Related Experiment Video
Updated: Sep 30, 2025
Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System
Published on: July 30, 2018
A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions
Raphael L Tsoukas1,2, Wolfram Volkwein3, Jian Gao1
1Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University, 58453 Witten, Germany.
Abstract:
To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.
Insights
Understanding adenovirus infection mechanisms is key for gene therapy. This study used a human knockout model to identify specific adenovirus receptors, revealing CD46 and CAR dependencies for different adenovirus types.
Area of Science:
- Virology
- Gene Therapy
- Molecular Biology
Background:
- Adenoviral gene delivery requires understanding virus-host interactions.
- Key adenoviral attachment proteins include CD46, CAR, and sialic acid.
- Previous receptor studies often used non-human cells, limiting direct human application.
Purpose of the Study:
- To systematically analyze adenovirus receptor usage in human cells.
- To compare the side-by-side receptor dependency of various adenoviruses.
- To identify suitable adenovirus serotypes for gene therapy applications.
Main Methods:
- Utilized a reporter gene-tagged adenovirus library.
- Employed CRISPR/Cas9 to create CD46 and CAR knockout A549 cells (human lung epithelial carcinoma line).
- Infected knockout cells with 22 luciferase-expressing adenoviruses from species B, C, D, and E.
Main Results:
- Identified CD46-dependent adenoviruses (e.g., HAdV-B16, -B21, -B50, -B34, -B35).
- Identified CAR-dependent adenoviruses (e.g., HAdV-C5, -E4).
- Demonstrated dual CAR and CD46 roles for HAdV-B3, -B14, and species D viruses, suggesting additional receptors.
Conclusions:
- The human knockout cell model is effective for receptor usage analysis.
- This research aids in selecting appropriate adenoviruses for gene therapy.
- Provides insights into adenovirus infection biology and host cell interactions.
