PDF(Pubmed)
Abstract：
Virus-encoded replicases often generate aberrant RNA genomes, known as defective viral genomes (DVGs). When co-infected with a helper virus providing necessary proteins, DVGs can multiply and spread. While DVGs depend on the helper virus for propagation, they can in some cases disrupt infectious virus replication, impact immune responses, and affect viral persistence or evolution. Understanding the dynamics of DVGs alongside standard viral genomes during infection remains unclear. To address this, we conducted a long-term experimental evolution of two betacoronaviruses, the human coronavirus OC43 (HCoV-OC43) and the murine hepatitis virus (MHV), in cell culture at both high and low multiplicities of infection (MOI). We then performed RNA-seq at regular time intervals, reconstructed DVGs, and analyzed their accumulation dynamics. Our findings indicate that DVGs evolved to exhibit greater diversity and abundance, with deletions and insertions being the most common types. Notably, some high MOI deletions showed very limited temporary existence, while others became prevalent over time. We observed differences in DVG abundance between high and low MOI conditions in HCoV-OC43 samples. The size distribution of HCoV-OC43 genomes with deletions differed between high and low MOI passages. In low MOI lineages, short and long DVGs were the most common, with an additional cluster in high MOI lineages which became more prevalent along evolutionary time. MHV also showed variations in DVG size distribution at different MOI conditions, though they were less pronounced compared to HCoV-OC43, suggesting a more random distribution of DVG sizes. We identified hotspot regions for deletions that evolved at a high MOI, primarily within cistrons encoding structural and accessory proteins. In conclusion, our study illustrates the widespread formation of DVGs during betacoronavirus evolution, influenced by MOI and cell- and virus-specific factors.
摘要：
病毒编码的复制酶通常会产生异常的RNA基因组，称为缺陷病毒基因组（DVG）。当与提供必需蛋白质的辅助病毒共感染时，DVG可以繁殖和传播。虽然DVG依赖于辅助病毒的传播，在某些情况下，它们可以破坏感染性病毒的复制，影响免疫反应，并影响病毒的持久性或进化。在感染期间，了解DVG与标准病毒基因组的动力学仍不清楚。为了解决这个问题，我们对两种β-冠状病毒进行了长期的实验进化，人类冠状病毒OC43（HCoV-OC43）和鼠肝炎病毒（MHV），在高和低感染复数（MOI）的细胞培养物中。然后我们以固定的时间间隔进行RNA-seq，重建的DVG，并分析了它们的积累动态。我们的研究结果表明，DVG进化为表现出更大的多样性和丰度，删除和插入是最常见的类型。值得注意的是,一些高MOI缺失显示出非常有限的暂时存在，而其他人随着时间的推移变得普遍。我们在HCoV-OC43样品中观察到高和低MOI条件之间的DVG丰度的差异。具有缺失的HCoV-OC43基因组的大小分布在高和低MOI传代之间不同。在低MOI谱系中，短而长的DVG是最常见的，在高MOI谱系中有一个额外的集群，随着进化时间的推移变得更加普遍。MHV在不同的MOI条件下也显示出DVG大小分布的变化，尽管与HCoV-OC43相比，它们不太明显，这表明DVG大小分布更为随机。我们确定了在高MOI下进化的缺失的热点区域，主要在编码结构和辅助蛋白的顺反子内。总之,我们的研究说明了DVGs的广泛形成过程中，受MOI和细胞和病毒特异性因素的影响。
