Effect of G3BP2 gene knockout on Seneca Valley virus replication in the cells
In This study,we used the CRISPR/CAS9 gene editing technology to construct a G3BP2 gene knockout 293T cell line and ini-tially explored the impact of G3BP2 gene deletion on the proliferation of Senecavirus A(SVA).Laser confocal microscopy was employed to see whether virus infection induced the formation of stress granules.The px459-sgRNA expression vector targeting the G3BP2 gene was de-signed and constructed,and transfected into 293T cells.Cell lines were obtained through puromycin pressure screening and subcloning,and were identified by western blot and gene sequencing.Cell proliferation rates were determined using the CCK-8 assay.Western blot,RT-qPCR,and plaque assay were used to detect the expression level of viral protein VP1,the copy number of VP1,and the virus titer in the cell supernatant after SVA infection.A dual luciferase reporter system targeting the SVA IRES was designed and constructed,and the promoter activity of the virus was measured using a dual luciferasereporter assay.The results showed that virus infection induced the formation of stress granules.A G3BP2 gene knockout 293T cell line(HEK 293T G3BP2-/-)with a 10 bp deletion was obtained,and there was no significant difference in cell viability,compared with non-knockout cells.Western blot,RT-qPCR,and plaque assay all demonstrated that G3BP2 knockout significantly inhibited virus proliferation.The dual luciferase reporter assay indicated that G3BP2 knockout downregulated virus pro-moter activity.In summary,this study obtained a HEK 293T G3BP2-/-cell line that inhibits virus proliferation by downregulating virus pro-moter activity,which laid a foundation for further exploring the impact of G3BP2 on SVA replication.
万方数据
维普
