Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles

Abstract

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

Figure 1

The effect of HAdV3 on A549 viability. (a) Intracellular esterase activity of viable A549 cells. Cells were labeled with nonfluorescent calcein-AM dye which is converted to green fluorescent molecules as a result of ester hydrolysis. The images were acquired at the indicated time after incubation. (b) Postinfection quantification of viable A549 cells after incubation with 3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetra bromide after 24 h (c) 48 h, and (d) 72 h. The cells were incubated with dye solution at 37°C for 3-4 h; absorbance was read at 570 nm. Data shows the mean ± SEM from four independent experiments performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 2

Morphological characterization of HAdV3-infected A549-derived exosomes. (a) TEM analysis of HAdV3-infected A549-derived exosomes. Images showing morphologies of EVs isolated from uninfected and MOI 300. (b) NTA showing distribution pattern of uninfected EVs (c) at MOI 300. (d) The graph showing assessment of EVs' mean particle sizes after 48 h of infection and (e) 72 h of infection. (f) The graph showing quantification of HAdV3-infected A549-derived EVs per mL after 48 h and (g) 72 h of infection. Data show the mean ± SEM from four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 3

The effect of HAdV3 infection on intracellular trafficking GTPase. (a) Dot blot analysis showing expression of Rab5, Rab7, and Rab35 after 48 h and 72 h infection. EVs were probed for the presence of Rab proteins. (b) (i) Graphs showing quantification of Rab5 level after 48 h and (ii) 72 h of infection. (c) (i) Rab7 after 48 h and (ii) 72 h of infection. (d) (i) Rab35 after 48 h and (ii) 72 h of infection. Data show the mean ± SEM from three independent experiments. Dots shown in the figure are representative of four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 4

The effect of HAdV3 infection on intracellular trafficking GTPase in A549 lysate. (a) Quantification of dot blot analysis showing expression of Rab5, Rab7, and Rab35 after 48 h and 72 h infection. A549 cell lysate was probed for the presence of Rab proteins. (a) (i) Graphs showing quantification of Rab5 level after 48 h and (ii) 72 h of infection. (b) (i) Rab7 after 48 h and (ii) 72 h of infection. (c) (i) Rab35 after 48 h and (ii) 72 h of infection. Dots shown in the figure are representative of four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 5

The effect of HAdV3 infection on isolated EV classical markers. Quantification of dot blot analysis showing expression of CD9, CD63, TSG101, and GAPDH expression after 48 h and 72 h infection. EVs were probed for the presence of classical markers. (a) (i) Graph showing quantification of CD9 level after 48 h and (ii) 72 h of infection. (b) (i) CD63 after 48 h and (ii) 72 h of infection .(c) (i) TSG101 after 48 h and (ii) 72 h of infection. (d) (i) GAPDH after 48 h and (ii) 72 h of infection. Dots shown in the figure are representative of four independent experiments. Data show mean ± SEM from three independent experiments. Data show the mean ± SEM from four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 6

The effect of HAdV3 infection on heat shock protein, Toll-like receptor, and caspase. Quantification of dot blot analysis showing expression of Hsp70, Hsp100, TLR7, caspase 1, and H2A-X expression after 48 h and 72 h infection. EVs were probed for the presence of proteins. (a) (i) Graphs showing quantification of Hsp70 level after 48 h and (ii) 72 h of infection. (b) (i) Hsp100 after 48 h and (ii) 72 h of infection. (c) (i) TLR7 after 48 h and (ii) 72 h of infection. (d) (i) Caspase 1 after 48 h and (ii) 72 h of infection. (e) (i) H2A-X after 48 h and (ii) 72 h of infection. Dots shown in the figure are representative of four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 7

The effect of HAdV3 infection on proinflammatory interleukin and interferon. (a) Dot blot analysis showing expression of IL-1β and TLR7 after 48 h and 72 h infection. EVs were probed for the expression of IL-1β and TLR7 proteins. (b) Western blot analysis showing Hsp70, CD9, and IRF8 expression after 48 h and 72 h of infection. (c) (i) Graph showing quantification of IL-1β after 48 h and (ii) 72 h of infection. (d) (i) IRF-3 after 48 h and (ii) 72 h of infection. Dots shown in the figure are representative of four independent experiments. Data shows the mean ± SEM from four independent experiments and performed using one-way analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is indicated by the mean ± SD as follows:^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.