Distinct regulation of hepatitis B virus biosynthesis by peroxisome proliferator-activated receptor gamma coactivator 1alpha and small heterodimer partner in human hepatoma cell lines

Abstract

The human hepatoma cell lines HepG2 and Huh7 have been used extensively to study hepatitis B virus (HBV) transcription and replication. Both cell lines support transcription of the 3.5-kb viral pregenomic RNA and subsequent viral DNA synthesis by reverse transcription. The effects of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) on HBV transcription and replication mediated by nuclear receptors were examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells in an attempt to determine the relative contribution of the various nuclear receptors to viral biosynthesis in the hepatoma cells. PGC1alpha and SHP modulated viral biosynthesis differently in the human hepatoma cell lines HepG2 and Huh7, indicating distinct modes of transcriptional regulation. Consistent with this suggestion, it appears that retinoid X receptor alpha/farnesoid X receptor alpha and liver receptor homolog 1 or estrogen-related receptor beta (ERRbeta) may contribute to the majority of the viral replication observed in HepG2 cells, whereas ERRalpha and ERRgamma are probably responsible for the majority of viral biosynthesis in Huh7 cells. Therefore, this approach indicates that the transcriptional regulation of HBV biosynthesis in HepG2 and Huh7 cells is primarily controlled by different transcription factors.

FIG. 1.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human hepatoma cell line HepG2. Cells were transfected with the HBV DNA (4.1-kbp) construct alone (lane 1) or 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 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. 2.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing RXRα/FXRα. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the RXRα and FXRα expression vectors (lane 1) or the HBV DNA (4.1-kbp) construct plus the RXRα, FXRα, 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 chenodeoxycholic acid at 1 μM and 100 μM, respectively, were used to activate the nuclear receptors RXRα and FXRα.

FIG. 3.

Effect of PGC1α and SHP expression on HBV biosynthesis in the human embryonic kidney cell line 293T expressing LRH1. Cells were transfected with the HBV DNA (4.1-kbp) construct plus the LRH1 expression vector (lane 1) or the HBV DNA (4.1-kbp) construct plus the LRH1, 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. 4.

Theoretical evaluation of the nuclear receptor combinations governing HBV biosynthesis in HepG2 cells. (A) Correlation coefficient values were determined for optimal combinations of nuclear receptors based on their effects on viral replication in 293T cells compared with that in HepG2 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 HNF4α, RXRα/PPARα, ERRα, ERRβ, and ERRγ are included in the companion study (18a). 7% HNF4α plus 46% RXRα/FXRα plus 37% LRH1 plus 10% ERRβ, r = 0.977; 8% HNF4α plus 47% RXRα/FXRα plus 47% LRH1, r = 0.977; 50% RXRα/FXRα plus 50% LRH1, r = 0.976; 16% HNF4α plus 42% RXRα/FXRα plus 42% ERRβ, r = 0.974; 52% RXRα/FXRα plus 48% ERRβ, r = 0.963; 100% RXRα/FXRα, r = 0.900; 100% LRH1, r = 0.888; 100% RXRα/PPARα, r = 0.872; 100% ERRβ, r = 0.865; 100% ERRγ, r = 0.817; 100% HNF4α, r = 0.809; 100% ERRα, r = 0.758. (B) Example of the theoretical optimal best fit of the levels of viral replication obtained with RXRα/FXRα plus LRH1 in 293T compared with that in HepG2 cells in the presence of the different levels of the PGC1α and SHP coregulators. Trend lines were calculated using linear regression analysis. (C) Comparison of the levels of viral replication observed in HepG2 cells compared with those observed in Huh7 cells in the presence of the different levels of the PGC1α and SHP coregulators. Trend lines were calculated using linear regression analysis.