Cryo-EM reveals how adeno-associated virus engages its receptor AAVR and an investigation of the GPR108 ectodomain
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Adeno-associated virus is one of the leading delivery agents used in gene therapy today. While it is already used in the clinic, some aspects of its basic biology are still not yet fully understood. Only recently in 2017 was the primary receptor for most AAV serotypes discovered and there are still unknown factors in AAV cell entry, uncoating, and host protein interactions. Furthermore, its use in gene therapy can be optimized with more accurate cell targeting and immune evasion. Being a non-enveloped virus, the protein shell of AAV is responsible for all of the host interactions from cell entry to gene delivery. With further study of the AAV structure and how AAV engages with human cells and the immune system we hope to create a roadmap of the AAV capsid that can help direct AAV capsid mutations for a more efficient AAV capsid, which can more easily evade the immune system, while maintaining its essential cellular interactions. We have previously reported that AAV interacts with two domains of its primary cellular receptor AAVR, the domains named PKD1 and PKD2. Our previous studies of the AAV2 capsid with its cellular receptor AAVR allowed us to visualize the essential interactions of AAV with the PKD2 domain of AAVR. However, the PKD1 domain of AAVR was not revealed in our previous structure, although it had been shown to be involved in cellular entry for many AAV serotypes, including AAV2. Utilizing cryogenic electron microscopy (cryo-EM) we explore the AAV5-AAVR interactions, using an AAV5-AAVR complex, and visualize how the previously unseen PKD1 domain engages with some AAV serotypes. We then observe how a never-before visualized AAV serotype, AAVGo.1 engages with the PKD1 domain of AAVR using an AAVGo.1-AAVR complex. In both the AAV5-AAVR and AAVGo.1-AAVR complexes, the PKD1 domain is revealed but the PKD2 domain is not resolved. We then further expand on the unseen domain locations in our complexes, PKD1 in the AAV2-AAVR complex and PKD2 in the AAV5-AAVR complex, by using cryogenic electron tomography (cryo-ET). Our cryo-ET data shows that there are distinct locations for the unseen domains and that the PKD1 domain does not appear to engage AAV2 in a similar manner as seen in the AAVGo.1 and AAV5 complexes. Finally, we explore the possible interactions between AAV2 and an uncharacterized protein that has been shown to be important in AAV entry for some serotypes, GPR108. We show that AAV2 preferentially binds to our expressed GPR108 constructs, implying that there may be a direct interaction between AAV2 and GPR108.
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Ph. D.