Powers John M, Chang Xiao Lan, Song Zhen, Nakai Hiroyuki
Department of Molecular and Medical Genetics, Oregon Health & Science University.
Department of Molecular and Medical Genetics, Oregon Health & Science University; Department of Molecular Microbiology and Immunology, Oregon Health & Science University;
J Vis Exp. 2018 Jun 12(136):56766. doi: 10.3791/56766.
While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.
虽然腺相关病毒（AAV）作为一种有吸引力的基因治疗载体已被广泛接受，但它也是理解病毒生物学的一种模型病毒。在后一个方面，最近发现的一种非结构AAV蛋白，称为装配激活蛋白（AAP），为病毒衣壳VP蛋白装配成衣壳的过程提供了新的线索。尽管许多AAV血清型的装配需要AAP，但我们最近报道AAV4、5和11是该规则的例外。此外，我们证明了不同血清型的AAP和装配好的衣壳定位于不同的亚细胞区室。不同AAV血清型中AAP的生物学特性和功能作用存在这种意想不到的异质性，凸显了对源自不同血清型的AAP进行研究的重要性。本手稿详细介绍了一种用于AAV定量的简单斑点印迹分析方法及其在评估衣壳装配中AAP依赖性和血清型特异性方面的应用。为了证明这种斑点印迹分析方法的实用性，我们着手表征蛇AAV（一种先前未表征的爬行动物AAV）以及AAV5和AAV9的衣壳装配和AAP依赖性，此前已分别证明AAV5和AAV9是不依赖AAP和依赖AAP的血清型。该分析表明，蛇AAV衣壳装配需要蛇AAP，而不能由AAV5和AAV9的AAP促进。该分析还表明，与许多通过交叉互补促进异源衣壳装配的常见血清型AAP不同，蛇AAP不促进AAV9衣壳的装配。此外，我们表明核酸酶的选择会显著影响斑点印迹分析的读数，因此，选择最佳酶对于成功评估AAV滴度至关重要。
