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Published on: November 12, 2015
Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular
Ayodeji O Ipinmoroti1, Brennetta J Crenshaw1, Rachana Pandit1
1Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL 36104, USA.
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).
Insights
Adenovirus type 3 (Ad3) infection alters extracellular vesicle (EV) production and composition in lung cells. These Ad3-modified EVs may play a role in viral pathogenesis and offer potential diagnostic and therapeutic targets for adenovirus infections.
Area of Science:
- Virology
- Cell Biology
- Immunology
- Extracellular Vesicles
Background:
- Adenoviruses (Ad) cause respiratory infections and are used in gene therapy.
- Extracellular vesicles (EVs) are implicated in viral entry and pathogenesis.
- Adenovirus type 3 (Ad3) may influence EV biogenesis and immune responses.
Purpose of the Study:
- To investigate the impact of Ad3 infection on EV biogenesis and composition in A549 lung epithelial cells.
- To assess changes in EV quantity, size, protein content, and specific protein markers following Ad3 exposure.
Main Methods:
- A549 cells were infected with Ad3 at various multiplicities of infection (MOIs) and time points.
- Cell viability was assessed using MTT and calcein-AM assays.
- EVs were isolated via ultracentrifugation and characterized using nanoparticle tracking, TEM, SDS-PAGE, and immunoblotting.
Main Results:
- Ad3 infection decreased cell viability in a dose- and time-dependent manner.
- Increased MOIs led to significantly larger particle sizes, higher concentrations, and greater total EV protein content.
- Ad3-infected cells produced EVs with elevated levels of Rab5, Rab7, and Rab35, alongside standard EV markers.
Conclusions:
- Ad3 infection modulates EV biogenesis, composition, and trafficking in lung epithelial cells.
- These Ad3-induced EV alterations may contribute to viral infection pathogenesis and disease progression.
- EVs represent potential diagnostic and therapeutic targets for adenovirus and related viral infections.
