Department of Infectious Diseases, Institute of Virology, University of Veterinary Medicine Hannover, Germany.
Institut Pasteur, Unit of Structural Virology, Paris, France.
Genome Biol Evol. 2017 Apr 1;9(4):817-829. doi: 10.1093/gbe/evx046.
RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins.
RNA 重组是 RNA 病毒进化的主要驱动力，它在病毒适应新宿主、毒力变化以及新病毒的出现（包括耐药和逃逸突变体）中起着重要作用。然而，动物 RNA 病毒中重组的分子细节知之甚少。为了确定病毒 RNA 重组是否依赖于病毒蛋白的翻译，建立了一种非复制性的重组系统，该系统基于用合成的牛病毒性腹泻病毒（Flaviviridae 科）RNA 基因组片段共转染细胞，这些片段要么缺乏帽非依赖性翻译所需的内部核糖体进入位点，要么缺乏几乎完整的多蛋白编码区。许多重组病毒的出现表明，IRES 介导的病毒蛋白翻译对于高效重组是可有可无的，并表明在没有病毒蛋白的情况下，RNA 重组可以发生。对 58 个独立出现的病毒进行分析，发现了在开放阅读框的 5'端具有重复、缺失和插入的重组基因组，导致可通过 Western blot 分析检测到的核心融合蛋白增大。这证明了瘟病毒核心蛋白具有显著的灵活性。用含有荧光素酶基因的加帽和不加帽基因组片段进行进一步实验，以监测蛋白翻译水平，结果表明，即使病毒蛋白翻译水平提高了约 1000 倍，也不会增加 RNA 重组的频率。总之，这项研究强调了非复制性 RNA 重组不需要翻译病毒蛋白。
