Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway
Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway
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外壳蛋白突变通过 ÏX174 早期组装途径改变形态发生中间体的通量
DOI：
10.1128/jvi.01384-17
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发表时间：
2017
影响因子：
5.4
通讯作者：
Fane, Bentley A.
中科院分区：
文献类型：
--
作者：
Blackburn, Brody J.;Li, Shuaizhi;Roznowski, Aaron P.;Perez, Alexis R.;Villarreal, Rodrigo H.;Johnson, Curtis J.;Hardy, Margaret;Tuckerman, Edward C.;Burch, April D.;Fane, Bentley A.
两个支架蛋白协调ϕx174的形态发生。内部支架蛋白B介导五聚体组装中间体的形成，而外部支架蛋白D将这些中间体中的12个组织成原衣壳。芳香氨基酸侧链介导了大多数外壳-内部支架蛋白的相互作用。一个残基在内部支架蛋白中，三个残基在外壳蛋白中，构成了B蛋白结合裂缝的核心。三个外壳基因密码子分别随机排列，以确定编码氨基酸的化学需求和突变的形态发生后果。由此产生的突变体表现出广泛的隐性表型，这通常可以在结构背景下解释。带有苯丙氨酸、酪氨酸和蛋氨酸替换的突变体在表型上与野生型没有区别。然而，色氨酸的替代在两个位置是有害的。带电残基的耐受性很差，具有极端温度敏感和致命的表型。18个致命性和条件性致命性突变体进行了遗传和生化特征分析。与错义替换相关的主要缺陷包括：内部支架蛋白B结合效率低下，以及外部支架蛋白D介导的错误的Proapsid伸长反应。通过外源克隆基因表达将B蛋白浓度提高到野生型水平以上，补偿无效的B蛋白结合，抑制基因B内的突变也是如此。同样，将D蛋白浓度提高到野生型水平以上或基因D内的补偿性突变可以抑制错误的伸长。一些亲本突变是多效性的，影响了多种形态发生反应。这逐渐减少了通过该途径的中间体的流量。因此，可能无关的多种机制可以恢复生存能力。遗传学分析在破译许多生化途径的时间事件方面发挥了重要作用。然而，多效性效应可能会使分析变得复杂。相对于病毒粒子的形态发生，早期组装中间体中不适当的蛋白质-蛋白质相互作用会影响所有后续反应的效率。因此，组装中间体的通量随着途径的进展而累积减少。在形态发生过程中，ϕx174外壳蛋白至少参与了四个明确的反应，每个反应的特点是与支架或结构蛋白相互作用。在这项研究中，遗传分析、生化特征和生理分析，即提高与外壳蛋白相互作用的蛋白质水平，被用来阐明可能通过形态发生途径改变中间产物通量的多效性效应。
Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.IMPORTANCEGenetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.
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