Zhang Hu, Fischer Douglas K, Shuda Masahiro, Moore Patrick S, Gao Shou-Jiang, Ambrose Zandrea, Guo Haitao
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
J Med Virol. 2022 Jun;94(6):2438-2452. doi: 10.1002/jmv.27650. Epub 2022 Feb 19.
The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggest that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.
持续的新型冠状病毒肺炎大流行严重影响全球公共卫生和经济。为促进对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)病毒学和抗病毒药物发现的研究,需要一个在生物安全2级防护下运行的非感染性病毒复制子系统。我们在此报告两种SARS-CoV-2微型基因组复制子系统的构建和特性。首先,我们构建了IVT-CoV2-Rep互补DNA模板,通过体外转录(IVT)生成带有纳米荧光素酶(NLuc)报告基因的复制子信使核糖核酸(mRNA)。复制子mRNA转染试验证明IVT-CoV2-Rep在多种细胞系中能快速短暂复制,已知的SARS-CoV-2复制抑制剂可完全消除这种复制。我们的数据还表明,IVT-CoV2-Rep的短暂表型并非由于宿主先天抗病毒反应。此外,我们开发了一种DNA启动的复制子BAC-CoV2-Rep,它支持将复制子mRNA作为初始复制模板进行细胞内转录。与IVT-CoV2-Rep版本相比,BAC-CoV2-Rep瞬时转染系统表现出更强、更长的复制子信号。我们还发现,一部分NLuc报告基因信号来自剪接后的BAC-CoV2-Rep mRNA,并且对抗病毒治疗有抗性,尤其是在转染后的早期阶段。总之,已建立的SARS-CoV-2瞬时复制子系统适用于基础研究和抗病毒研究,通过进一步优化有望开发出稳定的复制子细胞系。