478. An Essential and Ubiquitous Protein Receptor for AAV; Glycans as Attachment Receptors

Motivated by unsuccessful attempts to observe physical binding between AAV-2 and heterologously expressed domains of previously reported co-receptors, we set out to identify novel protein receptor(s) for AAV2 through an unbiased genome-wide knockout screen in human cells. Using an mCherry AAV vector, resistant cells were iteratively selected by FACS for gene trap screening in a library of mutagenized haploid cells. Upon deep sequencing, refractory cells had significantly high frequencies of mutation in genes encoding glycan synthesis and retrograde transport, but most significantly in a hitherto poorly characterized transmembrane protein, now termed AAVR. Genetic confirmation of AAVR's role in the entry of multiple AAV serotypes has come through CRISPR-Cas9 knockouts in multiple cell lines then restoration of susceptibility through complementation; infection of poorly permissive cells following AAVR transduction; and creation of a mouse knockout with greatly diminished susceptibility.Various AAVR ectodomain constructs have been heterologously expressed and purified as fusion proteins, and these have been shown to inhibit in vitro viral transduction at concentrations consistent with effective nM binding constants (between AAV & AAVR) measured by surface plasmon resonance (SPR). Pre-incubation with antibodies to AAVR also inhibits infection or transduction. AAVR is transiently expressed on the plasma membrane. Expression of chimeric constructs suggests that AAV takes advantage of its trafficking to the perinuclear trans Golgi network as the dominant, but non-exclusive, entry pathway. Identification of AAVR and its apparently ubiquitous use has interesting implications for AAV's cell specificity. Progress towards structure of complexes will be reported.AAVR exhibits the classic characteristics of a viral receptor, casting the roles ascribed to glycan “primary” receptors in new light. Electron microscopy has been used to visualize AAV-DJ in complex with various heparin analogs at increasingly high resolution. A structure at 2.8 A resolution, as a pentasaccharide complex, shows some disorder in the glycan, but the side chains of viral amino acids are clearly resolved and in different conformations from those seen in a sucrose octasulfate complex. With little change to the backbone, the binding site accommodates diverse glycan sequences through adjustments to side chains, consistent with SPR binding assays of AAV-2 to a library of heparanoids. This, together with comparisons of heparan and AAVR cell knock-outs, indicates a more accessory role for glycans than is implied by the term “primary”. As for several other viruses, in AAV-2 at least, the glycan is an attachment receptor that likely elevates the AAV concentration proximal to the membrane, improving the efficiency with which the virus then binds to AAVR.