Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India.
SASTRA University, Thanjavur, India.
FEBS J. 2019 Dec;286(24):4964-4981. doi: 10.1111/febs.15013. Epub 2019 Aug 1.
Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMOylation (13%) and acetylation (11%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had >7 post-translational modifications in their capsid protein. About 82% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~ 100-fold vs. AAV2 wild-type vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. ENZYMES: Trypsin, EC 3.4.21.4.
病毒衣壳的翻译后修饰被认为可以微调并调节宿主中几种病毒感染生命周期的几个方面。在特定的生产细胞系中产生的重组病毒在其衣壳蛋白的细胞内成熟过程中很可能继承独特的翻译后修饰。关于重组腺相关病毒血清型（AAV1-rh10）衣壳中的这种翻译后修饰的数据有限。我们采用液相色谱和质谱分析来表征 AAV1-rh10 衣壳蛋白的翻译后修饰。我们的分析共鉴定出 AAV2-AAVrh10 衣壳中的 52 种翻译后修饰，包括泛素化（17%）、糖基化（36%）、磷酸化（21%）、SUMO 化（13%）和乙酰化（11%）。虽然 AAV1 没有检测到翻译后修饰，但至少有四种 AAV 血清型的衣壳蛋白有超过 7 种翻译后修饰。这些翻译后修饰中有 82%是新的。用 MALDI-TOF 和 Western blot 分析对 AAV2 衣壳进行的有限验证表明 AAV2 衣壳的糖基化和泛素化程度很低。为了进一步验证这一点，我们破坏了 AAV2 衣壳中鉴定出的一个糖基化位点（AAV2-N253Q），这严重降低了其包装效率（与 AAV2 野生型载体相比约 100 倍）。为了确认其他检测到的翻译后修饰，如 SUMO 化，我们对 AAV2 中的一个 SUMO 化位点（K258Q）进行了突变。与眼部基因转移后 2 周，这种突变载体显示 SUMO-1/2/3 蛋白水平降低，转导能力显著降低。我们的研究强调了 AAV 载体中翻译后修饰的异质性。这里提供的数据应该有助于进一步研究，以了解 AAV 生命周期中翻译后修饰的生物学意义，以及为基因治疗应用开发新型生物工程 AAV 载体。酶：胰蛋白酶，EC 3.4.21.4。
