PDF(Pubmed)
Abstract：
Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.
摘要：
副本,来自RNA病毒，是保留必需病毒酶基因而缺乏关键结构蛋白基因的遗传构建体。一旦引入细胞，复制子RNA携带的基因被表达，RNA自我复制，然而，病毒颗粒生产不发生。通常,RNA复制子在体外转录,然后在细胞中电穿孔。然而,在DNA转染而不是RNA转染后在细胞中产生复制子将是有利的。在这项研究中,将在T7启动子控制下编码SARS-CoV-2复制子的细菌人工染色体（BAC）DNA转染到HEK293T细胞中，这些细胞被工程化以功能性表达T7RNA聚合酶（T7RNAP）。转染BACDNA后，我们观察到低，但该复制子携带的报告蛋白GFP和荧光素酶的可重复表达。报道蛋白的表达需要在转染之前使BACDNA线性化。此外，表达独立于T7RNAP。基因表达也对remdesivir治疗不敏感，这表明它不涉及复制子RNA的自我复制。在高度允许SARS-CoV-2感染的Calu-3细胞中获得了类似的结果。引人注目的是，SARS-CoV-2N蛋白的先前表达增强了转染的SARS-CoV-2RNA复制子的表达，但不增强复制子BACDNA的表达。总之,编码冠状病毒复制子的大DNA的转染通过未知的机制导致可重复的复制子基因表达。这些发现突出了从转染的复制子cDNA中表达复制子基因的新途径，为开发基于DNA的RNA复制子应用方法提供有价值的见解。
