From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
the Department of Pharmaceutical Chemistry, University of Innsbruck, A-6020 Innsbruck, Austria.
J Biol Chem. 2011 Mar 11;286(10):8414-8424. doi: 10.1074/jbc.M110.205534. Epub 2010 Dec 23.
To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.
为了开发适用于所有甲型流感病毒的新型衰减策略，我们针对病毒聚合酶亚基 PA 和 PB1 之间高度保守的蛋白-蛋白相互作用。我们假设 PA 和 PB1 之间结合的受损会严重影响三聚体聚合酶复合物的形成，从而导致体内病毒复制效率降低。作为概念验证，我们在 PB1 或 PA 的蛋白-蛋白结合域中引入单个或多个氨基酸取代，以显著降低结合亲和力和聚合酶活性。正如预期的那样，在产生含有这些突变的重组甲型流感病毒（SC35M 株）后，出现了许多假性回复突变体，部分恢复了 PA-PB1 结合和聚合酶活性。这些聚合酶组装突变体在细胞培养和小鼠中表现出严重的衰减。在 IFNα/β 受体敲除小鼠中，聚合酶组装突变体的衰减得以维持。例如，使用 H5N1 聚合酶组装突变体为例，这种衰减策略也可以应用于其他高致病性甲型流感病毒株。因此，我们提供了原理性证明，即靶向突变 PA 和 PB1 的高度保守相互作用域代表了一种衰减甲型流感病毒的新策略。
