Sun Ying, Overman Stacy A, Thomas George J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA.
Virology. 2007 Sep 1;365(2):336-45. doi: 10.1016/j.virol.2007.02.040. Epub 2007 May 9.
The podovirus P22, which infects O-antigen strains of Salmonella, incorporates a dsDNA translocating channel (portal dodecamer) at a unique vertex of the icosahedral capsid. The portal subunit (gp1, 82.7 kDa) exhibits multiple S-Hcdots, three dots, centeredX hydrogen bonding states for cysteines 153, 173, 283 and 516 and these interactions are strongly perturbed by portal ring formation. Here, we analyze in vivo activities of wild type (wt) and Cys-->Ser mutant portals, demonstrate that in vivo activity is correlated with in vitro assembly kinetics, and suggest mechanistic bases for the observed assembly defects. The C283S portal protein, which assembles into rings at about half the rate of wt, exhibits significantly diminished infectivity ( approximately 50% of wt) and manifests its defect prior to DNA packaging, most likely at the stage of procapsid assembly. Conversely, the C516S mutant, which assembles at twice the rate of wt, is more severely deficient in vivo ( approximately 20% of wt) and manifests its defect subsequent to capsid maturation and DNA packaging. Both C153S and C173S portals function at levels close to wt. The results suggest that C283S and C516S mutations may be exploited for improved characterization of the folding and assembly pathway of P22 portal protein.
噬菌体P22可感染沙门氏菌的O抗原菌株，它在二十面体衣壳的一个独特顶点处整合了一个双链DNA转运通道（门户十二聚体）。门户亚基（gp1，82.7 kDa）的半胱氨酸153、173、283和516呈现出多个中心为X的S-H···氢键结合状态，并且这些相互作用会因门户环的形成而受到强烈干扰。在此，我们分析了野生型（wt）和半胱氨酸突变为丝氨酸的突变体门户在体内的活性，证明体内活性与体外组装动力学相关，并为观察到的组装缺陷提出了机制基础。C283S门户蛋白组装成环的速率约为野生型的一半，其感染力显著降低（约为野生型的50%），并且在DNA包装之前就表现出缺陷，很可能是在原衣壳组装阶段。相反，C516S突变体组装速率是野生型的两倍，但其在体内的缺陷更为严重（约为野生型的20%），并且在衣壳成熟和DNA包装之后才表现出缺陷。C153S和C173S门户蛋白的功能水平均接近野生型。结果表明，C283S和C516S突变可用于更好地表征P22门户蛋白的折叠和组装途径。
