PRP19 Regulates Transcription And Replication Of Hepatitis B Virus

Background and objectiveThe infection of hepatitis B virus(HBV)is closely related to hepatitis,liver cirrhosis and liver cancer,which is a threat to human life and health.In the face of the resistance,rebound,relapse and side effect of antiviral therapy,exporling the molecular mechanism of interaction between HBV and host protein is the key to develop new antiviral drugs and even cure the infection of HBV.With the development of molecular biology and the application of new techniques,the research of targeting viral life cycle has become a hot spot.The interaction between host proteins and viral entry,assembly and release have made great progress.However,the interaction between host proteins and Core promoter regulating viral transcription has rarely been reported.Core promoter plays an important role in the transcription of HBV.It mainly regulates the transcription of precore m RNA(pre C m RNA)and pregenomic m RNA(pg RNA).Pre C m RNA can be translated into C protein,which is involved in the formation of viral nucleocapsid.Pg RNA can be used as a reverse transcription template to synthesize viral DNA and participate in viral replication.Pre-m RNA processing 19(PRP19)is an important host protein involved in DNA damage repair,protein transport and cell proliferation.PRP19 has been shown to interact with the NSI protein of influenza A virus to regulate viral replication.In the previous study,PRP19 was found to be a host protein interacting with Core promoter by immunoprecipitation and mass spectrometry.Therefore,we choose PRP19 as the study object to explore the effect and mechanism of PRP19 on viral transcription and replication.This will shed light on the interaction between host proteins and HBV.This will also provide clues for the development of new antiviral drugs.Methods1.PRP19 was overexpressed to investigate the effect on transcription and replication of HBV.Huh7 cells were co-transfected with PRP19 overexpression plasmid and HBV genotype B expression plasmid.Western blot was used to detect the expression level of PRP19.ELISA was used to detect the expression levels of HBs Ag and HBe Ag.The transcription level of pg RNA was detected by RT-q PCR.The replication level of HBV DNA was detected by q PCR.2.PRP19 was knocked down to investigate the effect on transcription and replication of HBV.Huh7 cells were transfected with PRP19 si RNA and NC si RNA.Subsequently,HBV genotype B rexpression plasmid was transfected.Western blot was used to detect the expression level of PRP19.ELISA was used to detect the expression levels of HBs Ag and HBe Ag.The transcription level of pg RNA was detected by RT-q PCR.The replication level of HBV DNA was detected by q PCR.3.PRP19 was overexpessed to investigate the activity of Core promoter.We also investigated the possible target regions of PRP19 on Core promoter.We constructed luciferase reporter plasmids driven by Core promoter(nt.900-1817)and Enh?/BCP(nt.1627-1817).Huh7 cells were co-transfected with PRP19 overexpression plasmid and two aforementioned luciferase reporter plasmids.The effect of PRP19 on Core promoter and Enh?/BCP activity was detected by the rennin luciferase reporter assay.4.The interaction between PRP19 and Enh?/BCP domain was explored.We transfected the plasmid which upregulated the expression of PRP19 into Huh7 cells.Then,we extracted the nuclear protein of Huh7 cells.We constructed a biotin-labeled probes of PRP19.Huh7 cell nuclear protein was incubated with probe to obtain the product of DNA pull down.Western blot was used to detect the expression of PRP19 to clarify the relationship between Enh?/BCP and PRP19.Results1.Western blot showed that compared with the control group,the expression level of PRP19 in the experimental group was significantly increased.ELISA showed that overexpression of RPR19 could decrease the expression level of HBs Ag and HBe Ag.RT-q PCR showed that overexpression of PRP19 could decrease the transcription level of pg RNA.q PCR showed that overexpression of PRP19 could decrease the replication level of HBV DNA.The differences were statistically significant(P < 0.05).2.Western blot showed that compared with the control group,the expression level of PRP19 in the experimental group was significantly decreased.ELISA showed that knocking down the expression of PRP19 could increase the expression level of HBs Ag and HBe Ag.RT-q PCR showed that knocking down the expression of PRP19 could increase the transcription level pg RNA.q PCR showed that knocking down the expression of PRP19 could increase the replication level of HBV DNA.The differences were statistically significant(P < 0.05).3.The luciferase reporter assay showed that overexpression of PRP19 could inhibit the activity of Core promoter and Enh?/BCP.The differences were statistically significant(P < 0.05).4.DNA pull down and Western blot showed that Enh?/BCP probe could bind to PRP19.Conclusion1.Overexpression of PRP19 can inhibit transcription and replication of HBV in Huh7 cells.2.Knocking down the expression of PRP19 can promote transcription and replication of HBV in Huh7 cells.3.Overexpression of PRP19 can inhibit the activity of Core promoter in Huh7 cells.4.The Enh?/BCP region is the target region where PRP19 acts on the Core promoter.PRP19 regulates the activity of Core promoter by binding to the Enh?/BCP domain.