Negative Regulatory Role Of Human RNase P Proteins On Hepatitis C Virus Replication

Objective:In this study,we wanted to assay that if certain protein subunits of human RNase P(RPP) could bind directly with the 5’ untranslated region of hepatitis C virus(HCV) genomic RNA in vitro, and if the certain RPP proteins possess a regulatory role for HCV multiplication in host cell.Methods and Results: Part 1:Assays of humanized RPP protein binding with HCV5’UTR in vitro. Firstly, the lentiviral vectors recombined with a certain RPP gene were constructed, and were then transfected into the HEK293 cell line. By flow cytometry screening, four cell lines that stably expressed RPP protein were obtained(i.e. Rpp20,Rpp21, Rpp25 and Rpp29-stably expressed cell lines). The four RPP-expressed cell lines were amplified, and purified by using Bio-Rad protein purification metry, and then detected by SDS-polyacrylamide electrophoresis,results showed that all the four RPP proteins could expressed in the cell line, and the purity of RPP proteins were quite good(more than 90%)。Moreover, by in vitro transcription that incorporated with32P-UTP,a isotope labeling RNA fragment that including 5’ UTR(nt 1-341) of HCV genome was prepared as the target RNA. The target RNA was separately mixed with the four humanized RPP proteins, the results of EMSA showed that all the four RPP proteins could directly bind with HCV 5’UTR, the binding activity of Rpp20 was weak, whereas the binding activity of Rpp29 was comparatively strongest. Past 2:Investigating the regulatory role of host RRP protein on HCV multiplication.Huh7.5.1 cells were infected with the four constructed lentivirus above-metioned,then infected with HCV strain(JFH1), and detected the viral titers of HCV in the cell and the culture supernatant by fluorescent quantitive PCR, results showed that overexpression the RPP proteins in host cells would lead to significantly inhibition of HCV replication. Thereinto the repressive role of Rpp29 was strongest comparatively.Meanwhile, through silencing the RPP proteins in host cells by using RNAi technique,we found that silencing Rpp20 did not affect HCV titers significantly, but silentcing Rpp21, Rpp25 or Rpp29 would extremely promote the multiplication HCV.Conclusions:Rpp21, Rpp25 and Rpp29 as the subunits of RNase P complex might be negative regulators in host cell to affect the life cycle of HCV, and the functional mechanism of RPP proteins was possibly through binding with the 5’UTR of HCV genomic RNA. Our results were help to expand the understanding of biologic functions of RPPs, and to uncover a new regulating mechanism of HCV replication.