In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine

Abstract

2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC₅₀) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 μmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovir-resistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.

Keywords: Antiviral; HBV; Hepatitis B; Nucleoside analogues; Woodchuck hepatitis virus (WHV).

Fig. 1

Southern blot and Northern blot analyses of anti-HBV effect of DoG. HepAD38 cells were incubated with DoG in the absence of Tet for 5 days. Northern or Southern blot was conducted to analyze the total RNA and viral core DNA with a HBV-specific probe. Relative HBV DNA level of each group was quantified by Quantity One software (Bio-Rad, Hercules, CA, USA). Representative results from three experiments are presented. 28S, and 18S rRNA, 28S and 18S ribosomal RNA; pgRNA, pregenomic RNA; env RNA, mRNAs encoding envelope proteins; dslDNA, double-stranded linear DNA; rcDNA, relaxed circular DNA; ssDNA, single stranded DNA.

Fig. 2

Southern blot and Northern blot analyses of antiviral effect of DoG on DHBV. Dstet 5 cells were exposed to DoG for 3 days. A Total RNA and DHBV DNA were examined by Northern or Southern blot with DHBV-specific probes. Relative HBV DNA level in each group was quantified with Quantity One software (Bio-Rad). Ribosomal RNAs served as loading controls. B The core particles were extracted as previously described (Zhang et al.2016). Capsid-associated RNA was analyzed by Northern blotting. Representative results are presented from three experiments. 28S, and 18S rRNA, 28S and 18S ribosomal RNA; pgRNA, pregenomic RNA; env RNA, mRNAs encoding envelope proteins; dslDNA, double-stranded linear DNA; rcDNA, relaxed circular DNA; ssDNA, single stranded DNA.

Fig. 3

Southern analysis of antiviral effect of DoG on WHV. pCMV-WHV plasmids were transfected into HepG2 cells with Lipofectamine 2000. At 6 h post-transfection, the cells were incubated with DoG and 10 μmol/L 3TC for 3 days. Core DNA was examined by Southern blotting. Relative HBV DNA level in each group was quantified by Quantity One software (Bio-Rad). Representative results from three experiments are presented. rcDNA, relaxed circular; dslDNA, double-stranded linear DNA; ssDNA, single-stranded viral DNA.

Fig. 4

Southern blot analysis of antiviral effect of DoG on mutant HBV. HepG2 cells transfected with rtA181V (A), or rtN236T (B). HBV mutant plasmids were treated with DoG and 10 μmol/L3TC for 3 days. Viral core DNA was detected by Southern blot. Viral core DNA was quantified by Quantity One software (Bio-Rad) and the relative percentages compared to the negative control are presented. Representative results from two experiments are presented. rcDNA, relaxed circular; dslDNA, double-stranded linear DNA; ssDNA, single-stranded viral DNA.

Fig. 5

Sequencing of 3′ ends of encapsidated viral RNA caused by DoG. HepAD38 cells were treated with 10 μmol/L 3TC or 40 μmol/L DoG for 3 days. The poly-free viral RNA was collected, their 3′ termini were determined by a 3′-RACE procedure as previously described (Zhang et al.2016). The positions of the 3′ termini of these novel RNA species were confirmed by sequencing.