DOI: 10.5061/dryad.d2547d89h
title: Data from: Alpha-defensin binding expands human adenovirus tropism

description: Mammalian α-defensins are a family of abundant effector peptides of the
 mucosal innate immune system. Although primarily considered to be
 antimicrobial, α-defensins can increase rather than block infection by
 certain prominent bacterial and viral pathogens in cell culture and in
 vivo. We have shown previously that exposure of mouse and human
 adenoviruses (HAdVs) to α-defensins is able to overcome competitive
 inhibitors that block cell binding, leading us to hypothesize a
 defensin-mediated binding mechanism that is independent of known viral
 receptors. To test this hypothesis, we used genetic approaches to
 demonstrate that none of several primary receptors nor integrin
 co-receptors are needed for human α-defensin-mediated binding of HAdV to
 cells; however, infection remains integrin dependent. Thus, our studies
 have revealed a novel pathway for HAdV binding to cells that bypasses
 viral primary receptors. We speculate that this pathway functions in
 parallel with receptor-mediated entry and contributes to
 α-defensin-enhanced infection of susceptible cells. Remarkably, we also
 found that in the presence of α-defensins, HAdV tropism is expanded to
 non-susceptible cells, even when viruses are exposed to a mixture of both
 susceptible and non-susceptible cells. Therefore, we propose that in the
 presence of sufficient concentrations of α-defensins, such as in the lung
 or gut, integrin expression rather than primary receptor expression will
 dictate HAdV tropism in vivo. In summary, α-defensins may contribute to
 tissue tropism not only through the neutralization of susceptible viruses
 but also by allowing certain defensin-resistant viruses to bind to cells
 independently of previously described mechanisms.
description: # Alpha-defensin binding expands human adenovirus tropism
 [https://doi.org/10.5061/dryad.d2547d89h](https://doi.org/10.5061/dryad.d2547d89h) ## Description of the data and file structure Files are provided for each figure as follows: Figure 1A: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif) from 2 independent replicates (Rep##). Experimental variables include human defensin 5 (HD5) and two human adenovirus (HAdV) serotypes, C5 and D64. Samples were arranged as indicated on the plate map (.pdf). Figure 1B: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif) from 3 independent replicates (Rep##). Samples were arranged as indicated on the plate map (.pdf). Experimental variables include wheat germ agglutinin (WGA) and human defensin 5 (HD5). From these images, total well fluorescence was enumerated using Fiji (.xlsx) by applying a rolling ball background subtraction and measuring integrated density limited to the threshold indicated in the .xlsx file. Sample wells were normalized to control wells (no WGA, no HD5), and the normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 1C: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif files) from 3 independent replicates (Rep##). Samples were arranged as indicated on the plate map (.pdf). Experimental variables include neuraminidase (NA) and human defensin 5 (HD5). From these images, total well fluorescence was enumerated using Fiji (.xlsx) by applying a rolling ball background subtraction and measuring integrated density limited to the threshold indicated in the .xlsx file. Sample wells were normalized to control wells (no NA, no HD5), and the normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 2B: Paired end reads were obtained from an Illumina platform (.fastq.gz) for an amplicon from the coxsackievirus and adenovirus receptor (CAR) knockout clone used in this study. Amplicon and sgRNA sequences (.rtf) are provided. Figure 2C: Archival cytometry standard (.ACS) files containing flow cytometry data and FlowJo 10.7.1 analysis is provided for 3 independent replicates (Rep##). A table (.xlsx) of the tube number in the ACS files corresponding to each sample is provided. Experimental variables include antibody (anti-CAR vs isotype) and A549 cell genotype: wild type (WT) or CAR knockout (KO). Figure 2D: A scan of a 96-well plate of cells infected with a 3-fold dilution series (top to bottom) of green fluorescent protein-expressing viruses was obtained using a multi-mode plate scanner (*_platescan.tif). Experimental variables include two human adenovirus (HAdV) serotypes, C5 and B35, and A549 cell genotype: wild type (WT) or CAR knockout (KO). Samples were arranged as indicated on the plate map (.pdf). Figure 2E: Archival cytometry standard (.ACS) files containing flow cytometry data and FlowJo 10.7.1 analysis is provided for 3 independent replicates (Rep##). A table (.xlxs) of the tube number in the ACS files corresponding to each sample and analysis to calculate fold change in geometric mean fluorescence (GMF) is provided. This data was analyzed using GraphPad Prism 10.0.2 (.prism). Experimental variables include human defensin 5 (HD5), two human adenovirus (HAdV) serotypes (C5/D64-HVR1 and C5/D64-HVR1/RGE), and A549 cell genotype: wild type (WT) or CAR knockout (KO). Red values in columns C, J, and Q of the .xlxs table correspond to the geometric mean fluorescence of control samples (without HD5) that were used as the denominator to calculate fold change for other samples. Figure 3A: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif files) from 5 independent replicates (Rep##). Samples were arranged as indicated on the corresponding plate maps (.pdf). Figure 3B: Analysis of the images in Figure 3A. From these images, total well fluorescence was enumerated using Fiji (.xlsx) by measuring integrated density limited to the threshold indicated in the .xlsx file. Experimental variables include human defensin 5 (HD5) and three human adenovirus (HAdV) serotypes (C5, C5/D64-HVR1, and C5/D64-HVR1/RGE). Data for WT A549 cells (*top) and CAR knockout A549 cells (*bottom) were both normalized to WT A549 cells infected in the absence of HD5 for each virus. The normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 4A: Archival cytometry standard (.ACS) files containing flow cytometry data and FlowJo 10.7.1 analysis is provided for 3 independent replicates (Rep##). A table (.xlxs) of the tube number in the ACS files corresponding to each sample and analysis to calculate fold change in geometric mean fluorescence (GMF) is provided. This data was analyzed using GraphPad Prism 10.0.2 (.prism). Experimental variables include the E21 methyl mutant of human defensin 5 (E21me HD5), two human adenovirus (HAdV) serotypes (C5/D64-HVR1 and C5/D64-HVR1/RGE), and A549 cell genotype: wild type (WT) or CAR knockout (KO). Red values in columns C, J, and Q of the .xlxs table correspond to the geometric mean fluorescence of control samples (without E21me HD5) that were used as the denominator to calculate fold change for other samples. Figure 4B: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif files) from 4 independent replicates (Rep##). Samples were arranged as indicated on the corresponding plate maps (.pdf). Figure 4C and 4D: Analysis of the images in Figure 4B. From these images, total well fluorescence was enumerated using Fiji (.xlsx) by measuring integrated density limited to the threshold indicated in the .xlsx file. Experimental variables include the E21 methyl mutant of human defensin 5 (E21me HD5), three human adenovirus (HAdV) serotypes (C5, C5/D64-HVR1, and C5/D64-HVR1/RGE), and A549 cell genotype: wild type (WT) or CAR knockout (KO). Data for WT A549 cells (Figure 4C) and CAR knockout A549 cells (Figure 4D) were both normalized to WT A549 cells infected in the absence of HD5 for each virus. The normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 5A: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif files) from 9 independent replicates (Rep##). Samples were arranged as indicated on the corresponding plate maps (.pdf). Figure 5B: Analysis of the images in Figure 4B. From these images, total well fluorescence was enumerated using Fiji (.xlsx) by measuring integrated density limited to the threshold indicated in the .xlsx file. Experimental variables include the E21 methyl mutant of human defensin 5 (E21me HD5), three human adenovirus (HAdV) serotypes (C5, C5/D64-HVR1, and C5/D64-HVR1/RGE), and A549 cell genotype: wild type (WT) or CAR knockout (KO). Data for WT A549 cells (*top) and CAR knockout A549 cells (*bottom) were both normalized to WT A549 cells infected in the absence of HD5 for each virus. The normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 6B: Scans of 96-well plates of A549 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif) from multiple independent replicates (Rep##). Samples were arranged as indicated on the plate map (.pdf). Experimental variables include human defensin 5 (HD5) and human adenovirus (HAdV) serotype: HAdV-B35 (Ad35), HAdV-D37 (Ad37), and HAdV-B35/D37 Vertex/HVR1 (VH). Viruses studied in parallel have the same replicate number. From these images, total well fluorescence was enumerated using Fiji (.xlsx) by applying a rolling ball background subtraction and measuring integrated density limited to the threshold indicated in the .xlsx file. Sample wells were normalized to control wells (no HD5), and the normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 6C: Analysis of the images in Figure 6D. From these images, total well fluorescence was enumerated using Fiji (.xlsx) by measuring integrated density limited to the threshold indicated in the .xlsx file. Experimental variables include human defensin 5 (HD5) and HAP1 cell genotype: wild type (WT) or CD46 knockout (KO). Data for CD46 KO cells was normalized to WT HAP1 cells infected in the absence of HD5 for each virus. The normalized data from all replicates was analyzed using GraphPad Prism 10.0.2 (.prism). Figure 6D: Scans of 96-well plates of HAP1 cells infected with green fluorescent protein-expressing viruses were obtained using a multi-mode plate scanner (*_platescan.tif) from multiple independent replicates (Rep##). Samples were arranged as indicated on the plate map (.pdf). Figure 7: Archival cytometry standard (.ACS) files containing flow cytometry data and FlowJo 10.7.1 analysis is provided for 3 independent replicates (Rep##). A table (.xlxs) of the tube number in the ACS files corresponding to each sample and analysis to calculate %GFP positive is provided. This data was analyzed using GraphPad Prism 10.0.2 (.prism). Experimental variables include human defensin 5 (HD5), cell type (A549 and CTM-93), and culture format (monoculture or mixed).