Alignments of HKU1 and HKU14 sequences with HCoV-OC43, and alignment of PHEV ORF2 sequences

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2 to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. In contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which arose via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remains largely intact, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes and extending these findings to viruses currently adapting to humans.

冠状病毒具备多种遗传创新机制，例如通过捕获宿主细胞基因或复制自身现有病毒基因来获得附属基因（accessory genes）。附属基因能够调控病毒的宿主范围与细胞嗜性，但目前学界对选择压力如何作用于病毒基因组的此类可变区域仍了解有限。本研究以编码宿主源性AKAP7磷酸二酯酶与失活型天然磷酸二酯酶NS2的鼠肝炎病毒（mouse hepatitis virus, MHV）为模型，模拟附属基因的演化命运。经过多轮连续传代感染后，编码失活型NS2的基因ORF2意外保持完整，这表明其受到与NS2功能无关的隐蔽选择约束。与之形成对比的是，AKAP7在强选择压力下得以保留，却在松弛选择压力下快速丢失。实验演化还使病毒复制呈现细胞类型特异性改变，并改变了噬斑纯化病毒分离物中亚基因组病毒RNA（subgenomic viral RNA）的相对占比，揭示了额外的适应机制。受ORF2的保留现象以及相关β冠状病毒（betacoronaviruses）中类似模式的启发，我们对SARS-CoV-2的ORF8进行了分析：该基因通过基因复制产生，且在全球多个优势流行谱系中存在提前终止密码子（premature stop codons）。与MHV的ORF2类似，存在编码缺陷的SARS-CoV-2 ORF8基因整体仍保持完整，这与MHV实验演化中观测到的模式相符。该结果挑战了学界此前关于病毒基因组基因丢失动态的固有假设，并将相关研究发现拓展至当前正在适应人类宿主的病毒领域。