AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.

METHODS: 2.2.15 cells were plated at a density of 5 × 10⁴ per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end, 5 μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindIII. Both DNAs were subjected to Southern blot, hybridized with a ³²P-labeled HBV probe and autoradiographed. Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED₅₀ was calculated. Then Hybond-N membrane was washed and rehybridized with a ³²P-labeled mtDNA-specific probe, and effect of β-L-D4A on mitochondrial DNA was studied. 2. 2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. ID₅₀ was calculated. Structure-activity relationships between D2A and D4A were also studied as above.

RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. ED₅₀ was 0.2 μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID₅₀ at 200 μM.

CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

• Dideoxyadenosine/analogs & derivatives
• Dideoxyadenosine/chemistry
• Dideoxyadenosine/pharmacology
• Dose-Response Relationship, Drug
• Hepatitis B Surface Antigens/drug effects
• Hepatitis B e Antigens/drug effects
• Hepatitis B virus/drug effects
• Hepatitis B virus/physiology
• Humans
• Structure-Activity Relationship
• Tumor Cells, Cultured
• Virus Replication/drug effects

AIM: To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro.

METHODS: HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5’-cis-Rz and 3’-cis-Rz. ³²p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. ³²p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T₇ RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with ³²p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis.

RESULTS: HpRz has the specific ability of cleavage of target RNA at 37 °C and 12 mM MgCl₂. K_m = 26.31 nmol/L, K_cat = 0.18/min. These results revealed that the design of HpRz was correct.

CONCLUSION: HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.

• Base Sequence
• Enzyme Activation
• Genetic Therapy
• Hepatitis B/therapy
• Hepatitis B/virology
• Hepatitis B Core Antigens/genetics
• Hepatitis B virus/genetics
• Humans
• In Vitro Techniques
• Kinetics
• Molecular Sequence Data
• Plasmids
• RNA, Catalytic/metabolism
• RNA, Viral/metabolism
• Transcription, Genetic