Receptor Switching in Newly Evolved Adeno-associated Viruses

Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduc-tion are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake, and transduction, but through dis-tinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycos-aminoglycans, while another displays receptor switching from AAVR to integrin beta 1 (ITG beta 1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITG beta 1 re-ceptor over AAVR. Visualization of the AAVhum.8 capsid by cryoelectron microscopy at 2.49-angstrom resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. IMPORTANCE Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines, and gene transfer vectors. Here, we demonstrate that distinct epitopes on the sur -face of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the three-dimensional structure of one such newly evolved AAV capsid, which provides a road -map for designing viruses with improved attributes for gene therapy applications.