Smorodintsev Research Institute of Influenza, Ministry of Health of Russian Federation, Prof. Popova 15/17, Saint Petersburg 197376, Russian Federation; Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, Lavrentiev Avenue 8, Novosibirsk 630090, Russian Federation.
Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, Lavrentiev Avenue 8, Novosibirsk 630090, Russian Federation.
Gene. 2022 Jan 30;809:146024. doi: 10.1016/j.gene.2021.146024. Epub 2021 Oct 19.
Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6 mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6 cells significantly exceeded the virus titer in the original HEK293FT cells.
利用源自人类的细胞培养物来繁殖流感病毒是一种很有吸引力的方法，可以保持其糖基化谱和抗原特性，这对于流感监测和疫苗生产至关重要。然而，只有少数细胞系对流感病毒高度允许，而且它们都不是源自人类。这种障碍可能与宿主限制因子有关，这些因子抑制流感的生长，例如 AnxA6 蛋白可以拮抗流感病毒包膜的形成过程。在本研究中，我们探索了 CRISPR-Cas9 介导的 ANXA6 基因敲除，以克服宿主限制障碍并提高人源细胞系对流感感染的敏感性。通过 CRISPR-Cas9 基因组编辑，我们修饰了 HEK293FT 细胞，并获得了几个 ANXA6 基因缺陷的克隆。在生长曲线实验中比较了流感 A 病毒在原始 HEK293FT 细胞和 HEK293FT-ANXA6 突变细胞中的复制。通过包括 TCID 测定和流式细胞术在内的方法组合，我们表明突变型 HEK293FT-ANXA6 细胞中流感 A 病毒的积累明显超过了原始 HEK293FT 细胞中的病毒滴度。
