Parent Kristin N, Suhanovsky Margaret M, Teschke Carolyn M
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA.
Mol Microbiol. 2007 Sep;65(5):1300-10. doi: 10.1111/j.1365-2958.2007.05868.x. Epub 2007 Aug 3.
Eighteen single amino acid substitutions in phage P22 coat protein cause temperature-sensitive folding defects (tsf). Three intragenic global suppressor (su) substitutions (D163G, T166I and F170L), localized to a flexible loop, rescue the folding of several tsf coat proteins. Here we investigate the su substitutions in the absence of the original tsf substitutions. None of the su variant coat proteins displayed protein folding defects. Individual su substitutions had little effect on phage production in vivo; yet double and triple combinations resulted in a cold-sensitive (cs) phenotype, consistent with a defect in assembly. During virus assembly and maturation, conformational switching of capsid subunits is required when chemically identical capsid subunits form an icosahedron. Analysis of double- and triple-su phage-infected cell lysates by negative-stain electron microscopy reveals an increase in aberrant structures at the cs temperature. In vitro assembly of F170L coat protein causes production of polyheads, never seen before in phage P22. Purified procapsids composed of all of the su coat proteins showed defects in expansion, which mimics maturation in vitro. Our results suggest that a previously identified surface-exposed loop in coat protein is critical in conformational switching of subunits during both procapsid assembly and maturation.
噬菌体P22外壳蛋白中的18个单氨基酸取代会导致温度敏感型折叠缺陷（tsf）。三个定位在柔性环上的基因内全局抑制（su）取代（D163G、T166I和F170L）可挽救几种tsf外壳蛋白的折叠。在这里，我们在不存在原始tsf取代的情况下研究su取代。没有一个su变体外壳蛋白表现出蛋白质折叠缺陷。单个su取代对体内噬菌体产生的影响很小；然而，双重和三重组合导致了冷敏感（cs）表型，这与组装缺陷一致。在病毒组装和成熟过程中，当化学性质相同的衣壳亚基形成二十面体时，衣壳亚基需要进行构象转换。通过负染色电子显微镜对双重和三重su噬菌体感染的细胞裂解物进行分析，发现在cs温度下异常结构增加。F170L外壳蛋白的体外组装导致多聚头部的产生，这在噬菌体P22中从未见过。由所有su外壳蛋白组成的纯化原衣壳在膨胀方面存在缺陷，这在体外模拟了成熟过程。我们的结果表明，先前确定的外壳蛋白表面暴露环在原衣壳组装和成熟过程中衣壳亚基的构象转换中至关重要。
