled in the different structures. White text on black background: regions not modeled. Gray background: regions modeled, but not in all independent copies of IX. Red boxes: N-terminal extra residues in HAdV-F41, triskelion hydrophobic core, and poly-Ala stretch (absent in BAdV-3). Blue boxes highlight the two Phe residues discussed in the text. Blue arrows: the two flexible bends. (C) N-terminal domain of HAdV-F41 protein IX rainbow colored from blue (N terminus) to red (last traced residue), with the density map in gray mesh and a zoom into the hydrophobic residues discussed in the text. (D) Comparison of the triskelions in HAdV-C5 (PDB ID: 6B1T), HAdV-F41, and HAdV-D26 (PDB ID: 5TX1). Top row: triskelions as seen from outside the capsid. Bottom: a view rotated by 90°, so that the hexon shell surface would be located at the bottom.

Fig. 4. Organization of protein IX.

(A) External remnant density (green and yellow, unsharpened, 0.2σ). Modelled triskelions are in cyan (I3 axis, I3 NT) and magenta (L3 axes, L3 NT). Dotted rectangles: location of helix bundles in HAdV-C5. (B) Interpretation of the remnant map. Apart from elements shown in (A), density protruding at the L3 axes is in blue, and the proposed path for the rope domains corresponding to each triskelion in dashed lines. (C) Cartoon representing the hypothesis that the C-terminal domains associate forming a mobile, spike-like structure whose density is averaged away. Hexon HVR2 loops would constrain the flexibility of IX, producing the most evident blurry density (dark blue splotch). Less intense dark blue represents weaker density observed in some localized reconstruction classes (fig. S5B). (D) The rope domain of the IX molecules forming the I3 triskelion follows different paths in HAdV-C5/D26 and HAdV-F41. One of the IX monomers forming the HAdV-D26 I3 triskelion is in green; the equivalent monomer in HAdV-F41 in cyan; and the HAdV-F41 L3 triskelion in magenta. Grey surface: HAdV-F41 remnant map. Hexons (H3, H3′, H4) in semi-transparent surface. Top: view from outside the capsid as in (A). Bottom: side view after rotating as indicated. Notice that the blurry density at L3 protrudes above the hexon towers. (E) Zoom in on the region where the triskelion ends and the rope domain turns. Green, overlapped structures of IX in HAdV-C5 and HAdV-D26. HAdV-D26 residue labels are underlined. Dashed lines: untraced rope domains in HAdV-C5 and HAdV-F41. Grey mesh: HAdV-F41 remnant density corresponding to the rope domain. Hexon amino acids with RMSD above 2 Å are in red.

Fig. 5. Proteins IIIa and VIII.

(A) Superposition of HAdV-C5 IIIa (PDB ID: 6B1T, chain N) in yellow and HAdV-F41 IIIa colored by RMSD as in Fig. 2. The GOS glue, connecting helix, VIII-binding, and core proximal domains are indicated. (B) Detail of the connecting helix to show its fit into the cryo-EM map. (C) Schematics showing the alignment of protein IIIa sequences in HAdV-C5, HAdV-D26, and HAdV-F41. Traced domains are indicated below, in different colors. APD is the appendage domain traced in HAdV-D26, but not in the other two viruses. The N-terminal extension in HAdV-F41 (N-term extra) and maturation cleavage site (scissors) are also indicated. (D) Superposition of HAdV-C5 protein VIII (PDB ID: 6B1T Chain O) in orange and HAdV-F41 VIII colored by RMSD. The body, neck, and head domains are indicated, as well as the gap corresponding to the peptide cleaved during maturation (scissors). (E) Sequence alignment showing the two central peptides of protein VIII cleaved by AVP (AVPa and AVPb) in HAdV-C5, HAdV-D26, and HAdV-F41. Amino acid and mean pairwise identity histogram color schemes are the same as in Fig. 2.

Fig. 6. Additional internal components of the HAdV-F41 capsid.

(A) Left: remnant densities in one facet (red), not occupied by traced proteins hexon (light blue), penton (light pink), IIIa (yellow), or VIII (orange). Right: remnant map components color-coded as RD1 to RD4 (see text). Densities correspond to the unsharpened map contoured at 1.5σ. (B) N-terminal peptides of proteins VI (pVIn, two copies) and VII (pVIIn₂) tentatively traced in RD1, inside the hexon cavity. (C) Proposed interpretation of remnant densities RD3 and RD4. Except for the inset at the right hand side, RD in the sharpened map is shown, contoured at 2.5σ. Orange thin ribbon: traced part of the two copies of protein VIII (VIII vertex and VIII central plate). Orange thick worms: poly-Ala peptides modeled in the unassigned densities proposed to correspond to the central peptide of VIII. Left inset: possibly α-helical shape of one of the remnant densities in RD3. This density is much weaker in RD4, where a black star indicates its position. Scissors: AVP cleavage site in protein VIII. Peptide pVIn is represented as a gray ribbon, with the possible location of its four untraced N-terminal residues as a black dotted line. In the RD3 panel, the IIIa connecting helix is in yellow, the first residue traced in the penton base protein (E 35) is colored pink, and a dotted pink line indicates the possible trajectory of the untraced 34 residues. Right framed zoom: the IIIa connecting helix is shown overlapped with the HAdV-C5 helix in red, and the unsharpened map contoured at 1σ is shown as a gray mesh, to emphasize low-resolution density proposed to correspond to the penton base N-terminal arm.