Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein.</p><p><b>METHODS</b>Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP.</p><p><b>CONCLUSION</b>Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.</p>

Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles.</p><p><b>METHODS</b>Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium.</p><p><b>CONCLUSION</b>After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.</p>

Replication and encapsidation of HBV mutants with the truncated C gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the replication and encapsidation of HBV mutants with the truncated C gene.</p><p><b>METHODS</b>The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome.</p><p><b>RESULTS</b>The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay.</p><p><b>CONCLUSION</b>The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.</p>

The anti-HBV effect and mechanism of C gene truncated mutant in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effect and mechanism on HBV replication in C gene truncated mutant.</p><p><b>METHODS</b>Protein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot.</p><p><b>RESULTS</b>The recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C.</p><p><b>CONCLUSION</b>C gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication</p>

Approach to transforming hepatitis B virus as a gene therapeutic vector / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.</p><p><b>METHODS</b>A fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.</p><p><b>RESULTS</b>The HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.</p><p><b>CONCLUSION</b>It is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.</p>

Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein.</p><p><b>METHODS</b>Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium.</p><p><b>CONCLUSIONS</b>It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.</p>