Phenotype of mutants in pH-sensitive regions of the AAV capsid and evidence for a pH-induced capsid protease

Adeno-Associated Virus (AAV) is a small, non-pathogenic parvovirus that has gained popularity as a gene therapy vector in clinical trials and a gene transfer tool in research. A greater understanding of the basic biology of this virus could allow for the optimization of this vector system, and thus may have far-reaching implications in both research and clinical settings. The virus is known to enter the cell by receptor-mediated endocytosis, and traffics to the nucleus where it uncoats and transcribes its genes. The requirement for endosomal acidification to facilitate AAV infection has been known for some time; however, the exact role of pH in AAV biology has not been clearly determined. Using crystallographic data that demonstrated transitions in two pHsensitive regions of the capsid, we made individual alanine mutants and where able to identify Y704, E562 and E564 as critical for AAV infection. We characterized the intracellular trafficking of these mutants and were able to show evidence of a novel role for the AAV capsid in the nucleus. We were also able to identify a previously undescribed pH-sensitive protease within the AAV capsid. Our work has shown that the pH-sensitive elements of the AAV capsid are critical for AAV infection and govern previously unknown aspects of AAV biology.