Structural and Biophysical Analysis of Adeno-Associated Virus Serotype 2 Capsid Assembly Variants
Antonette Bennett1, Joseph Gargas1, Austin Kansol1
1Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida, USA.
Journal of Virology
|June 13, 2023
Summary
Viral protein symmetry interactions are crucial for adeno-associated virus (AAV) capsid assembly, genome packaging, and infectivity. Understanding these interactions aids in designing better AAV gene therapy vectors.
Area of Science:
- Virology
- Structural Biology
- Gene Therapy
Background:
- Adeno-associated viruses (AAVs) are promising vectors for gene therapy, with numerous clinical trials underway.
- AAV2 is the most studied serotype, but the role of viral protein (VP) symmetry interactions in capsid function remains unclear.
Purpose of the Study:
- To investigate how viral protein (VP) symmetry interactions influence adeno-associated virus type 2 (AAV2) capsid assembly, genome packaging, stability, and infectivity.
- To characterize structural and functional implications of rationally designed AAV2 VP variants.
Main Methods:
- Generated 25 AAV2 VP variants targeting 2-fold, 3-fold, and 5-fold symmetry interfaces.
- Assessed capsid assembly using native immunoblots and ELISAs.
- Evaluated capsid stability through thermal stability measurements.
- Quantified genome packaging efficiency.
- Determined viral infectivity.
- Resolved structures of wild-type and variant AAV2 capsids using cryo-electron microscopy and 3D image reconstruction.
Main Results:
- Several VP variants failed to assemble capsids, while others exhibited reduced stability.
- Specific 3-fold variants showed significant defects in genome packaging.
- A 5-fold variant demonstrated impaired viral infectivity, linked to capsid regions important for genome ejection.
- Cryo-EM structures revealed the impact of stabilizing interactions on capsid assembly, stability, packaging, and infectivity.
Conclusions:
- VP symmetry interactions are critical for AAV2 capsid integrity and function.
- Alterations at specific symmetry interfaces can disrupt assembly, stability, packaging, and infectivity.
- Structural insights from this study provide a foundation for rational design of improved AAV vectors for gene therapy.
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