National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM108753
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM122564
United States
Citation
Journal: J Virol / Year: 2022 Title: Adeno-Associated Virus Receptor-Binding: Flexible Domains and Alternative Conformations through Cryo-Electron Tomography of Adeno-Associated Virus 2 (AAV2) and AAV5 Complexes. Authors: Guiqing Hu / Mark A Silveria / Grant M Zane / Michael S Chapman / Scott M Stagg / Abstract: Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is ...Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.
Model: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 15.0 nm
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: 4ul of AAV-2 at concentration 0.2 mg/ml was was applied on grid and incubated for 1.5 minutes. The grid was gently blotted on the side with filter paper, and another 4ul of PKD1-2 at ...Details: 4ul of AAV-2 at concentration 0.2 mg/ml was was applied on grid and incubated for 1.5 minutes. The grid was gently blotted on the side with filter paper, and another 4ul of PKD1-2 at concentration 1.5 mg/ml was applied and allowed to incubate for another 1.5 minutes followed by plunge-freeze using a Vitrobot Mark IV..
Details
4ul of AAV-2 at concentration 0.2 mg/ml was was applied on grid and incubated for 1.5 minutes. The grid was gently blotted on the side with filter paper, and another 4ul of PKD1-2 at concentration 1.5 mg/ml was applied and allowed to incubate for another 1.5 minutes followed by plunge-freeze using a Vitrobot Mark IV.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average electron dose: 1.6 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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