A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes

Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.反向拷贝型缺陷病毒基因组（copy-back defective viral genomes, cbDVGs）是呼吸道合胞病毒（respiratory syncytial virus, RSV）感染过程中，体内与体外触发抗病毒免疫应答的核心起始因子。然而，调控cbDVG生成的具体分子机制仍未阐明，这极大限制了我们通过操纵cbDVG组成以调节宿主感染应答的能力。本研究报道了一种特异性基因组信号，其可介导RSV cbDVG部分亚型的生成。借助定制化生物信息学工具，我们鉴定出RSV基因组中在感染过程中被频繁用于生成cbDVG的区域。随后，我们构建微型基因组系统，对其中一段序列的功能进行验证，并探究该位置的特定核苷酸是否为cbDVG生成所必需。进一步地，我们通过在该序列中引入突变，构建了无法生成部分cbDVG亚型的重组病毒。经鉴定的序列同时也是自然RSV感染过程中cbDVG生成的位点，且在多名感染患者的样本中，均发现了源自该位点的共性cbDVG。上述实验数据表明，病毒基因组编码的序列决定了cbDVG的形成位点，因此cbDVG的生成并非随机过程。本研究发现为通过基因工程手段操纵cbDVG生成以调节感染结局提供了可行路径。