Icosahedral Virion Scaffolding Proteins

本研究的主要目的是阐明病毒组装过程中的关键相互作用，并确定微病毒科病毒中负责的大分子（支架蛋白）的结构域。将采用遗传、生物化学和结构方法（包括X射线晶体学）相结合的方法。这些分析的结果可能会提供进一步的见解组装的病毒和重组蛋白的设计。微病毒科的内部支架蛋白gpB与分子伴侣具有许多共同的性质。先前的结果表明，内部支架蛋白要么具有固有的灵活性或与其基板以非特异性的方式，也许通过接口相互作用。确定含有外来支架蛋白的杂交原衣壳的原子结构将直接解决这个问题。对于其中外来支架蛋白不交叉功能的那些情况，将采用遗传分析来定义可赋予底物特异性的结构域。获得病毒外壳蛋白的功能突变，允许利用外来支架蛋白，已被分离，并将进行检查。 微病毒科的外部支架蛋白gpD的原子结构表明它们也与分子伴侣共享许多特征。它们使用各种表面以许多不同的方式结合其他蛋白质，因此对特定病毒外壳蛋白的特异性可能存在于特定结构域中。与在一级结构中表现出大量差异的内部支架蛋白不同，外部支架蛋白共享75%的序列同一性。Phi X174蛋白不能有效地指导其他微病毒科病毒粒子的组装因此可归因于位于其NH 2-末端的发散残基，其形成大的α-螺旋结构。将用嵌合多肽测试该假设，并将使用基于质粒的交叉互补系统。 *** 所有病毒都必须通过多种大分子相互作用来组装自己，这种组装通常依赖于称为支架蛋白的蛋白质。 类似于建筑物中使用的支架，支架蛋白存在于病毒组装中间体中，但不在成熟病毒中。两种不同的支架蛋白，外部和内部，需要为 微病毒科病毒的组装。用这些病毒可以纯化仍然包含外部支架蛋白的病毒中间体。通过检查外部支架蛋白的原子结构并对内部和外部蛋白进行遗传分析，已经确定支架蛋白的不同区域可能具有特异性和特异性。 可识别的功能。本研究的主要目的是完善这些蛋白质的原子结构，并通过构建杂交微病毒科支架蛋白来测试关于其各种功能的假设。这些分析的结果将为病毒组装提供进一步的见解。

9604680 Fane The main objectives of this study are to elucidate the critical interactions during viral assembly, and to define the structural domains of the responsible macromolecules, the scaffolding proteins, in Microviridae viruses. A combination of genetic, biochemical and structural approaches including X-ray crystallography will be employed. The results of these analyses may provide further insights into the assembly of viruses and the design of recombinant proteins. The Microviridae internal scaffolding proteins, gpB, share many properties with molecular chaperones. Prior results indicate that the internal scaffolding proteins either possess inherent flexibility or interact with their substrates in nonspecific manners, perhaps via interfaces. Determining the atomic structures of hybrid procapsids, containing foreign scaffolding proteins, will directly address this question. For those instances in which foreign scaffolding proteins do not cross function, genetic analyses will be employed to define domains which may confer substrate specificity. Gain of function mutations in viral coat proteins, allowing for the utilization of foreign scaffolding proteins, have been isolated and will be examined. The atomic structure of the external scaffolding proteins, gpD, of Microviridae suggests that they also share many features with molecular chaperones. They bind other proteins in many different ways using various surfaces, the specificity for a particular viral coat protein may thus reside in specific domains. Unlike the internal scaffolding proteins which exhibit a great deal of divergence in primary structure, the external scaffolding proteins share 75% sequence identity. The inability for Phi X174 protein to productively direct the assembly of other Microviridae virions is therefore attributable to the divergent residues localized its NH2-termini, which forms a large alpha-helical structure. This hypothesis will be tested with chimeric polypeptides, and the plasmid-based cross complementation system will be used. *** All viruses must assemble themselves by means of multiple macromolecular interactions, the assembly is often dependent on proteins known as scaffolding proteins. Analogous to scaffoldings used in the construction of buildings, scaffolding proteins are found in virus assembly intermediates but not in the mature viruses. Two different scaffolding proteins, external and internal, are required for the assembly of the Microviridae family of viruses. With these viruses it is possible to purify viral intermediates which still include the external scaffolding protein. By examining the atomic structure of the external scaffolding protein and performing genetic analyses with both the internal and external proteins, it has been determined that different regions of the scaffolding proteins may have specific and identifiable functions. The main objectives of this research are to refine the atomic structures of these proteins and to test hypotheses regarding their various functions by constructing hybrid Microviridae scaffolding proteins. The results of these analyses will provide further insights into viral assembly.