Van Vliet Kim, Blouin Veronique, Agbandje-McKenna Mavis, Snyder Richard O
Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA.
Mol Ther. 2006 Dec;14(6):809-21. doi: 10.1016/j.ymthe.2006.08.1222. Epub 2006 Sep 27.
The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.
此前，已通过X射线晶体学或冷冻电子显微镜确定了三种腺相关病毒（AAV）血清型病毒衣壳的三维结构。这些对AAV的研究以及对自主细小病毒的类似研究，已得出了关于处于低能量构象的病毒粒子的重要结构信息。然而，关于生理条件下溶液中AAV病毒粒子的结构特性的信息却很少。我们证明，用胰蛋白酶对AAV2病毒粒子进行蛋白水解消化会导致在衣壳表面的一个特定位点发生切割，而衣壳仍保持完整。利用独特的抗体、蛋白质测序、质谱和三维结构建模，将消化产物定位到所有三种AAV2结构蛋白共有的一个表面环上的一个区域。空的AAV2衣壳可与完整（含DNA）衣壳区分开来，其VP2对胰蛋白酶的敏感性增加，且被胰凝乳蛋白酶消化得更快。如通过不同的敏感性和独特的片段模式所见，利用胰蛋白酶或胰凝乳蛋白酶进行的蛋白水解分析也能够区分AAV2与AAV1和AAV5。这些数据证明了一种研究溶液中AAV衣壳结构的新方法，在用于基因转移的AAV载体的测试和工程中应具有重要价值。
