Department of Biochemistry & Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, College of Medicine, University of Floridagrid.15276.37, Gainesville, Florida, USA.
Institute of Molecular Biophysics, Florida State Universitygrid.255986.5, Tallahassee, Florida, USA.
J Virol. 2021 Sep 9;95(19):e0084321. doi: 10.1128/JVI.00843-21. Epub 2021 Jul 14.
Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.
腺相关病毒（AAV）是一种小型无包膜单链 DNA（ssDNA）病毒，目前正在被开发为基因治疗生物制剂。进入细胞后，AAV 通过内体溶酶体途径运输到细胞核。随后 pH 值的下降引发衣壳的构象变化，使衣壳蛋白（VP）N 端外露，包括小衣壳蛋白 VP1 的独特结构域（VP1u），这允许衣壳溶酶体出芽所需的磷脂酶活性。在这里，我们报告了在 pH 值（7.4、6.0、5.5 和 4.0）为内体 pH 值时确定的 AAV9 衣壳结构，以及在 pH 值为 7.4 和 5.5 时用末端半乳糖结合的 AAV9 衣壳的冷冻电镜和三维图像重建。总之，这些研究提供了在 5 倍孔内体运输过程中 AAV9 衣壳构象变化的见解，无论是在存在还是不存在其细胞聚糖受体的情况下。我们首次观察到，酸化诱导 VP3 和可能的 VP2 N 端的外露，推测是在 pH 值为 4.0 时 VP1u 外露之前，这对于溶酶体膜的破坏是必不可少的。此外，AAV9-半乳糖相互作用的结构研究表明，AAV9 在晚期内体 pH 值 5.5 时仍与聚糖受体结合。这种相互作用显著改变了 VP 可变区 I 的构象稳定性，以及与 VP N 端外露相关的动力学。有 13 种不同的腺相关病毒（AAV）血清型在结构上同源，其衣壳蛋白（VP1 至 VP3）在氨基酸序列上相似。然而，AAV9 是研究最多的病毒之一，被用作基因治疗载体。这在一定程度上是因为 AAV9 能够穿过血脑屏障，很容易转导广泛的组织，包括中枢神经系统。在这项研究中，我们提供了 AAV9 衣壳在细胞内运输过程中的结构见解。虽然已经表明 AAV 衣壳会使 VP 的 N 端外露，从而在低 pH 值下酶促破坏溶酶体膜，但没有结构证据证实这一点。通过利用 AAV9 作为我们的模型，我们提供了第一个结构证据，证明外排过程发生在二十面体 5 倍对称轴的蛋白界面上，并可以通过降低 pH 值触发。
