A Genome-Wide Knock-Out Screen Identifies Novel Host Cell Entry Factor Requirements for Divergent Adeno-Associated Virus Serotypes

Adeno-Associated Virus (AAV) is a non-pathogenic virus that has been harnessed as a vector system for therapeutic gene transfer. Despite decades of research on AAV as a vector, little is known about the molecular determinants required for cellular entry of different AAV capsid serotypes and how this may lead to unique cell and tissue tropism for these viral vectors. This work presents and characterizes a novel AAV entry pathway used by an evolutionarily related subset of divergent AAVs that is independent of the canonical AAV receptor, AAVR, both in vitro and in vivo. This subset of AAV capsids, comprised of rh32.33 and AAV4, are unable to use AAVR for entry or to bind AAVR in vitro. We further characterize the role of AAVR for AAVR dependent serotypes, suggesting a predominantly post-attachment function for this receptor. Mutagenesis of both capsid and AAVR investigate the role human and animal variation may have in the success and translation of AAV-based gene therapies to the clinic. Additionally, a genome-wide CRISPR/Cas9 based entry screen identifies multiple cellular factors required for entry of AAVR dependent and independent serotypes. First, we describe a minimal alternate receptor complex comprised of NEU1 and CTSA that is uniquely required for entry of AAVR independent serotypes and acts at a post-attachment step in the entry pathway. We also identify and describe a previously uncharacterized protein, required by all AAV serotypes except AAV5. Chimeric capsids suggest both a capsid binding-site as well as an endosomal escape function located within the VP1 unique region of capsid. We therefore present a model in which most AAVs use a canonical entry pathway requiring two conserved entry factors, yet the highly divergent AAV serotypes AAV5, AAV4, and rh32.33 have unique alternate entry factor requirements. Our basic virology studies of AAV aim to both inform currently unanswered questions on the implementation of AAV-based gene therapies in the clinic, as well as allow for better design of AAV-based gene therapies in the future.