Peroxisome proliferator-activated receptor gamma Coactivator 1alpha and small heterodimer partner differentially regulate nuclear receptor-dependent hepatitis B virus biosynthesis

Abstract

Hepatitis B virus (HBV) biosynthesis involves the transcription of the 3.5-kb viral pregenomic RNA, followed by its reverse transcription into viral DNA. Consequently, the modulation of viral transcription influences the level of virus production. Nuclear receptors are the only transcription factors known to support viral pregenomic RNA transcription and replication. The coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) have central roles in regulating energy homeostasis in the liver by modulating the transcriptional activities of nuclear receptors. Therefore, the effect of PGC1alpha and SHP on HBV transcription and replication mediated by nuclear receptors was examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells. This analysis indicated that viral replication mediated by hepatocyte nuclear factor 4alpha, retinoid X receptor alpha (RXRalpha) plus peroxisome proliferator-activated receptor alpha (PPARalpha), and estrogen-related receptor (ERR) displayed differential sensitivity to PGC1alpha activation and SHP inhibition. The effects of PGC1alpha and SHP on viral biosynthesis in the human hepatoma cell line Huh7 were similar to those observed in the nonhepatoma cells expressing ERRalpha and ERRgamma. This suggests that these nuclear receptors, potentially in combination with RXRalpha plus PPARalpha, may have a major role in governing HBV transcription and replication in this cell line. Additionally, this functional approach may help to distinguish the transcription factors in various liver cells governing viral biosynthesis under a variety of physiologically relevant conditions.

FIG. 1.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human hepatoma cell line Huh7. Cells were transfected with the HBV DNA (4.1-kbp) construct alone (lane 1) or with the HBV DNA (4.1-kbp) construct plus the PGC1α and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from four independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from four independent experiments. Trend lines were calculated using linear regression analysis.

FIG. 2.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing HNF4α. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the HNF4α expression vector (lane 1) or the HBV DNA (4.1-kbp) construct plus the HNF4α, PGC1α, and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from three independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from three independent experiments. Trend lines were calculated using linear regression analysis.

FIG. 3.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing RXRα/PPARα. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the RXRα and PPARα expression vectors (lane 1) or the HBV DNA (4.1-kbp) construct plus the RXRα, PPARα, PGC1α, and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from three independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from three independent experiments. Trend lines were calculated using linear regression analysis. All-trans-retinoic acid and clofibric acid at 1 μM and 1 mM, respectively, were used to activate the nuclear receptors RXRα and PPARα.

FIG. 4.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing ERRα. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the ERRα expression vector (lane 1) or the HBV DNA (4.1-kbp) construct plus the ERRα, PGC1α, and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from three independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from three independent experiments. Trend lines were calculated using linear regression analysis.

FIG. 5.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing ERRβ. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the ERRβ expression vector (lane 1) or the HBV DNA (4.1-kbp) construct plus the ERRβ, PGC1α, and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from three independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from three independent experiments. Trend lines were calculated using linear regression analysis.

FIG. 6.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing ERRγ. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the ERRγ expression vector (lane 1) or the HBV DNA (4.1-kbp) construct plus the ERRγ, PGC1α, and SHP expression vectors (lanes 2 to 16), as indicated. (A) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (B) Quantitative analysis of the 3.5-kb HBV RNA results from three independent experiments. Trend lines were calculated using linear regression analysis. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. (D) Quantitative analysis of the HBV replication intermediate results from three independent experiments. Trend lines were calculated using linear regression analysis.

FIG. 7.

Theoretical evaluation of the nuclear receptor combinations governing HBV biosynthesis in Huh7 cells. (A) Correlation coefficients were determined for optimal combinations of nuclear receptors based on their effects on viral replication in 293T cells compared with that in Huh7 cells in the presence of the different levels of the PGC1α and SHP coregulators. The combinations of nuclear receptors are reported in a descending order, with respect to their correlation coefficient values. Data used to determine the correlation coefficient values for RXRα/FXRα and LRH1 are included in the companion study (27a). 32% RXRα/PPARα plus 46% ERRα plus 22% ERRγ, r = 0.969; 37% RXRα/PPARα plus 57% ERRα plus 6% ERRβ, r = 0.968; 40% RXRα/PPARα plus 60% ERRα, r = 0.967; 49% ERRα plus 51% ERRγ, r = 0.962; 30% RXRα/PPARα plus 70% ERRγ, r = 0.957; 81% ERRα plus 19% ERRβ, r = 0.955; 11% RXRα/FXRα plus 89% ERRα, r = 0.954; 9% LRH1 plus 91% ERRα, r = 0.952; 5% HNF4α plus 95% ERRα, r = 0.952; 6% HNF4α plus 94% ERRγ, r = 0.952; 100% ERRα, r = 0.951; 100% ERRγ, r = 0.950; 100% RXRα/PPARα, r = 0.903; 100% ERRβ, r = 0.855; 100% LRH1, r = 0.761; 100% RXRα/FXRα, r = 0.682; 100% HNF4α, r = 0.585. (B) Example of the theoretical optimal best fit of the levels of viral replication obtained with RXRα/PPARα plus ERRα in 293T compared with that in Huh7 cells in the presence of the different levels of the PGC1α and SHP coregulators. Trend lines were calculated using linear regression analysis.