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Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob
Hongjie Wang1, Yen-Chywan Liaw, Daniel Stone
1Division of Medical Genetics, University of Washington, Box 357720, Seattle, WA 98195, USA.
Abstract:
Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a KD (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-A resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.
Insights
Researchers identified key residues in species B adenovirus (Ad35) that are critical for binding to the CD46 receptor. Understanding this interaction is vital for developing Ad35 as a gene therapy vector and for designing antiviral strategies.
Area of Science:
- Virology
- Structural Biology
- Gene Therapy
Background:
- Species B adenoviruses (Ads) are linked to severe illnesses in immunocompromised patients.
- Species B Ads, utilizing CD46 as a receptor, are being explored for gene therapy applications.
- Adenovirus serotype 35 (Ad35) is a species B Ad of particular interest.
Purpose of the Study:
- To investigate the molecular interactions between the Ad35 fiber knob domain and its receptor, CD46.
- To identify critical residues within the Ad35 knob essential for CD46 binding.
- To provide structural insights into the Ad35 knob-CD46 complex for vector design and therapeutic development.
Main Methods:
- Generated a mutant library of Ad35 knob domains and screened for CD46 binding.
- Validated critical residues using surface plasmon resonance and competition binding assays.
- Determined the Ad35 knob structure via X-ray crystallography and modeled its interaction with CD46.
Main Results:
- Identified four key residues (Phe242, Arg279, Ser282, Glu302) in the Ad35 knob that are essential for CD46 binding.
- Mutations in these residues abolished CD46 binding without affecting knob trimerization.
- Structural modeling revealed that Ad35 binds CD46 in a manner where one CD46 molecule interacts with two knob monomers.
Conclusions:
- The identified residues are crucial for Ad35-CD46 interaction, impacting viral entry and potential signaling.
- Findings are fundamental for characterizing species B Ads and designing targeted antiviral therapies.
- This research supports the development of Ad35-based vectors for gene transfer applications.
