Eliminating the requirement of an essential gene product in an already very small virus:: Scaffolding protein B-free ⊘X174, B-free
Eliminating the requirement of an essential gene product in an already very small virus:: Scaffolding protein B-free ⊘X174, B-free
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DOI：
10.1016/j.jmb.2007.07.064
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发表时间：
2007-10-19
影响因子：
5.6
通讯作者：
Fane, Bentley A.
中科院分区：
文献类型：
--
作者：
Chen, Min;Uchiyama, Asako;Fane, Bentley A.
与大多数病毒组装系统不同，两种支架蛋白B和D介导噬菌体oX174的形态发生。外部支架蛋白D的原子结构高度有序，其正常功能对突变非常敏感。相比之下，内部支架蛋白B相对无序，广泛的改变不会消除功能。尽管存在这种遗传松弛性，但病毒组装绝对需要蛋白质B。因此，这个系统，其重叠阅读框架的复杂安排，可以被视为一个“不可简化的复杂性”的例子。为了解决双支架蛋白系统的生化功能和复杂性的进化，采用渐进和靶向遗传选择来减少并最终消除B蛋白依赖性。在整个分析过程中，生物化学和遗传基础被表征，导致具有b独立表型的六重突变体，通过野生型细胞中的斑块形成进行评估。主要的适应似乎是外部支架蛋白突变体的过度表达。在抗裂解细胞中进行后代生产。产生感染性病毒粒子的能力并不需要所有六种突变。然而，随着突变的积累，后代产生前的滞后期缩短。结果表明，内部支架蛋白的主要功能可能是降低原衣壳形成所需的外部支架蛋白的临界浓度。此外，他们还展示了一种新的机制，通过这种机制，即使在一个只编码8种严格必需蛋白质的系统中，严格需要的基因产物也可以被绕过。(C) 2007 Elsevier Ltd.版权所有。
Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage oX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins. (C) 2007 Elsevier Ltd. All rights reserved.