Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics |
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Authors: | Vamseedhar Rayaprolu Shannon Kruse Ravi Kant Balasubramanian Venkatakrishnan Navid Movahed Dewey Brooke Bridget Lins Antonette Bennett Timothy Potter Robert McKenna Mavis Agbandje-McKenna Brian Bothner |
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Institution: | Department of Chemistry and Biochemistry, Montana State University, Bozeman Montana, USAa;Department of Biochemistry and Molecular Biology and Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, Florida, USAb |
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Abstract: | Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production. |
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