Zhu Yinxing, Sullender Meagan E, Campbell Danielle E, Wang Leran, Lee Sanghyun, Kawagishi Takahiro, Hou Gaopeng, Dizdarevic Alen, Jais Philippe H, Baldridge Megan T, Ding Siyuan
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
Department of Medicine, Division of Infectious Diseases and Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, MO, USA.
NPJ Vaccines. 2024 Nov 6;9(1):211. doi: 10.1038/s41541-024-01007-7.
Rotaviruses pose a significant threat to young children. To identify novel pro- and anti-rotavirus host factors, we performed genome-wide CRISPR/Cas9 screens using rhesus rotavirus and African green monkey cells. Genetic deletion of either SERPINB1 or TMEM236, the top two antiviral factors, in MA104 cells increased virus titers in a rotavirus strain independent manner. Using this information, we optimized the existing rotavirus reverse genetics systems by combining SERPINB1 knockout MA104 cells with a C3P3-G3 helper plasmid. We improved the recovery efficiency and rescued several low-titer rotavirus reporter and mutant strains that prove difficult to rescue otherwise. Furthermore, we demonstrate that TMEM236 knockout in Vero cells supported higher yields of two live-attenuated rotavirus vaccine strains than the parental cell line and represents a more robust vaccine-producing cell substrate. Collectively, we developed a third-generation optimized rotavirus reverse genetics system and generated gene-edited Vero cells as a new substrate for improving rotavirus vaccine production.
轮状病毒对幼儿构成重大威胁。为了鉴定新的促轮状病毒和抗轮状病毒宿主因子，我们使用恒河猴轮状病毒和非洲绿猴细胞进行了全基因组CRISPR/Cas9筛选。MA104细胞中排名前两位的抗病毒因子丝氨酸蛋白酶抑制剂B1（SERPINB1）或跨膜蛋白236（TMEM236）的基因缺失，以轮状病毒株独立的方式提高了病毒滴度。利用这一信息，我们通过将敲除SERPINB1的MA104细胞与C3P3-G3辅助质粒相结合，优化了现有的轮状病毒反向遗传学系统。我们提高了拯救效率，并拯救了几种低滴度的轮状病毒报告基因和突变株，否则这些毒株很难被拯救。此外，我们证明，Vero细胞中TMEM236的敲除比亲代细胞系支持两种减毒活轮状病毒疫苗株产生更高的产量，并且代表了一种更强健的疫苗生产细胞底物。总体而言，我们开发了第三代优化的轮状病毒反向遗传学系统，并生成了基因编辑的Vero细胞作为改善轮状病毒疫苗生产的新底物。
