| Hepatitis B virus infections remain to present a major infectious disease in our country. Patients with chronic HBV infection may develop acute or chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellular carcinomas. An estimated 120 million Chinese are hepatitis B antigen-positive carriers and about 200 million of them suffering from chronic hepatitis B virus. HBV infections and HBV related diseases are serious threats to patients and bring them great economic burden. At present, the mechanisms of HBV infection remain unclear and there is no specific medicine for it. Current clinical therapeutics for HBV infection include interferon(IFN) and nucleoside analogues. But IFN has adverse effects such as flu-like symptoms in the beginning of treatment and later on for instance fatigue and low blood counts, and the major limitation associated with nucleoside analogue is the emergence of antiviral drug resistance. Therefore, it is urgent to research for novel medicine with high potency and low side-effect. Phospholipid scramblase1(PLSCR1) was originally identified as a type Ⅱ plasma membrane protein that disrupts the phospholipid symmetry of the plasma membrane lipid bilayer in a Ca2+ dependent manner. PLSCR1 expression is robustly induced in response to interferon(IFN) treatment. IFN induces transcription of PLSCR1 through STAT1 depends upon sequential activation of protein kinase Cdelta and JNK. Not only are IFN a critical component of the innate antiviral response, but it also has important immune modulating capacity and is one of the major drugs for the treatment of HBV. It was found that the antiviral activity of IFN was depressed when PLSCR1 deficiency or siRNA against PLSCR1 was used, while the antiviral activity against encephalomyocarditis and vesicular virus of IFN was strengthened by over-expressed PLSCR1 by increasing levels of antiviral genes. Previous study in our lab has showed the interaction between PLSCR1 and HBsAg through co-immunoprecipitation and the antiviral activities against HBV by PLSCR1 proteins in vitro and vivo. The results suggest that PLSCR1 might play the important roles in HBV infection but it is not clear. In this study, we will further study to validate the involvement of PLSCR1 in HBV replication based on these data. In order to clarify the molecular mechanism of the inhibition of HBV replication by PLSCR1, the effects of PLSCR1 on HBV replication cycles, HBV replication pathways and host cellular functions, and the effects of the interaction between PLSCR1 and HBsAg on HBV replication were determined. And this study will provide new insight into the mechanism associated with HBV-host interactions and facilitate the development of novel anti-HBV therapeutic strategies.Objective: To clarify the molecular mechanism of the inhibition of HBV replication by PLSCR1. Methods: detected HBV-DNA in cell and cell culrure after transfected PLSCR1 and HBV1.3; siRNA directly against PLSCR1 was designed and synthetized. Then the inhibitory effect of siRNA was determined by using semi-quantitative PCR and Western-blot. HepG2 cells treated with IFN were transfected with plasmids expressing HBV1.3 and PLSCR1 siRNA targeting PLSCR1. Total RNA of HepG2 cells was isolated and the mRNA level of PLSCR1 was measured by reverse transcription semi-quantitative PCR. The expressions of HBsAg and HBeAg in culture supernatant were determined by ELISA. After being respectively transfected with plasmids PLSCR1, HBV1.3 and siRNA targeting PLSCR1, cell proliferation of HepG2 was analyzed using a Cell Counting Kit-8. In addition, cell cycle and were assayed by flow cytometry. Transfected plasmids PLSCR1 and plasmids contain both HBV promoter gene and Gluc gene into HepG2 cell for 48 h, then detect Gluc active of cell culture. Detect the mRNA relative with HBV and protein of innate immune. Results: PLSCR1 can inhibited HBV-DNA in cell and cell culture;The expression of PLSCR1 was inhibited by siRNA in positive cells treated with IFN leading to decreased antiviral effect of IFN against HBV without significant effect on cell function. HBV promoter was inhibited by PLSCR1. after luciferase reporter gene expression vectors containing HBV promoter by gene recombinant technology and eukaryotic expression vector. The results of PCR showed that PLSCR1 could reverse changes of several genes caused by HBV, and PLSCR1 could interact with other genes. The results of western blotting showed than after Transfected HBV1.3, protein of TLR9 was inhibited and the protein of TLR9 recovered when Transfected both HBV1.3and PLSCR1. Conclusions: PLSCR1 can inhibited HBV-DNA in cell and cell culture; The antiviral mechanism of IFN may be through inhibiting the expression of HBV promoter by PLSCR1. Inhibition or overexpression of PLSCR1 had no significant effect on cell function. In addition, the PLSCR1 protein located in cytoplasm around cell nucleus of HepG2 cells and PLSCR1 interacted with various cytokines to inhibit HBV. PLSCR1 inhibited HBV promoter though PPARA. PLSCR1 can enhance TLR9 protein to improve sensitive of HepG2 for HBV. |
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