Screening Of Host Factors Regulating Rabies Virus Neurotropism

Rabies is a zoonotic disease caused by rabies virus infection.Most clinical manifestations are specific phobia,water phobia,pharyngeal muscle spasm,and irreversible encephalomyelitis.Although rabies is now 100% preventable,the number of reported deaths from rabies has consistently ranked among the top in China for notifiable infectious diseases.There is currently no effective treatment for rabies.Once it occurs,the disease is almost fatal and poses a serious threat to human life and health.In recent years,some progress has been made in rabies research,but the research on the pathogenesis of rabies virus infection is not thorough.Rabies virus(RV)is a strict intracellular parasitic virus.The entire life cycle of the virus requires the participation of multiple host proteins.Therefore,exploring host proteins that interact with the viral life cycle and understanding its mechanism of action will help us explain the viral life cycle,provide new targets for the prevention and treatment of rabies,and lay the foundation for efficient oral vaccine research.The high-throughput screening technology based on CRISPR technology that has emerged in recent years can systematically study all aspects of the virus life cycle,and can quickly find key host proteins involved in biological processes such as virus invasion and replication.Therefore,CRISPR/Cas9 technology will provide strong technical support for the further exploration of the rabies virus life cycle,the design and treatment of targeted drugs.In this study,a N2a-Cas9 monoclonal cell line expressing Cas9 protein was constructed on N2 a cells by the method of lentivirus transduction.Western Blot verified that the Cas9 gene was stably expressed on N2 a cells,and the Cas9 had good cleavage activity;In addition,after detection with CellTiter-Glo reagent,the expression of Cas9 gene had no effect on the proliferation activity of N2 a cells.Then,78,637 guide RNA(gRNA)plasmid libraries of 19,674 mouse protein-coding genes were amplified by electrotransformation.The second-generation sequencing verified that the coverage of the amplified plasmid library met the requirements.Then the lentiviral transduction system was further used to express these gRNAs in N2 a cells expressing Cas9,and a mouse-based genome-wide knockout library based on the CRISPR system was amplified.After high-throughput sequencing analysis,the amplified cell library also had good coverage.The mouse-derived genome-wide cell knockout library based on the CRISPR/Cas9 was infected ERA-GFP rabies virus constructed by our laboratory,and GFP-negative cells were collected using a flow cytometer.After five infections and sorting,each the negative cells selected in the sorting are subjected to whole-genome sequencing and bioinformatics analysis to obtain candidate genes corresponding to enriched sgRNA,and to obtain host factors that potentially affect the replication of rabies virus.Analysis by DAVID bioinformatics website revealed that these genes were mainly concentrated in the membrane components of cells,as well as the biological processes of signal transduction and cell differentiation.In this study,the N2 a cell line expressing Cas9 protein and the screening platform of mouse-derived genome-wide library based on CRISPR system were successfully constructed,and the important host genes regulating the replication of rabies virus were screened,providing a new theoretical basis for the exploration of rabies virus life cycle,targeted drug design and drug treatment.