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Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain
S C Stevenson1, M Rollence, B White
1Department of Molecular and Cell Biology, Genetic Therapy, Inc., Gaithersburg, Maryland 20878, USA.
Abstract:
The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered.
Insights
Adenovirus fiber proteins bind to cell receptors. Researchers found the fiber head domain determines receptor specificity, enabling potential engineering of adenovirus vectors for targeted gene delivery.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Adenovirus fiber proteins mediate viral attachment to host cells.
- The specific cellular receptors for group C (adenovirus type 5 [Ad5]) and group B (Ad3) adenoviruses are not fully identified.
- Evidence suggests distinct receptors interact with Ad5 and Ad3.
Purpose of the Study:
- To identify the determinants of adenovirus receptor specificity.
- To investigate the role of the fiber protein's head domain in receptor binding.
- To explore the potential for engineering adenovirus vectors with altered receptor specificities.
Main Methods:
- Competition binding experiments using recombinant native and chimeric fiber proteins from Ad5 and Ad3.
- Radiolabeling and in vitro synthesis of fiber proteins.
- Assessment of specific binding using radiolabeled proteins and competition analysis with baculovirus-expressed proteins.
- Flow cytometry assay to determine Ad5 receptor distribution on various cell lines.
Main Results:
- Only assembled fiber protein trimers, not monomers, exhibited receptor binding activity.
- Competition data indicated that Ad5 and Ad3 interact with different cellular receptors.
- Chimeric fiber proteins demonstrated that the head domain dictates receptor specificity.
- The isolated Ad3 fiber head domain could trimerize and bind to cellular receptors.
Conclusions:
- The head domain of the adenovirus fiber protein contains the determinants for cellular receptor binding.
- The isolated fiber head domain is sufficient for receptor binding and can mediate altered receptor specificity.
- Engineering the fiber head domain offers a strategy for developing adenovirus vectors with targeted gene delivery capabilities.