Trottier M, Guo P
Department of Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA.
J Virol. 1997 Jan;71(1):487-94. doi: 10.1128/JVI.71.1.487-494.1997.
Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions. Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly. This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components. It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes. The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding. Two methods were used for stoichiometry determination. Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids. The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation [EQUATION IN TEXT] where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to [FORMULA IN TEXT]. With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit. It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging. Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions. Components with known stoichiometries served as standard controls. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 would indicate multiple-copy involvement. By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12. These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers. However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.
由于生物反应速度很快，很难分离中间体或确定中间反应中参与者的化学计量关系。本研究没有确定每种成分的绝对量，而是使用了相对参数，如稀释因子、百分比概率和滴定曲线的斜率，这些参数可以更准确地量化，以确定参与噬菌体phi29组装的成分的化学计量关系。这项工作利用了敏感的体外噬菌体phi29组装系统，在该系统中，可以从八种纯化成分组装出每毫升10^8个无背景的感染性病毒粒子。它为定量包装成分的化学计量关系提供了一种方便的检测方法，这些成分包括病毒原衣壳、基因组DNA、DNA包装pRNA以及其他结构蛋白和酶。pRNA中存在原衣壳结合结构域和另一个重要的功能结构域，这使其成为构建用于竞争性原衣壳结合的致死突变体的理想成分。使用了两种方法来确定化学计量关系。方法1是确定与原衣壳结合的突变型和野生型pRNA的结合概率。用二项式方程[文本中的方程]预测具有一定数量突变型和一定数量野生型pRNA且结合亲和力相等的原衣壳的概率，其中Z是每个原衣壳中pRNA的总数，M是与一个原衣壳结合的突变型pRNA的数量，且(Z - M)等于[文本中的公式]。在体外病毒组装中，用不同比例的突变型与野生型pRNA，绘制突变型pRNA百分比与病毒粒子产量的关系图，并与一系列预测曲线进行比较以找到最佳拟合。确定一次DNA包装事件需要五个或六个拷贝的pRNA，而每个原衣壳仅一个突变型pRNA就足以阻断包装。方法2涉及比较稀释因子曲线与病毒粒子产量的斜率。具有已知化学计量关系的成分用作标准对照。成分的化学计量关系越大，稀释因子对反应的影响就越显著。斜率为1表明一个拷贝的该成分参与一个病毒粒子的组装。斜率大于1表明有多个拷贝参与。通过这种方法，确定phi29颗粒中gp11的化学计量关系约为12。这些方法对于确定参与病毒组装的功能单元的化学计量关系很有用，无论它们是单分子还是寡聚体。然而，如果功能单元在组装前由多个亚基组成，这些方法不适用于确定单个分子的确切拷贝数。
