Screening Host Genes Associated with RSV Replication by VBIM
Objective:To employ the validation-based insertional mutagenesis(VBIM)technology in screening host factors asso-ciated with respiratory syncytial virus(RSV).Methods:HEp-2 cells were transduced with VBIM lentiviruses to generate a mutant cell pool.RSV-resistant cell clones were obtained after RSV infection.After extracting genomic DNA from these cells,we used reverse PCR to identify inserted sites of the VBIM vector in the resistant clones.Corresponding gene siRNAs were synthesized and used to suppress the gene expression.The replication of RSV was then analyzed using RT-qPCR and western blotting,the viral titers were determined as well.Results:Six RSV-resistant cell clones were obtained and eight potential host genes regulating RSV replication including AKR1C3,DDX18,GFI1B,TAPBP,MEF2A,SEMA3C,KCNIP4,GRID2 were identified.After preliminary verification,RSV RNA level,protein level and viral titer decreased by 21％,60％and 68％respectively after MEF2A knockdown.After SEMA3C knockdown,RSV RNA level decreased by 23％,protein level decreased by 28％,and viral titer decreased by 40％.Conclusions:By utilizing the VBIM technol-ogy,we successfully obtained host factors affecting RSV replication,providing a theoretical basis for the study of virus-host interaction as well as RSV prevention and treatment.
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