Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants

Abstract

During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.

FIG. 1.

Schematic illustration of the replication-competent 1.28-fold-HBV vectors with restriction enzyme digestion sites. In the polymerase gene (striped), either wt or LMV resistance mutations (rtM204I or rtL180M+rtM204V) were present. In the BCP and the PC region (shaded), either wt sequences or the BCP (A1762T plus G1764A) or the PC (C1858T plus G1896A) mutation was introduced.

FIG. 2.

(A) Representative Northern blot analysis of HBV-specific RNA, 4 days after transient transfection with HBV wt or mutant constructs. Fifteen micrograms of total cellular RNA was loaded per lane. The Northern blot was also incubated with a probe directed against β-Gal RNA in order to control transfection efficiency. (B) 28S and 18S RNA of the same Northern blot. (C) Quantification of HBV-S RNA and pregenomic-PC RNA normalized to β-Gal RNA and 28S. Means and standard deviations are based on three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk. (D) Representative semiquantitative triplex RT-PCR with primer pairs detecting the HBV-S gene (and the overlapping polymerase gene), the HBV-C region (of the pregenomic-pC RNA), and GAPDH as a cellular housekeeping gene. Results after 18 cycles are shown where the PCR was calculated to be within the linear mode of amplification. (E) Quantification of the S product and the C product relative to GAPDH expression. Means and standard deviations are based on more than three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk. SM, single mutant (rtM204I); DM, double mutant (rtL180M/rtM204V); pBS, pBluescript plasmid; pg/pc RNA, pregenomic-PC RNA (3.5-kb band).

FIG. 2.

(A) Representative Northern blot analysis of HBV-specific RNA, 4 days after transient transfection with HBV wt or mutant constructs. Fifteen micrograms of total cellular RNA was loaded per lane. The Northern blot was also incubated with a probe directed against β-Gal RNA in order to control transfection efficiency. (B) 28S and 18S RNA of the same Northern blot. (C) Quantification of HBV-S RNA and pregenomic-PC RNA normalized to β-Gal RNA and 28S. Means and standard deviations are based on three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk. (D) Representative semiquantitative triplex RT-PCR with primer pairs detecting the HBV-S gene (and the overlapping polymerase gene), the HBV-C region (of the pregenomic-pC RNA), and GAPDH as a cellular housekeeping gene. Results after 18 cycles are shown where the PCR was calculated to be within the linear mode of amplification. (E) Quantification of the S product and the C product relative to GAPDH expression. Means and standard deviations are based on more than three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk. SM, single mutant (rtM204I); DM, double mutant (rtL180M/rtM204V); pBS, pBluescript plasmid; pg/pc RNA, pregenomic-PC RNA (3.5-kb band).

FIG. 3.

(A) Progeny DNA with dot blot analysis after immunoprecipitation of intracellular HBV capsids, 4 days after transient transfection with HBV wt or mutant constructs. For wt HBV, increasing amounts of transfected DNA (1, 3, and 5 μg) are shown; all other constructs were transfected with 5 μg. This experiment has been performed more than three times with consistent results; one representative dot blot analysis is shown. (B) Protein concentration of the cell lysates used for progeny DNA analysis as measured by Bio-Rad protein assay. (C) Quantification of HBV progeny DNA (per protein concentration of cell lysates). Means and standard deviations are based on more than three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk.

FIG. 4.

(A) Endogenous HBV DNA polymerase assay visualizing polymerase activity of intracellular encapsidated HBV, 4 days after transient transfection with 5 μg of HBV wt or mutant constructs. One representative experiment is shown. (B) Quantification of the HBV polymerase activity. Means and standard deviations are based on three independent experiments; values are given relative to wt. Significant differences compared with wt expression are indicated by an asterisk.

FIG. 5.

Quantification of HBV copy number from supernatant by quantitative real-time PCR, 4 days after transient transfection with HBV wt or mutant constructs and after PEG precipitation of secreted virions. Mean values and standard deviations from three independent experiments are shown. Values are normalized to HBV copy number of the wt construct (100%). Significant differences compared with wt expression are indicated by an asterisk.

FIG. 6.

Mean HBsAg (A) or HBeAg (B) concentrations (from six independent experiments) measured in the supernatant, 4 days after transient transfection with HBV wt or mutant constructs. Values are normalized to the HBsAg (A) or HBeAg (B) level of wt. Significant differences compared with wt expression are indicated by an asterisk.

FIG. 7.

(A) Drug sensitivity testing for LMV. Progeny DNA with dot blot analysis after immunoprecipitation of intracellular HBV capsids, 4 days after transient transfection with HBV wt or mutant constructs. Medium contained different LMV concentrations as indicated in the figure. One representative experiment is shown. (B) Quantification of HBV progeny DNA (per protein concentration of cell lysates) after incubation with 1 and 10 μM LMV. Means and standard deviations are based on three independent experiments; values are given relative to incubation without LMV. Differences between the wt-SM/PC-SM/BCP-SM or wt-DM/PC-DM/BCP-DM constructs did not reach statistical significance. (C) Quantification of HBV copy number from supernatant 4 days after transient transfection with HBV wt or mutant constructs and after PEG precipitation of secreted virions. Log fold reduction in HBV copy number due to 1 or 10 μM LMV condition compared with no treatment is shown, as are means and standard deviations based on three independent experiments.

FIG. 8.

(A) Drug sensitivity testing for ADV. The figure shows progeny DNA with dot blot analysis after immunoprecipitation of intracellular HBV capsids, 4 days after transient transfection with HBV wt or mutant constructs. Medium contained either 0 or 5 μM ADV. One representative experiment is shown. (B) Quantification of HBV copy number from supernatant, 4 days after transient transfection with HBV wt or mutant constructs and after PEG precipitation of secreted virions. Log fold reduction in HBV copy number in 5 μM ADV condition compared with no treatment is shown, as are means and standard deviations based on three independent experiments.