Institute of Molecular Biophysics, Florida State Universitygrid.255986.5, Tallahassee, Florida, USA.
Department of Biochemistry, University of Missourigrid.134936.a, Columbia, Missouri, USA.
J Virol. 2022 Jul 13;96(13):e0010622. doi: 10.1128/jvi.00106-22. Epub 2022 Jun 8.
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.
腺相关病毒（rAAV）的重组形式是许多遗传疾病治疗开发的首选载体。为了提高效率和特异性，需要更深入地了解 AAV 的分子病毒学。最近的进展包括鉴定出一个近乎普遍的进入受体 AAVR，以及通过冷冻电子显微镜（cryo-EM）单颗粒分析（SPA）检测到的结构，这些结构以高分辨率揭示了与 AAV 紧密结合的 AAVR 的仅有结构域。在这里，应用低温电子断层扫描（cryo-ET）来揭示灵活受体的相邻结构域。对于 AAV5，其 PKD1 结构域强烈结合，在远离病毒的三个位置观察到 PKD2。AAV2 紧密结合在 PKD2 结构域上的独特位置，而 cryo-ET 现在揭示了 PKD1 的四个构象，都与 AAV5 不同。AAV2 受体复合物的内表面也显示出未建模的特征，似乎是一种平衡的替代构象。其他 AAV 结构从 5 倍轴附近开始，但现在β-链 A 是次要构象，对于主要构象，靠近 2 倍轴的部分有序 N 末端与经典衣壳果冻卷折叠在βA-βB 转折处连接。低温电子断层扫描的加入揭示了未被注意到的复杂性，这可能与病毒进入和改进基因治疗载体的发展有关。有 150 项针对 30 种疾病的临床试验正在进行中，AAV 是一种领先的基因治疗载体。高剂量使用以克服低效转导的免疫毒性偶尔会证明是致命的，并突出了基础病毒学的差距。AAV 通过不同的结构域与 AAVR 受体的不同结构域相互作用进入细胞，根据 AAV 族来决定。单结构域在低温电子显微镜结构中得到解决。在这里，通过 AAV2 和 AAV5 复合物的 cryo-ET 揭示了相邻结构域。它们处于灵活的构象中，与 AAV 的相互作用最小，尽管 AAV2 的转导明显依赖于两个结构域。
