Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, Massachusetts, USA.
Ocular Genomics Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2019 Mar 21;93(7). doi: 10.1128/JVI.02013-18. Print 2019 Apr 1.
The adeno-associated virus (AAV) serves as a broadly used vector system for gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly. Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.
腺相关病毒 (AAV) 作为基因传递的广泛使用的载体系统。AAV 衣壳组装的过程仍然知之甚少。病毒辅助因子装配激活蛋白 (AAP) 是最大 AAV 产量所必需的，并且在衣壳组装中具有多种作用，即，将结构蛋白 (VP) 运送到组装的核部位，促进 VP 抵抗多种降解途径的稳定性，并促进 VP 单体之间的稳定相互作用。AAP 的 N 端 60 个氨基酸 (AAPN) 对于这些功能是必不可少的。推测 AAP 必须与 VP 物理相互作用才能执行其多种功能，但是 AAP-VP 相互作用的分子性质尚不清楚。在这里，我们查询了结构相关的 AAV 如何从 AAV 血清型 2 (AAP2) 发挥功能作用，以进行衣壳装配。这些研究导致鉴定了在不兼容 AAP2 的变体的腔内腔表面上对 AAP-VP 和 VP-VP 相互作用很重要的关键残基。用富含谷氨酰胺的模体替换变体的保守 VP β-桶结构上的一簇谷氨酸残基会产生与 AAP2 兼容的功能获得突变体。相反，突变 AAP2 中的带正电荷的残基和 AAPN 中的保守核心结构域会产生功能获得的 AAP2 突变体，可挽救不兼容变体的组装。我们的结果表明了一种衣壳组装模型，其中表面电荷/中性决定了 AAPN 与腔内腔 VP 表面之间的相互作用，从而引发衣壳组装。为了获得基因治疗的理想特性而对 AAV 衣壳进行工程改造（例如，趋向性，降低免疫原性和提高效力），需要衣壳修饰不影响颗粒组装。促进其组装的辅助因子 VP 和辅助因子之间的关系是组装保存和载体生产的核心。了解这种兼容性的要求可以为生产策略提供信息，以最大程度地提高产量并降低成本。此外，同时检查大量衣壳变体的基于文库的方法将受益于普遍功能的 AAP，这可以防止由于与同源 AAP 不兼容而在载体文库生产过程中丢失具有潜在有价值表型的变体。研究 AAV 结构和非结构成分之间的相互作用可以增强我们对衣壳组装机制和形成二十面体衣壳所需的蛋白质-蛋白质相互作用的基本认识。
