PDF(Pubmed)
Abstract：
The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.
摘要：
高级结构（HOS）是重组腺相关病毒（rAAV）的关键质量属性。由于VP1独特（VP1u）和VP1/VP2共同区域的灵活性和/或较少折叠性质，因此评估整个rAAV衣壳的HOS具有挑战性。这是这些区域在病毒感染后发挥其功能所必需的结构特征。在这项研究中,氢/氘交换质谱(HDX-MS)用于全和空rAAV8衣壳的结构分析。我们获得了代表85%序列覆盖率的486个肽。令人惊讶的是,VP1u区域显示出快速的氘吸收，即使该区域包含主要由α-螺旋组成的磷脂酶A2结构域。满和空衣壳之间氘吸收的比较显示，在5倍对称轴的通道结构处，全衣壳中的氢/氘交换具有显着的保护作用。这对应于低温电子显微镜研究，其中仅在完整的衣壳中观察到扩展的密度。此外,氘的摄取在全衣壳的VP1u区域减少，提示该区域与衣壳化基因组的折叠和/或相互作用。这项研究证明HDX-MS是探测整个rAAV衣壳结构的强大方法。
