PDF(Pubmed)
Abstract：
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.
摘要：
严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）诱导直接的细胞病变效应，复杂的低细胞毒性细胞培养模型的建立研究其复制。我们最初开发了一种基于DNA载体的复制子系统，该系统利用CMV启动子产生带有报告基因的重组病毒基因组。然而,该系统经常导致耐药性和细胞毒性，阻碍模型建立。在这里，我们提出了一种通过Cre/LoxP介导的DNA重组诱导SARS-CoV-2复制的新型细胞培养模型。将工程化的SARS-CoV-2转录单位亚克隆到细菌人工染色体（BAC）载体中。为了加强生物安全，病毒刺突蛋白基因被删除，核衣壳基因被报告基因取代。外源序列作为在Cre/LoxP介导的DNA重组和随后的RNA剪接后可去除的调节盒插入NSP1中。使用PiggyBac转座子策略，转录单元整合到宿主细胞染色质中，产生能够诱导重组SARS-CoV-2RNA复制的稳定细胞系。该模型对潜在的抗病毒剂连翘酯苷A和维替泊芬表现出敏感性。引入了一种创新的诱导型SARS-CoV-2复制子细胞模型，以进一步探索病毒的复制和发病机理，并促进抗SARS-CoV-2疗法的筛选和评估。
