ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro
ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro
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X174 衣壳组装：抑制性外部支架蛋白和抗性外壳蛋白的体外作用
DOI：
10.1128/jvi.01878-16
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发表时间：
2016
影响因子：
5.4
通讯作者：
Sandri-Goldin, Rozanne M.
中科院分区：
文献类型：
--
作者：
Cherwa, James E.;Tyson, Joshua;Bedwell, Gregory J.;Brooke, Dewey;Edwards, Ashton G.;Dokland, Terje;Prevelige, Peter E.;Fane, Bentley A.;Sandri-Goldin, Rozanne M.
在BX174的形态发生过程中，240个外部支架蛋白D的拷贝将12个五聚体组装中间体组织成原衣壳，这是一种体外重建的反应。在以前的研究中，实验进化了对外源表达的显性致死D基因具有抗性的X174菌株。抗性是通过逐步获得外壳蛋白突变来实现的。一旦抗性建立，刺激性D蛋白突变，大大增加了菌株适应性。在这项研究中，在体外生物物理和生物化学方法被用来阐明的机制的细节和进化的权衡所产生的耐药突变。原衣壳形成的动力学进行了分析，在vitrousing野生型，抑制，实验进化的外壳和支架蛋白。我们的数据表明，病毒的健身是相关的vitroassembly动力学，并证明thatin vivoexperimental进化可以在anin vitrobiophysical context.IMPORTANCEExperimental进化分析是一个非常有价值的工具。祖先和进化的基因型之间的比较提出了关于适应机制的假设。然而，并不总是能够在体内严格检验这些假设。我们应用了体外生物物理和生物化学方法来阐明机制细节，使实验进化的病毒对抗病毒蛋白产生抗性，然后进化出该蛋白的生产性用途。此外，我们的研究结果表明，支架和外壳蛋白的各自的作用可能已经重新分配的两个支架蛋白系统的进化过程中。在单支架蛋白病毒组装系统中，外壳蛋白混杂地相互作用以在不存在支架蛋白的情况下形成异质异常结构。因此，支架蛋白控制保真度。在BX174组装过程中，外部支架蛋白的作用类似于外壳蛋白，在没有外壳蛋白的情况下自缔合成大的异常球形结构，而外壳蛋白似乎控制保真度。
During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstitutedin vitro. In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study,in vitrobiophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzedin vitrousing wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated within vitroassembly kinetics and demonstrate thatin vivoexperimental evolution can be analyzed within anin vitrobiophysical context.IMPORTANCEExperimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypothesesin vivo. We appliedin vitrobiophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.
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