E1(+)/GFP Human Adenovirus (Serotype 5)

Using T84 cells as a model of polarized epithelium, researchers investigated the role of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. Initial Ad5 infection in T84 cell cultures was inefficient. However, during the progression of Ad5 infection, cell-cell junctions gradually distorted, accompanied by fiber release. Incubation of T84 cell cultures with virion-free supernatant from Ad5-infected cultures resulted in distorted intercellular junctions and reduced infectivity of Ad5-GFP vector. Supernatants containing virion-free fibers were isolated from Ad5-infected culture supernatants. Fractions containing fibers were further characterized, including their ability to inhibit infection with GFP Human Adenovirus (Serotype 5) (Ad5-GFP) vector, their composition in adenoviral structural proteins identified using Western blotting and LC-MS/MS, and their ability to remodel intercellular junctions. Fiber molecules in complexes containing penton bases and hexons or containing mainly hexons were identified. Only fiber complexes with a relatively high penton base content, but not fiber hexon complexes with a low penton base content, could penetrate into T84 cells and cause deformation of intercellular junctions. These findings suggest that the two types of fiber complexes may play different roles in adenovirus infection.

To identify components released by infected cells that cause distortion of intercellular junctions, virion-free supernatants of A549 cultures were prepared at day 3 post-infection with Ad5 at an MOI of 1 (conditioned medium, CM). CM was functionally titrated to determine its ability to inhibit infection of A549 cells with Ad5-GFP vector. In control cultures, CAR was localized to the apical portion of TJs between T84 cells and DSG-2 was localized below the CAR molecules with a clear and distinct staining pattern (Figure 1A). Incubation of T84 cells with CM from the apical surface for 6 h resulted in diffuse localization of CAR to deeper layers below the apical surface and internalization of CAR (Figure 1A). Co-staining of CAR and DSG-2 further supported the distortion of intercellular junctions (Figure 1A). DSG-2 staining became diffuse and distorted after treatment with CM compared to control cultures. For the same culture time, application of CM from the basolateral side resulted in a more pronounced distortion of CAR localization than application of CM from the apical surface (Figure 1B). Fibrin was clearly bound to the cell membrane beneath the basolateral side (Figure 1B).

Figure 1. Remodeling of cell-cell junction between T84 epithelial cells by virion-free supernatant from Ad5 infected culture. (Zhang B, et al., 2015)