Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.
Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.
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腺相关病毒 Rep68 的寡聚特性反映了其多功能性。
DOI：
10.1128/jvi.02441-12
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发表时间：
2013
影响因子：
5.4
通讯作者：
Escalante,CarlosR
中科院分区：
文献类型：
--
作者：
Zarate-Perez,Francisco;Mansilla-Soto,Jorge;Bardelli,Martino;Burgner2nd,JohnW;Villamil-Jarauta,Maria;Kekilli,Demet;Samso,Monserrat;Linden,RMichael;Escalante,CarlosR
腺相关病毒（AAV）编码四种称为Rep的调节蛋白。大型AAV Rep蛋白Rep68和Rep78是病毒生命周期的几乎每一步所需的基本因子。在结构上，它们共有两个结构域：AAA+结构域的修饰版本（表征SF3解旋酶家族）和N末端结构域（特异性结合DNA）。这两个结构域的组合赋予了非凡的多功能性，除了它们在位点特异性整合过程中的重要作用之外，还可以作为DNA复制的启动子和转录的调节子。尽管SF3家族的大多数成员在体外形成六聚环，但由于Rep68在溶液中聚集的倾向，其寡聚性质尚不清楚。我们在这里报告了一个全面的研究，以确定Rep68的低聚物的特点，使用的方法，包括沉降速度ultracentragation，电子显微镜，和流体动力学建模的组合。我们已经确定残基Cys151在体外诱导Rep68聚集。我们发现，Rep68显示浓度依赖性的动态寡聚行为，其特征在于存在两个群体：一个与单体和二聚体在缓慢的平衡和第二个组成的混合物的多环结构的七个和八个成员。ATP或ADP的存在诱导通过堆叠多个环形成的较大复合物的形成。总之，我们的结果支持Rep68分子的想法，该分子表现出执行AAV生命周期期间发生的广泛功能所需的灵活的寡聚体行为。
The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric ringsin vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregatein vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.