Abstract
The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based method to determine the amount of vector bound to the cell surface and the amount of subsequent virus internalization based on viral genome quantification. This protocol is optimized for studying AAV. Nevertheless, it can serve as a backbone for studying other viruses with careful modification.
Keywords: Adeno-associated virus, Binding, Internalization, Virus entry, Virus, Cell surface
Background
Studies that assess AAV biology generally use transgene expression as the experimental endpoint. However, there are a number of critical steps AAV must successfully navigate before it reaches the nucleus and transduces the cell. Therefore, there are multiple distinct steps in the AAV infectious pathway that could be disrupted individually or collectively, leading to altered transduction. Assessment of AAV binding and internalization are important first steps in determining the cause of transduction differences observed upon cellular modification by small molecules, CRISPR-based gene knockout, siRNA-based gene knockdown, or other experimental procedures.
Materials and Reagents
Equipment
Procedure
Data analysis
To calculate the number of vector genomes per cell (Table 3), use the following calculations:
Recipes
Acknowledgments
This study was supported by National Institutes of Health Grants R01HL089221 and P01HL112761 through the NHLBI. This work was also supported by National Institutes of Health Training Grant 5T32 GM007092 through the NIGMS. The protocol described herein was based on the following paper: Berry et al. (2016).
References
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