Chen Min, Uchiyama Asako, Fane Bentley A
Department of Veterinary Science and Microbiology, University of Arizona, Tucson AZ 85721, USA.
J Mol Biol. 2007 Oct 19;373(2):308-14. doi: 10.1016/j.jmb.2007.07.064. Epub 2007 Aug 10.
Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 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.
与大多数病毒组装系统不同，噬菌体øX174的形态发生由两种支架蛋白B和D介导。外部支架蛋白D在原子结构中高度有序，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，广泛的改变也不会消除其功能。尽管在遗传上较为宽松，但病毒组装绝对需要蛋白B。因此，这个具有重叠阅读框复杂排列的系统可被视为“不可简化的复杂性”的一个例子。为了研究双支架蛋白系统的生化功能以及复杂性的进化，采用了渐进式和靶向基因筛选来减少并最终消除对B蛋白的依赖。在整个分析过程中，对适应的生化和遗传基础进行了表征，最终得到了具有不依赖B表型的六重突变体，通过在野生型细胞中形成噬菌斑进行评估。主要的适应似乎是突变的外部支架蛋白的过度表达。在抗裂解细胞中追踪子代产生情况。产生感染性病毒粒子的能力并不需要所有六个突变。然而，随着突变的积累，子代产生前的延迟期会缩短。结果表明，内部支架蛋白的主要功能可能是降低成核前衣壳形成所需的外部支架蛋白的临界浓度。此外，它们还展示了一种新机制，即使在一个仅编码八种严格必需蛋白的系统中，一个严格必需的基因产物也可以被绕过。
