The HAdV-7 DNA-binding protein (DBP) increases viral infectivity and replication competence compared with HAdV-3
Description
Human adenovirus type 7 (HAdV-7) causes more severe disease than HAdV-3. The adenoviral DNA binding protein (DBP) is a key regulatory protein involved in the viral replication cycle. However, the role of DBP in HAdV-7-induced severe disease remains poorly understood. rAd3E-7DBP was generated by replacing the DNA-binding protein (DBP) in recombinant HAdV-3 (rAd3E) with HAdV-7 DBP, and rAd7E-3DBP was constructed by replacing the DBP in recombinant HAdV-7 (rAd7E) with HAdV-3 DBP. We compared infectivity, cellular tropism, and replication competence between wild-type HAdV-3 and HAdV-7, and among recombinant strains rAd3E, rAd3E-7DBP, rAd7E-3DBP, and rAd7E, in human airway organoids.Wild-type HAdV-7 showed significantly higher infection rates and viral titers than HAdV-3. HAdV-7 infected more ciliated cells, club cells, basal cells, and goblet cells than HAdV-3 in human airway organoids. Additionally, compared with rAd3E, rAd7E infected more ciliated cells, basal cells, club cells, and goblet cells in human airway organoids. rAd7E infected more ciliated and basal cells than rAd7E-3DBP, whereas rAd3E infected fewer ciliated and basal cells than rAd3E-7DBP in human airway organoids. rAd7E showed significantly higher infection rates and viral titers than rAd3E in human airway organoids. rAd7E showed significantly higher infection rates and viral titers than rAd7E-3DBP, whereas rAd3E showed significantly lower infection rates and viral titers than rAd3E-7DBP in human airway organoids.This study suggest that HAdV-7 exhibited a significantly higher infection rate, cellular tropism, and replication competence than HAdV-3 in human airway organoids, and that these differences were due in large part to DBP.
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Steps to reproduce
Human airway organoid infection The 3D airway organoids were sheared by repeated pipetting with a glass Pasteur pipette to expose the apical surface for viral inoculation and resuspended in basal medium containing Advanced DMEM/F12 (Gibco, Cat#12634010), 1% HEPES (Gibco, Cat#15630056), 1% GlutaMax (Gibco, Cat#35050061), and 1% penicillin-streptomycin (Gibco, Cat# 15140122). Then, the airway organoids were infected with HAdV-3, HAdV-7, rAd3E, rAd3E-7DBP, rAd7E-3DBP, or rAd7E at 0.01 MOI for 2 h at 37°C. After washing with phosphate-buffered saline (PBS) 3 times to remove unbound viruses, airway organoids were re-embedded in 70% Matrigel in 24-well tissue plates and cultured in 500 μL proximal differentiation medium per well. At 24 h, 48 h, 72 h, and 96 h, the cell-free culture medium was collected for viral titration by TCID50 assay, as described above. The infected airway organoids were collected to quantify the percentage of infected cells.