Figure 1.
Sensitivity to neutralization by alpha-defensins is AdV species specific.
HAdVs were incubated with 15 µM HD5 (A) or HNP1 (B) and assessed for infectivity on A549 cells. Data are the mean of the percent infectivity compared to control cells infected with each virus in the absence of defensin for at least three independent experiments ± SD. The upper limit for quantification of this assay is 200%. Virus serotypes are grouped by species (A–F).
Figure 2.
A) HD5 binding to a representative defensin-sensitive serotype (HAdV-5) was quantified by an equilibrium-binding assay. Data are the mean of the number of HD5 molecules bound per virion at the indicated HD5 concentrations from at least three independent experiments ± SD. Binding curves were fitted using Prism software. B) Binding of HD5 to additional defensin-sensitive (HAdV-7p) and resistant (HAdV-19c, -25p, -51p) serotypes expressed as a percent of HD5 bound to HAdV-5. Data are the mean of two or three independent experiments ± SD.
Figure 3.
HD5 antiviral activity is structure dependent.
A) Ribbon representation of HD5 (PDB 1ZMP). Three disulfide bonds are numbered, and the two residues comprising the conserved salt bridge (R6 and E14) are indicated. B) Ad5.eGFP was incubated with 15 µM of each of the indicated defensins. HD5-Abu is HD5 with the six cysteines replaced with L-α-aminobutyric acid. Data are the mean percent of eGFP positive cells compared to control cells infected in the absence of defensin for at least three independent experiments ± SD. C) Binding of Alexa Fluor 488-labeled HAdV-5 to cells was assessed after incubation with or without 20 µM HD5 or HD5-Abu and in competition with 100 nM 5FK or 16 FK. Data are the mean fold increase in geometric mean fluorescence compared to cells alone and are of at least 10,000 cells from each of three independent experiments ± SD.
Figure 4.
CryoEM structures of Ad5.F35 and Ad5.F35+HD5.
A) Reconstructions viewed along icosahedral 2-fold axes and shown radially color-coded (blue = 405 Å; cyan = 425 Å; green = 445 Å, yellow = 465 Å; red = 485 Å). Inset, enlarged views of the vertex regions. B) and C) Ad5.F35 and Ad5.F35+HD5 difference maps. The density representations of the docked hexon and penton base/fiber complex are in blue, the Ad5.F35 difference map is in yellow, and the Ad5.F35+HD5 difference map is in red. Two threshold levels are shown for the Ad5.F35+HD5 difference map, one showing only the strongest density (middle) and a second at just above the noise level (right). Only one threshold level is shown for the Ad5.F35 difference map at just above the noise level. B) Four unique hexons, numbered 1–4, within the asymmetric unit of the icosahedral capsid. C) Penton base and fiber viewed at a 45° angle. Scale bars, 100Å.
Figure 5.
HD5 binding regions on the penton base and fiber.
(A) Alignment of the N-terminal sequences of fiber from serotypes that were studied for defensin sensitivity. Several sequences are representative of multiple serotypes within a species, as indicated. HAdV-F serotypes have a short (S) and long (L) fiber. No sequence information is available for this region for HAdV-23 and -51. Sequences for defensin-resistant serotypes are in red and those for defensin-sensitive serotypes are in black. The variable region is in bold with the key residues underlined for HAdV-C and HAdV-D. Sequences shown correspond to residues 10 to 35 in HAdV-2. B-D) The strongest density in the Ad5.F35+HD5 difference map (black mesh) is shown together with the Ad5.F35 difference map (colored in gold for the RGD loop of the penton base and green for the fiber shaft). Also shown are the docked penton base (gold ribbon) and N-terminal fiber peptide (green ribbon) from the HAdV-2 penton crystal structure (PDB 1X9T). The side chains of fiber residues Asp-18 and Thr-19 (DT of DTET) are shown in a space filling representation. The penton complex is shown in both top (B) and side (C) views. In (D), the crystal structure of an HD5 monomer (PDB 1ZMP, blue ribbon) is included for scale.
Figure 6.
Neutralization determinants are located in fiber and penton base.
A) Schematic of chimeric viruses. Capsid proteins are depicted in the order in which they are encoded in the virus genome for HAdV-5 (white) and HAdV-19c (grey). The variable residues in fiber (GYAR and DTET) are indicated for each construct. B) Each of the chimeric viruses was incubated with 5 µM (grey) or 10 µM (black) HD5 and assessed for infectivity on A549 cells. Data are the mean of the percent infectivity compared to control cells infected with each virus in the absence of defensin for at least three independent experiments ± SD. The upper limit for quantification of this assay is 200%.