ABSTRACT
Objective To identify the intracellular host factors necessary for the hepatitis C virus (HCV) infection process so as to provide a scientific basis for the discovery of new antiviral targets.Methods In total 1×108 human liver cancer cells Huh7.5 were infected with a CRISPR Cas9 lentiviral small guide RNA (sgRNA) library which covers 19 050 human genes.Cell clonal populations with genes knockout were screened using blasticidin and puromycin resistance markers,and then were infected with HCV JFH1 strain (multiplicity of infection of 0.30).By comparing the infection levels with the unknocked Huh7.5 cells (control group),the cell populations with significantly reduced infection levels were screened and obtained.Finally,the cloned gene sequence of cell clonal populations was obtained by Illumina sequencing,and the single gene was separately verified by short hairpin RNA (shRNA) technology to identify the intracellular host factor necessary for HCV infection.Results The gene knockout cell populations by CRISPR-Cas9 library were successfully screened by blasticidin and puromycin resistance markers.Cell populations with significantly decreased HCV infection levels compared with the control group were obtained by immunofluorescence assay.These populations were sequenced by Illumina and the results suggest that there are at least 10 genes that may be associated with a reduction in HCV infection.The shRNA knockdown single gene verification results showed that compared with the unknocked Huh7.5 cells,the HCV infection rates were decreased in MTCP1 or RPS14 genes knockout Huh7.5 cells,and the differences were statistically significant (all P＜0.05).That indicates that MTCP1 and RPS14 are two intracellular host factors essential for HCV infection.Conclusion A new method for identifying intracellular host factors essential for HCV infection was established.The two intracellular host factors essential for HCV infection,MTCP1 and RPS14,were screened.This study lays a foundation for further studies on related action mechanism,and also provides a scientific basis for discovering new antiviral targets.