Adeno-associated virus (AAV) is a leading gene therapy vector for delivery of DNA to correct genetic errors or predispositions to disease. Our objective is an understanding of the virus-host interactions that mediate cell entry. This foundation will support the widely sought goal of manipulating cell specificity, efficiently transducing desired cells and reducing off-target side effects. This goal has motivated extensive genetic and phenotypic characterization, which, with structure, provide an exceptional paradigm for general insights into viral entry. Our binding and siRNA studies cast doubt on published co-receptors, motivating a gene trap screen in a haploid cell line. The gene most frequently hit in AAV-resistant mutant cells, encoded a protein that we named AAVR which binds with nM affinity, inhibits transduction, and is essential for all AAV serotypes and cell types tested. We have expressed the AAV-binding domains, and will determine a structure of the complex by cryo- electron microscopy (EM) before further characterizing the interactions through mutagenesis. Other genes were implicated by the screen. We will determine which encoded proteins interact with AAV or AAVR through photo-induced cross-linking, pull-down, and mass spectrometric identification of interacting domains. These will be expressed for binding analysis, and hybrid x-ray/EM structure. The gene trap indicates potential partners, not just at the cell surface, but during AAV's trafficking to the peri-nuclear trans Golgi network, and which will illuminate how AAV's phospholipase A2 domain is released for endosomal escape. EM methods, applicable to AAV, will be developed for general use. For refinement of hybrid structures, our map-fitting optimization will incorporate parsimony restraints on model flexibility to avoid the common problem of over-fitting at intermediate resolution. Difference map analysis will be improved, through model-based calibration, to enhance the sensitivity with which small ligands and subtle conformational changes can be analyzed by high resolution EM. This will be applicable to studies of AAV's attachment to extracellular glycans, or to the binding of ligands to ion channels, ribosomes or enzyme complexes in other laboratories. Within the MIRA framework, contributions to collaborative studies of protein dynamics will continue. These are integrating crystal structure with NMR relaxation dispersion and residual dipolar couplings to characterize rate- limiting milli-/micro-second protein motions in arginine kinase as it turns over. Our model system is illuminating poorly understood general principles of intrinsic motions and conformational selection. These fundamental questions are the primary goal, but the research also feeds back into methods development. The NMR relaxation exchange data allows validation tests that our parsimonious model parameterization captures real conformational changes. In summary, we will continue to work on challenging structural biology with both fundamental and applied goals that drive the development of widely applicable methodology.
Structure-function analysis of the molecular interactions of Adeno-associated Virus (AAV) on cellular entry will lay a key foundation, enabling development of more efficient vectors for gene therapy treatments of genetic disorders, such as hemophilia, and other diseases, including cancer. Modulation of these interactions will be needed to improve the targeting of therapeutic delivery to specific cells. Basic studies of conformational change and dynamics are directed towards understanding the transitions that occur during molecular interactions, a foundation for understanding normal mechanism, molecular pathology and potential pharmaceutical interventions.
|Davulcu, Omar; Peng, Yu; Brüschweiler, Rafael et al. (2017) Elevated ?s-ms timescale backbone dynamics in the transition state analog form of arginine kinase. J Struct Biol 200:258-266|
|Pillay, Sirika; Zou, Wei; Cheng, Fang et al. (2017) AAV serotypes have distinctive interactions with domains of the cellular receptor AAVR. J Virol :|