Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

Abstract

Aim: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model.

Methods: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively.

Results: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups.

Conclusion: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

Figure 1

Effect of APOBEC3G on HBV replication in cotransfected hepatoma cells. Cells were treated with lipofectamine 2000 reagents, and viral replicative DNA intermediates were analyzed with southern blotting 3 d after transfection. Numbers at the end of the lines indicate the amount of transfected plasmid DNA in micrograms. HBV, hepatitis B virus; RC, relaxed circular DNA; SS, single -stranded DNA.

Figure 2

Western blot analysis of cytoplasmic extracts from Huh7 cells cotransfected with indicated plasmids.

Figure 3

Effect of APOBEC3G on HBsAg and HBeAg secretion in the media of cotransfected Huh7 cells 3 d after transfection. HBsAg and HBeAg levels (S/N) were significantly reduced (error bar indicate standard error). Numbers at the end of the lines indicate the amount of transfected plasmid DNA in micrograms.

Figure 4

Effect of APOBEC3G on HBV RNA expression. Northern blot analysis of HBV transcripts from Huh7 cells co-transfected with indicated plasmids. The same blot was hybridized with a glyceraldehyde phosphate dehydrogenase (GAPDH) DNA probe as an internal control.

Figure 5

Serum HBsAg levels in APOBEC3G treated mice. HBsAg levels(S/N) in the sera of BALB/c mice were significantly reduced after treatment with the APOBEC3G expression plasmids (error bar indicate standard error) in the indicated time points, n = 6 per treatment group.

Figure 6

Real time PCR quantification of HBV DNA in the sera of mice treated with APOBEC3G expression plasmid. BALB/c mice were coinjected with pHBV1.3 vector (10 μg ) and APOBEC3G expression plasmid (10 μg) 3 d after injection. HBV DNA levels (mean ± SD) of sera were determined by quantitative PCR, n = 6 per treatment group.

Figure 7

Real time PCR quantification of core-associated HBV RNA in the liver of mice treated with indicated plasmids. BALB/c mice were coinjected with pHBV1.3 vector (10 μg) and APOBEC3G expression plasmid (10 μg) 3 d after injection. The levels of liver HBV RNA were determined by quantitative RT-PCR, normalized to GAPDH mRNA and are reported as a ratio of HBV mRNA/GAPDH mRNA (mean ± SD). n = 6 per treatment group.