Korean J Hepatol.  2009 Mar;15(1):15-24. 10.3350/kjhep.2009.15.1.15.

Inhibition of in vitro hepatitis B virus replication by lentivirus-mediated short-hairpin RNA against HBx

• Kim JW
• Lee SH
• Park YS
• Jeong SH
• Kim N
• Lee DH

• KMID: 1776811
• DOI: http://doi.org/10.3350/kjhep.2009.15.1.15

Abstract

BACKGROUNDS/AIMS
Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV.
METHODS
Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR.
RESULTS
Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05).
CONCLUSIONS
Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.

Keyword

Hepatitis B virus; Lentivirus; MicroRNAs; RNA interference; siRNA

MeSH Terms

Cell Line, Tumor
Genetic Vectors
Hepatitis B virus/*genetics/growth & development/physiology
Humans
Lentivirus/*genetics
*RNA Interference
RNA, Viral/metabolism
Trans-Activators/*antagonists & inhibitors/genetics/metabolism
*Virus Replication