Jarvis T C, Kirkegaard K
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
EMBO J. 1992 Aug;11(8):3135-45. doi: 10.1002/j.1460-2075.1992.tb05386.x.
Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.
通过聚合酶链反应（PCR）直接定量检测重组RNA分子，为研究RNA病毒的重组提供了一种无需选择存活子代的新方法。本研究中使用的亲代脊髓灰质炎病毒株含有相距约600个碱基的多态性标记位点；两者均表现出野生型生长特性。我们建立了PCR产物量与输入模板量呈线性比例且重现性高的条件。重组子代主要是同源的，出现频率高达2×10⁻³。重组事件在整个复制过程中频率增加，这表明不存在早期遗传学研究中提出的病毒RNA隔离或感染后期重组抑制现象。先前的研究表明，脊髓灰质炎病毒重组是通过复制选择机制发生的，即病毒聚合酶在负链合成过程中切换模板。改变输入亲代病毒的相对量会显著改变相互重组频率。这与动力学数据一起表明，受体模板浓度是模板切换频率的一个决定因素。由于在脊髓灰质炎病毒感染过程中，正链数量远多于负链，这就解释了负链合成过程中重组的偏向性。
