This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed research will characterize adeno-associated virus (AAV) in complex with analogs of its cellular attachment receptor, heparan sulfate proteoglycan (HSPG). AAV is of interest as one of the leading vectors being developed for human gene therapy. Our group previously predicted the receptor binding site accurately for the type-species, AAV-2, from electrostatics calculated from the native crystal structure. This was recently confirmed in an low resolution cryo-EM structure of the complex. Native crystal structures of AAV-3B and AAV-6, related serotypes that also bind heparan, were recently determined in our lab. From these structures, we hypothesize that the determinants of receptor binding in these serotypes are distinct from those in AAV-2, presumably a result of selective pressure from the host immune response. This has practical implications for the development of vectors targeted to different cell types, because AAV-2 would not alone serve as a good model for virus-receptor interactions. Crystallographic structures of virus/receptor complexes will be used to visualize the receptor binding sites at higher resolution than can be attained through other methods. These structures will also reveal differences in receptor binding among serotypes. We currently have native crystals of both AAV-3B and AAV-6, which will be soaked with short oligosaccharide analogs of the heparan receptor. In addition, AAV-3B has been co-crystallized with a sulfated small molecule heparan analog. These crystals have a distinct morphology from the native crystals. Biochemical experiments are underway to confirm the presence of sulfated oligosaccharides in the soaked crystals.With the large unit cell of virus crystals (typically ~250 x ~250 x ~600), the diffraction intensities are much weaker than most protein crystals, and synchrotron x-ray sources are required for diffraction to modest resolution. In addition, the large unit cells result in finely separated diffraction spots. This problem is tractable with the larger detectors now available at beamlines, such as BioCARS. Finally, our crystals are of infectious forms of AAV, which are handled in BSL-2 facilities, for safety. The BioCARS beamlines would be ideal for handling such agents, and have facilities to collect data from crystals with large unit cells. The facilities at APS are ideal to test our hypotheses and reveal specific details of receptor attachment by natural variants of AAV.
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