| Hepatitis B virus belonging to the Hepadnaviridae family, is a major cause of liver diseases, such as viral hepatitis and liver cirrhosis. More than 400 million individuals worldwide have been infected by HBV. Current antiviral treatment for chronic hepatitis B is mainly used in the treatment of recombinant interferon and nucleoside analogues (such as, lamivudine and adefovir). However, considering the side effects of the current anti HBV therapies and the emergence of drug-resistant HBV strains, there is no definitive cure for chronic HBV infection. Therefore, developing new compounds and novel strategies to inhibit HBV replication in hepatocytes is central for Hepatitis B treatment.Ribonuclease P (RNase P) is a kind of ribonuclease which is widely existed in various tissues. It is mainly used to catalyze the maturation of tRNA precursors. EGS RNA can be complementary with the target mRNA to form a structure similar to the tRNA precursor, and intracellular RNase P for specific degradation of the target mRNA. In our study, we screened out the target site for EGS binding in the overlapping region of the S mRNA, pre-S/L mRNA and pregenomic RNA (pgRNA) of hepatitis B virus by DMS transformation experiment. We constructed EGS variant S-C386 according to the previous studies. We have got the best concentration of EGS and target mRNA in vitro binding assay. In vitro splicing experiments the EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA..Attenuated Salmonella, as an effective drug carrier for oral delivery, has been widely used in the research of DNA vaccines and anti-cancer drugs. Liver is one of the main organs of Salmonella systemic infection, and Salmonella can invade and infect liver cells. Therefore, we can use attenuated Salmonella to effectively delivery EGS to liver cells for gene targeting expression. This will improve the efficiency of EGS in inhibiting HBV replication and expression. We have proved that Salmonella can efficiently delivery all kinds of EGS expressing in HepG2 cells, and does not have any obvious cytotoxicity. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene targeting applications such as anti-HBV therapy.TP protein is an important component of the HBV polymerase, which act as a primer in reverse transcription of HBV. Using yeast two-hybrid system, we have get 14 host proteins interacting with TP protein. By studying the interaction of TP protein with these host proteins, it is helpful to understand the mechanism of HBV genome replication, and provide a new direction for the treatment of HBV. |
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