Icosahedral Virion Scaffolding Proteins

二十面体病毒颗粒支架蛋白

• 批准号: 9604680
• 负责人: Bentley Fane
• 金额: $ 11万
• 依托单位: University of Arkansas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604680&HistoricalAwards=false
• 关键词: Icosahedral; Virion; Scaffolding; Proteins

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.

9604680 Fane 这项研究的主要目的是阐明病毒组装过程中的关键相互作用，并定义微病毒科病毒中负责大分子（支架蛋白）的结构域。将采用遗传、生化和结构方法（包括 X 射线晶体学）的结合。这些分析的结果可以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 与分子伴侣具有许多相同的特性。先前的结果表明，内部支架蛋白要么具有固有的灵活性，要么以非特异性方式（可能通过界面）与其底物相互作用。确定含有外来支架蛋白的混合原衣壳的原子结构将直接解决这个问题。对于外源支架蛋白不交叉功能的情况，将采用遗传分析来定义可赋予底物特异性的结构域。病毒外壳蛋白的功能突变获得，允许利用外源支架蛋白，已被分离出来并将进行检查。 微病毒科外部支架蛋白 gpD 的原子结构表明它们也与分子伴侣共享许多特征。它们利用不同的表面以多种不同的方式结合其他蛋白质，因此特定病毒外壳蛋白的特异性可能存在于特定的结构域中。与内部支架蛋白在一级结构上表现出很大差异不同，外部支架蛋白具有 75% 的序列同一性。因此，Phi X174 蛋白无法有效指导其他微病毒科病毒颗粒的组装，这可归因于位于其 NH2 末端的不同残基，从而形成大的 α 螺旋结构。该假设将用嵌合多肽进行测试，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进行自我组装，组装通常依赖于称为支架蛋白的蛋白质。 与建筑物建造中使用的脚手架类似，脚手架蛋白存在于病毒组装中间体中，但不存在于成熟病毒中。微病毒科病毒的组装需要两种不同的支架蛋白（外部和内部）。利用这些病毒，可以纯化仍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定的和可识别的功能。这项研究的主要目的是通过构建混合微病毒支架蛋白来完善这些蛋白质的原子结构，并测试有关其各种功能的假设。这些分析的结果将为病毒组装提供进一步的见解。