[The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP].
OBJECTIVE
Persistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication.


METHODS
The X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot.


RESULTS
The 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP.


CONCLUSIONS
The dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.
Institute of Liver Diseases, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Jia-Wu
JW
Song
Jiu-Sheng
JS
Lin
Xin-Juan
XJ
Kong
Kuo-Huan
KH
Liang
D006528:Carcinoma, Hepatocellular / Q000473:pathology; D003001:Cloning, Molecular; D004261:DNA Replication; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D017354:Point Mutation; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D054334:Viral Regulatory and Accessory Proteins; D014779:Virus Replication
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D049452:Green Fluorescent Proteins