PGC-1alpha controls hepatitis B virus through nutritional signals

Abstract

Hepatitis B virus (HBV) is a 3.2-kb DNA virus that replicates preferentially in the liver. Liver-enriched nuclear receptors (NRs) play a major role in the HBV life cycle, operating as essential transcription factors for viral gene expression. Notably, these NRs are also key players in metabolic processes that occur in the liver, serving as central transcription factors for key enzymes of gluconeogenesis, fatty acid beta-oxidation, and ketogenesis. However, the association between these metabolic events and HBV gene expression is poorly understood. Here we show that peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major metabolic regulator and a coactivator of key gluconeogenic genes, robustly coactivates HBV transcription. We further demonstrate that the liver-enriched NR hepatocyte nuclear factor 4alpha that binds HBV plays an important role in this process. Physiologically, we show that a short-term fast that turns on the gluconeogenic program robustly induces HBV gene expression in vivo. This induction is completely reversible by refeeding and depends on PGC-1alpha. We conclude that HBV is tightly regulated by changes in the body's nutritional state through the metabolic regulator PGC-1alpha. Our data provide evidence for nutrition signaling to control viral gene expression and life cycle and thus ascribe to metabolism an important role in virus-host interaction.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Overexpression of PGC-1α results in HBV coactivation. (A) A schematic representation of the liver-enriched NRs binding sites on EnhI and on EnhII of the HBV genome. RXR, retinoid X receptor; Cp, core promoter; PCp, precore promoter; Xp, X promoter. (B) HepG2 cells were seeded on 10-cm dishes and were transfected with 1.3x wtHBV plasmid (12 μg) together with increasing amounts (2–8 μg) of PGC-1α plasmid, with or without pSUPER PGC-1α plasmid. Three days after transfection, cells were harvested and RNA was analyzed by Northern blot for HBV transcripts. 18S and 28S rRNAs were analyzed for equal loading control. pg/pc, pregenomic and precore HBV RNAs; Surface, surface RNA; X, X RNA. (C) The same transfection protocol as in B was performed, this time with a HA-tagged PGC-1α construct. Three days after transfection, cells were harvested for protein analysis (Core, HBV core protein; IB, immunoblot). (D Upper) A schematic representation of the 1.3x HBV-Luc construct [P(A)S, poly (A) signal; Cp, core promoter; RV, EcoRV restriction site]. (D Lower) HepG2 cells were cotransfected with 1.3x HBV-Luc construct with the indicated plasmids. Sixty hours after transfection, cells were harvested and analyzed for luciferase activity. Results are presented as folds of activation after normalization to Renilla. The experiment was repeated three times in triplicate. HBc, HBV core protein; *, P < 0.05 (calculated by using Student's t test). (E) Huh7 cells were transfected with 10 μg of 1.3x wtHBV plasmid together with increasing amounts (1–6 μg) of PGC-1α plasmid with or without pSUPER PGC-1α (4–10 μg). Six days after transfection, cells were harvested and analyzed for HBV replicative-intermediates (RC, relaxed circular DNA; DL, double-stranded linear DNA; SS, single-stranded DNA).

Fig. 2.

Induction of the endogenous PGC-1α results in HBV coactivation. (A Left) HepG2 and HepG2 2215 cells were treated as indicated. Fourteen hours later, cells were harvested and RNA was analyzed by semiquantitative RT-PCR. (A Right) RNA from the same experiment was analyzed by real-time RT-PCR. (G2-HepG2, 2215-HepG2 2215 cell lines). (B) HepG2 cells were cotransfected with 1.3x wtHBV construct and the indicated plasmids. Twenty-four hours after transfection, treatment was performed as indicated. Fourteen hours later, cells were harvested and analyzed by semiquantitative RT-PCR. (C) HepG2 cells were cotransfected with 1.3xHBV-Luc construct and the indicated plasmids. After transfection (≈40 h), treatment was performed as indicated. Fourteen hours later, cells were harvested and analyzed for luciferase activity. The experiment was repeated three times in triplicate. Forsk, forskolin; Dex, dexamethasone.

Fig. 3.

Transcription coactivation of HBV by PGC-1α is mediated through HNF4α. (A) Chang cells were transfected with 10 μg of 1.3x wtHBV plasmid with or without HNF4α-expressing plasmid (200 ng) and PGC-1α-expressing plasmid (2–6 μg). Two days after transfection, cells were harvested and RNA was extracted and analyzed by Northern blot for HBV transcripts. HBV-transfected HepG2 cells were used as a positive control. Analysis of the GAPDH transcripts was used for equal loading control. pg/pc, pregenomic and precore HBV RNAs; le, long early RNA; Surface, surface RNA; X, X RNA. (B) Chang cells were transfected as indicated. Two days after transfection, cells were harvested for protein analysis. IB, immunoblot. (C) Chang cells were transfected with 1.3x HBV-Luc construct together with the indicated plasmids. Two days after transfection, cells were harvested and analyzed for luciferase activity. Results are presented as folds of activation after normalization to Renilla (open columns, no PGC-1α; filled columns, increasing amounts of PGC-1α).

Fig. 4.

Fasting-induced activation of HBV expression in vivo. (A) Mice were tail-injected with 6 μg of HBV-Luc plasmid. Forty-eight hours later, mice were divided into two groups: a control group (n = 5) that was allowed continuous free feeding and an experimental group (n = 8) that was subjected to a 7-h fast and to a subsequent 12-h refeeding. In vivo luciferase analysis of all mice was performed at baseline, after 7 h, and after an additional 12 h. Shown are the luciferase images of three representive animals from the experimental group at the indicated times. (B) A quantitative analysis of the experiment described in A. Red bars indicate the experimental (Exp) group and blue bars indicate the control (Cont) group. Error bars indicate SD. *, P = 0.002 (calculated by using Student's t test).

Fig. 5.

Fasting-induced coactivation of HBV in vivo is PGC-1α-dependent. (A) Semiquantitative RT-PCR analysis of total RNA extracted from livers of mice injected with the indicated constructs and subjected to either 14-h fast or normal feeding. (B) The same experiment described in Fig. 5A was performed. Total RNA extracted from mice livers was subjected to a Northern blot analysis. pg/pc, pregenomic and precore HBV RNAs; HBsAg, HBV surface antigen-encoding RNAs. (C) The same experiment described in Fig. 5A was performed. Protein was extracted and analyzed by Western blotting. IB, immunoblot. (D) A ChIP analysis of livers from either fasting or fed mice using either anti-PGC-1α antibody or only beads as a control. The immunoprecipitated chromatin was amplified by primers spanning the HNF4α-binding site region in EnhII (Right) or another unrelated region along the HBV genome (Left). Numbers on the arrows indicate nucleotide number (5′ to 3′, EcoRI site = nt 0) from which the primers originate. Cp, core promoter; PCp, precore promoter; RXRα, retinoid X receptor α; pX, X promoter.