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Science 338 (6108): 795-798

Copyright © 2012 by the American Association for the Advancement of Science

Coagulation Factor X Activates Innate Immunity to Human Species C Adenovirus

Konstantin Doronin1,*, Justin W. Flatt2,*, Nelson C. Di Paolo1,*, Reeti Khare1, Oleksandr Kalyuzhniy3,{dagger}, Mauro Acchione4, John P. Sumida4, Umeharu Ohto5,6, Toshiyuki Shimizu5,6, Sachiko Akashi-Takamura7, Kensuke Miyake7,8, James W. MacDonald9, Theo K. Bammler9, Richard P. Beyer9, Frederico M. Farin9, Phoebe L. Stewart2, and Dmitry M. Shayakhmetov1,{ddagger}

1 Department of Medicine, University of Washington, Seattle, WA 98195, USA.
2 Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
3 Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
4 Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
5 Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
6 RIKEN SPring-8 Center, Kouto 1-1-1, Sayo, Hyogo 679-5148, Japan.
7 Division of Innate Immunity, Department of Microbiology and Immunology, University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan.
8 Laboratory of Innate Immunity, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan.
9 Functional Genomics and Proteomics Core Facility, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98195, USA.


Figure 1
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Fig. 1. Cryo-EM structure of the FX-HAdv5 complex and simulation of the FX-hexon interface using MDFF. (A) The cryo-EM structure of HAdv5 in complex with FX. The density is shown with the hexon capsid in blue, penton base in gold, fiber in green, and FX in red. Scale bar, 100 Å. (B) An enlarged view of the FX-HAdv5 complex showing the network of the FX density above the hexon capsid. (C) The best rigid-body fit orientation of the zymogenic FX model (red ribbon) within FX cryo-EM density (transparent pink). This FX density is selected from above a hexon near the icosahedral threefold axis of the capsid. (D) Coordinates from a frame in the MDFF simulation that show hexon residues E424 and T425 surround residue K10 of the FX-GLA domain. The side chains of these three residues are shown in space-filling representation and colored by element. (E) FX-GLA domain and associated Ca2+ ions (green) in the central depression of the hexon trimer. (F) Residue K10 in the FX-GLA domain is in close enough proximity to E424 and T425 of hexon to engage in electrostatic interactions.

 

Figure 2
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Fig. 2. A single amino acid substitution (T425A) abrogates FX biding to HAdv5. (A) Kinetic response data and dissociation constant (Kd) for FX binding to the indicated mutant viruses obtained by using surface plasmon resonance analysis (Biacore, GE Healthcare Biosciences, Pittsburgh, PA). Black indicates experimentally obtained data. Orange indicates global fits of these data to 1:1 single-site interaction model. Representative data obtained from four independent experiments are shown. (B to D) In vivo analysis of hexon-mutated viruses. (B) Histological analysis of virus-encoded GFP expression in mouse hepatocytes 48 hours after intravenous infection of mice with WT HAdv5 (WT) or mutated viruses. Representative fields are shown (n = 5 biological replicates). GFP expression is observed as green fluorescence on fixed liver sections. Corresponding fields in 4',6-diamidino-2-phenylindole channel (blue, nuclei-specific staining) are shown. Scale bar, 100 μm. (C) Western blotting analysis of GFP expression in the livers of mice shown in (B). The biological duplicate samples for each virus are shown. (D) Colocalization of virus particles (red) with splenic CD169+ and MARCO+ marginal zone macrophages (green) observed 1 hour after infection for indicated viruses analyzed by means of confocal microscopy. Scale bar, 10 μm. Representative fields are shown. n = 5 biological replicates.

 

Figure 3
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Fig. 3. FX binding–ablated virus triggers blunted transcriptional response of NFKB1-dependent genes in vivo. (A) Gene Ontology pie chart. Genes that were differentially expressed more than 1.5-fold (P < 0.05) in the spleens of WT mice challenged with HAdv5 or mock (saline) were identified. The CateGOrizer tool was used to sort these genes in Gene Ontology categories and determine percentages of differentially expressed genes for each category. (B) Heatmap representation of the averaged gene expression levels for 34-gene set (coactivated in WT and Il1r1–/– mice with P < 0.0002) when WT and Il1r1–/– mice were infected with either HAdv5 or TEA mutant virus. In each experimental group, n = 3 biological replicates. The yellow and blue color legend shows log2-transformed fold changes. Heatmap was generated by using the Bioconductor gplots package (Seattle, WA). (C) z-score map of the transcription factor binding site frequencies in proximal promoters of indicated genes was generated by means of P-scan from the analysis of binding sites for 116 transcription factors (Transfac database). The five-gene set represents a subset of genes from (B) that are the most differentially inducted by HAdv5 and TEA mutant virus (1.5-fold cut off). Nr4a2, *P = 9.61 x 10–7; ATF3, *P = 6.77 x 10–6. The green and red color legend shows log2-transformed fold z-score changes.

 

Figure 4
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Fig. 4. HAdv5 binding to FX induces NF{kappa}-B1–dependent inflammatory cytokines and chemokines downstream of the TLR4-TRIF/MyD88-TRAF6 signaling axis in vivo. (A) Mouse cytokine array panel showing differences in inflammatory cytokines and chemokines in the spleens of WT mice 1 hour after infection with HAdv5 or TEA mutant, determined by means of proteome profiler antibody array. Representative blot from four independent experiments is shown. C, mouse was challenged with saline. (B) mRNA expression of IL-1β in the spleen of WT mice 30 min after challenge with indicated viruses. Graphs show mean ± SD, n = 4 biological replicates, **P < 0.01. AU, arbitrary units reflecting IL-1β to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios. (C and D) mRNA expression of IL-1β in the spleen of WT mice and mice deficient for indicated genes 30 min after challenge with HAdv5. Graphs show mean ± SD, n = 4 biological replicates, **P < 0.01. AU, arbitrary units reflecting IL-1β to GAPDH mRNA ratios. (E) Mouse cytokine array panel showing differences in inflammatory cytokines and chemokines in the spleens of WT and Myd88–/–, Ticam1–/–, Tlr4–/–, and Md2–/– mice 1 hour after challenge with HAdv5, determined by means of proteome profiler antibody array. Representative blot from four independent experiments is shown. C, mouse was challenged with saline. (F) Mouse cytokine array panel showing differences in inflammatory cytokines and chemokines in the spleens of WT mice 1 hour after challenge with WT human adenoviruses of indicated species, determined by means of proteome profiler antibody array. Representative blot from four independent experiments is shown. C, mouse was mock infected with saline.

 


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