STRUCTURAL STUDIES OF ADENO-ASSOCIATED VIRUS IN COMPLEX WITH ITS CELLULAR REC

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 拟议的研究将表征腺相关病毒（AAV）与其细胞附着受体硫酸乙酰肝素蛋白聚糖（HSPG）类似物的复合物。 AAV作为正在开发用于人类基因治疗的主要载体之一而受到关注。 我们的小组以前预测的受体结合位点准确的类型的物种，AAV-2，从静电计算的天然晶体结构。 这一点最近在复合物的低分辨率cryo-EM结构中得到证实。 我们的实验室最近测定了AAV-3B和AAV-6的天然晶体结构，这些血清型也结合乙酰肝素。 从这些结构中，我们假设这些血清型中受体结合的决定因素与AAV-2中的决定因素不同，推测是来自宿主免疫应答的选择性压力的结果。 这对靶向不同细胞类型的载体的开发具有实际意义，因为AAV-2单独不能作为病毒-受体相互作用的良好模型。 病毒/受体复合物的晶体结构将用于以比通过其他方法获得的分辨率更高的分辨率可视化受体结合位点。 这些结构还将揭示血清型之间受体结合的差异。 我们目前有AAV-3B和AAV-6的天然晶体，它们将用乙酰肝素受体的短寡糖类似物浸泡。 此外，AAV-3B已与硫酸化小分子乙酰肝素类似物共结晶。 这些晶体具有与天然晶体不同的形态。 生化实验正在进行中，以确认浸泡晶体中硫酸化寡糖的存在。由于病毒晶体的单位细胞很大（通常为~250 x ~250 x ~600），衍射强度比大多数蛋白质晶体弱得多，需要同步加速器X射线源才能衍射到中等分辨率。 此外，大的晶胞导致精细分离的衍射斑点。 这个问题是易于处理的较大的探测器，现在可在光束线，如BioCARS。 最后，我们的晶体是感染性形式的AAV，为了安全起见，在BSL-2设施中处理。 BioCARS光束线将是处理此类试剂的理想选择，并且具有从具有大单位细胞的晶体中收集数据的设施。 APS的设施非常适合测试我们的假设，并揭示AAV天然变体受体附着的具体细节。

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.