Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

Abstract

Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

Keywords: HepG2.RL1 cells; Hepatitis B virus; Lamivudine-resistant; N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-D- cytidine; rtL180M/M204V.

Fig. 1

Chemical structure of NCC. Molecular formula: C₁₂H₁₅FN₆O₄. Molecular weight: 326.28 g/mol.

Fig. 2

The PCR products of agarose gel electrophoresis analysis. (A) Full-length HBV genome amplified by PCR. Lane M: 250 bp DNA Ladder Marker; Lane 1: the full length HBV genome (3215 bp). (B) Gel purification of A, B, C and D PCR fragments. Lane M: DL2000 DNA Marker; Lane 1, 2, 3,4: gel purification of A (370 bp), B (1393) bp, C (1822 bp), and D (495 bp) PCR fragment, respectively. (C) Gel purification of A + B and C + D restriction enzyme digestion fragments. Lane M: 500 bp DNA Ladder Marker; Lane 1: gel purification of A + B fragment digested by Hind III and EcoR I; Lane 2: gel purification of C + D fragment digested by EcoR I and Not I. (D) Gel purification of double-enzyme digested pcDNA 3.1 plasmid. Lane M: DL15000 DNA Marker; Lane 1: gel purification of double-enzyme pcDNA 3.1 plasmid (5428 bp) digested by Hind III and Not I. (E) Restriction double-enzyme digestion identification of recombinant plasmid pcDNA3.1-1.3HBV by Hind III and Not I. Lane M: DL15000 DNA Marker; Lane 1: digestion of recombinant plasmid pcDNA3.1-1.3HBV by Hind III and Not I as pcDNA 3.1 plasmid (5428 bp) and A + B + C + D (4080 bp). (F) Enzyme digestion identification of recombinant plasmid pcDNA3.1-1.3HBV by Hind III. Lane M: DL15000 DNA Marker; Lane 1: digestion of plasmid pcDNA3.1by Hind III; Lane 2: digestion of recombinant plasmid pcDNA3.1-A + B by Hind III; Lane 3: digestion of recombinant plasmid pCDNA3.1-1.3HBV by Hind III.

Fig. 3

Construction of the pcDNA3.1-1.3HBV recombinant plasmid.

Fig. 4

Time-course of secretion of viral antigens and HBV DNA, and electron micrograph of HBV particles in the HepG2.RL1 cell line (A) Levels of HBeAg and HBsAg in the culture supernatants of the HepG2.RL1 cell line according to generation. OD_450/630, optical density at 450 nm and 630 nm. (B) Quantity of intracellular and extracellular DNA in HBV according to generation. (C) Shown here is a typical complex that includes some spherical HBsAg particles (diameter, 22 nm) (A) and a few Dane-like particles (42 nm) (B) (magnification 14,500×; bar: 200 nm; accelerating voltage: 160 kV).

Fig. 5

Drugs susceptibility to antigens secretion and viral replication. HepG2.2.15 cells and HepG2.RL1 cells were treated with lamivudine and adefovir for six days. Cell-culture supernatants were assayed using a specific ELISA kit. Viral DNA was extracted from the culture medium and cells, and then quantified by qPCR using a commercial kit. Experiments were done from wild-type and lamivudine-resistant HBV isolates, on the inhibitory effects of lamivudine (A) and adefovir (B) on HBeAg production; inhibitory efficiency of lamivudine (C) and adefovir (D) on extracellular production of HBV DNA; inhibitory efficiency of lamivudine (E) and adefovir (F) on intracellular production of HBV DNA. Mean values of inhibition from three independent measurements are shown as bars with standard-error values indicated at the top. **p < 0.01, *p < 0.05, compared with the no-drug control group. Black bar: wild-type HBV; gray bar: lamivudine-resistant HBV with rtL180M/M204V mutations.

Fig. 6

Effect of NCC on antigen secretion and viral replication. HepG2.2.15 cells and HepG2.RL1 cells were treated with the indicated concentrations of NCC for 6 days. HBeAg was tested using a specific ELISA kit (A). The intracellular DNA (B) and extracellular DNA (C) was quantified by qPCR using a commercial kit. Experiments on the inhibitory effects of NCC on HBeAg production and replication of HBV DNA from HepG2.2.15 cells (black bar) and HepG2.RL1 cells (gray bar) were performed in triplicate. Mean values of inhibition from three independent measurements are shown as bars with standard-error values indicated at the top. **p < 0.01, *p < 0.05, compared with the no-drug control group.