Icosahedral Virion Scaffolding Proteins

9604680费恩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。将结合遗传、生化和结构方法，包括X射线结晶学。这些分析的结果可能会为病毒的组装和重组蛋白的设计提供进一步的见解。微病毒科内部支架蛋白GPB与分子伴侣蛋白有许多共同的性质。先前的结果表明，内部支架蛋白要么具有固有的灵活性，要么以非特异性的方式与底物相互作用，可能是通过界面。确定含有外源支架蛋白的杂交Proapsids的原子结构将直接解决这个问题。对于那些外来支架蛋白没有交叉功能的情况，将使用遗传分析来定义可能赋予底物专一性的结构域。病毒外壳蛋白的功能突变的获得，允许利用外源支架蛋白，已经被分离出来，并将进行检查。微小病毒科的外部支架蛋白GPD的原子结构表明，它们也与分子伴侣蛋白有许多相似之处。它们使用不同的表面以许多不同的方式与其他蛋白质结合，因此特定病毒外壳蛋白的特异性可能驻留在特定的区域。与内部支架蛋白在一级结构上表现出很大的差异不同，外部支架蛋白有75%的序列同源性。因此，Phi x174蛋白不能有效地指导其他微病毒科病毒粒子的组装，这是由于其NH2末端存在分歧残基，形成了一个大的α-螺旋结构。这一假设将用嵌合多肽进行验证，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个大分子相互作用来组装自己，这种组装通常依赖于被称为支架蛋白的蛋白质。与建筑中使用的支架类似，支架蛋白在病毒组装中间体中发现，但在成熟病毒中没有。组装小病毒科病毒家族需要两种不同的支架蛋白，外部的和内部的。有了这些病毒，就有可能提纯病毒中间体，其中仍然包括外部支架蛋白。通过检查外部支架蛋白的原子结构并对内部和外部蛋白质进行遗传分析，已经确定支架蛋白的不同区域可能具有特定和可识别的功能。这项研究的主要目的是通过构建杂交的微病毒科支架蛋白来完善这些蛋白质的原子结构，并检验关于它们各种功能的假说。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.