Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes

• Published in: Journal of virological methods Vol. 180; no. 1-2; pp. 84 - 90
• Main Authors: Kan, Quancheng, Li, Duolu, Yu, Zujiang
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.03.2012; Elsevier
• Subjects: adverse effects; antisense RNA; Antiviral; Antiviral Agents - metabolism; Antiviral Agents - pharmacology; antiviral properties; Apoptosis; Biological and medical sciences; Cell Survival; Cells, Cultured; cytotoxicity; DNA fragmentation; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Gene regulation; gene transfer; Genetic Vectors; hepatitis B antigens; Hepatitis B Surface Antigens - genetics; Hepatitis B Surface Antigens - metabolism; Hepatitis B virus; Hepatitis B virus - drug effects; Hepatitis B virus - physiology; hepatocytes; Hepatocytes - immunology; Hepatocytes - virology; human cell lines; Humans; Interferon γ; interferon-gamma; Interferon-gamma - metabolism; Interferon-gamma - pharmacology; Microbiology; pcDNA3.1; Plasmids - genetics; ribosomes; Techniques used in virology; Virology; virus replication; Virus Replication - drug effects; Interferon γ; Antiviral; Hepatitis B virus; pcDNA3.1; Gene regulation; Human; Hepatitis B surface antigen; Cytokine; Orthohepadnavirus; Method; Virus; Gene; Hepatocyte; Hepadnaviridae; In vitro replication; Gamma interferon
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.12.016

Regulatory mode of pcDNA-SCIγ: (a) When HBsAg was secreted in HBV-infected cells, HBV S gene mRNA and transcripts from the HBV antisense S gene in the recombinant plasmid would form complementary sense-antisense dimers. Since the ligation of the HBV antisense S gene with the HCV core gene was performed by Soeing PCR, the termination sequence of the HBV antisense S gene was contiguous with the upstream region of the initiation AUG codon of the HCV core protein gene, with no additional bases. As a result, the formation of mRNA dimers would cover the AUG of the HCV core protein mRNA, thus decreasing expression of the HCV core protein, and relieving the inhibition of HCV IRES-dependent translation by the HCV core protein, resulting in enhanced capacity of HCV IRES to promote IFN-γ expression. (b) In contrast, little expression of IFN-γ should occur in non-HBV-infected cells because of inhibition of HCV IRES-dependent translation by the HCV core protein, which is abundantly expressed in the absence of mRNA dimers of the HBV S gene at the HCV core protein gene initiation codon. [Display omitted] ► A novel strategy for targeted delivery of human interferon gamma to the HBsAg-secreting hepatocytes mediated by pcDNA3.1 was developed. ► This strategy resulted in robust induction of interferon gamma expression in a cell-specific fashion with a very low background. ► The cell-specific interferon gamma expression controlled effectively HBV replication in HepG2.2.15 cells without cell toxicity. Interferon-gamma (IFN-γ) has been reported to have antiviral activity against Hepatitis B virus (HBV) and to suppress HBV replication noncytolytically in vivo. Since systemic administration of IFN-γ may cause severe adverse effects, studies of the effects of liver-specific IFN-γ expression from adenoviral vectors in vivo have been investigated. In this study, a novel strategy has been described that drives specific expression of human IFN-γ in HBsAg-secreting hepatocytes. A bicistronic expression vector has been developed, pcDNA3.1-HBV antisense S gene-HCV core protein gene-HCV internal ribosome entry sites (IRES)-IFN-γ (pcDNA-SCIγ), by inserting four DNA fragments into pcDNA3.1. Tight modulation of HCV IRES-dependent translation by the HCV core protein was achieved using an antisense RNA technique with a bicistronic expression vector. HepG2 cells and HepG2.2.15 cells stably expressing HBV were transduced with pcDNA-SCIγ to test the responsiveness of IFN-γ to HBsAg expression. Gene transfer resulted in a low background and a 30-fold induction of IFN-γ expression from pcDNA-SCIγ in a cell-specific fashion. Hepatocyte-specific IFN-γ expression controlled effectively HBV replication in HBsAg-secreting HepG2.2.15 cells without cell toxicity.