Vitamin D signaling inhibits HBV activity by directly targeting the HBV core promoter

Abstract

Clinical and epidemiological studies support a role for vitamin D in suppressing hepatitis B virus (HBV). This antiviral role of vitamin D is widely attributed to vitamin D receptor (VDR)/retinoid X receptor-mediated regulation of host immunomodulatory genes through vitamin D response elements (VDREs) in their promoters. Here, we investigated the ability of calcitriol (1α,25-dihydroxyvitamin D3, metabolically activated vitamin D) to directly regulate HBV activity through this signaling pathway. We observed that calcitriol selectively inhibited only the HBV core promoter without affecting the HBV-PreS1, HBV-PreS2/S, or HBx promoters. We then identified a VDRE cluster in the HBV core promoter that is highly conserved across most HBV genotypes. Disruption of this VDRE cluster abrogated calcitriol-mediated suppression of the HBV core promoter. Furthermore, we showed that VDR interacts directly with the VDRE cluster in the HBV core promoter independent of retinoid X receptor. This demonstrates that calcitriol inhibits HBV core promoter activity through a noncanonical calcitriol-activated VDR pathway. Finally, we observed that calcitriol suppressed expression of the canonical HBV core promoter transcripts, pregenomic RNA, and precore RNA in multiple HBV cell culture models. In addition, calcitriol inhibited the secretion of hepatitis B "e" antigen and hepatitis B surface antigen (HBV-encoded proteins linked to poor disease prognosis), without affecting virion secretion. Our findings identify VDR as a novel regulator of HBV core promoter activity and also explain at least in part the correlation of vitamin D levels to HBV activity observed in clinical studies. Furthermore, this study has implications on the potential use of vitamin D along with anti-HBV therapies, and lays the groundwork for studies on vitamin D-mediated regulation of viruses through VDREs in virus promoters.

Keywords: HBV core promoter; antiviral agent; cell signaling; gene regulation; hepatitis B virus; nuclear receptor; viral transcription; vitamin D; vitamin D receptor; vitamin D response element.

Figure 1

Calcitriol selectively suppresses the HBV core promoter in luciferase assays.A, each of the four HBV promoters (HBV-core, PreS1, PreS2/S, and HBx promoter) were cloned upstream of the luciferase reporter gene in the PGL3-basic construct. The activity of each of the promoters was tested in the presence of vehicle control (without calcitriol) or 10 nM calcitriol in hepatic cell lines, (B) HepG2, (C) Huh7, and (D) HepG2.2.15 by luciferase assay, 24 h after transfection. HBV core promoter activity was significantly supressed, whereas the activity of the remaining HBV promoters was unaffected in the presence of the ligand. All data are means ± SD for three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and NS (analyzed by paired Student's t test). HBV, hepatitis B virus; NS, not significant.

Figure 2

Identification of a VDRE cluster in the HBV core promoter that is conserved across most HBV genotypes.A, in silico analysis identified a cluster of overlapping VDREs on the sense strand (blue bars) in the HBV core promoter (1724–1748 in genotype “D,” highlighted with green box) in seven HBV genotypes (GT A, GT B, GT C, GT D, GT E, GT F, and GT H; nucleotide numbering may vary subtly across HBV genotypes). HBV genotype “G” was the exception with no VDREs in this region. HBV genotype “B” in addition has one VDRE on the antisense strand (orange bar). B, graphical representation of conservation of the HBV VDRE cluster within genotypes. The HBV core promoter sequence of genotypes A to H in the HBV database (n = 5757) was analyzed for the presence of VDREs. More than 94% of all sequences of seven HBV genotypes (GT A, GT B, GT C, GT D, GT E, GT F, and GT H) had three or more VDREs. Most of the sequences from HBV genotype “G” had no VDREs. C, majority of the positions of the 24-nucleotide region corresponding to the HBV VDRE cluster is highly conserved across HBV genotypes. All sequences from genotypes A to H in the HBV database (n = 5757) were analyzed for conservation using the HBV VDRE cluster of HBV genotype “D” (sequence on top). The height of each bar corresponds to the percentage of sequences with a conserved nucleotide at a given position when using the VDRE cluster from HBV genotype “D” as the reference sequence. This region was highly conserved across most HBV genotypes, with the exception of genotype “G”. ∗Majority (>85%) of the sequences in genotype “B” had an additional VDRE cluster (i.e., four VDREs in the cluster instead of three). HBV, hepatitis B virus; VDRE, vitamin D response element.

Figure 3

Mutations disrupting the HBV VDRE cluster abrogates calcitriol-mediated suppression of the HBV core promoter.A, a region of the HBV core promoter on which various nuclear receptor response elements (NRREs) have been mapped (green boxes), along with the identified overlapping cluster of putative VDREs (pVDREs, blue boxes bordered with orange). The high degree of overlap of the VDREs with other NRREs permits the alteration of only two base pairs (underlined). Mutations at these two positions (the yellow box shows the mutated core promoter sequence), performed as described in the Experimental procedures section, disrupt the sequence of the three VDREs in the cluster. Luciferase assays were performed to test the response of this mutated HBV core promoter (core mutant; with VDREs disrupted) as compared with the wildtype core promoter (core wildtype) in the presence of 10 nM calcitriol or vehicle control (without calcitriol) in (B) HepG2, (C) Huh7, and (D) HepG2.2.15 cells. Mutations disrupting the VDRE cluster abrogate the response of HBV core promoter to vitamin D signaling. All data are means ± SD for three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and NS (analyzed by paired Student's t test). COUP-TF1, chicken ovalbumin upstream promoter-transcription factor 1; HBV, hepatitis B virus; HNF3, hepatocyte nuclear factor 3; HNF4A, hepatocyte nuclear factor 4 alpha; NS, not significant; PPAR, proliferator–activated receptor; RXR, retinoid X receptor; TR4, testicular receptor 4; VDRE, vitamin D response element.

Figure 4

VDR binds to VDREs in the HBV core promoter independent of RXR.A, a fragment of the HBV core promoter carrying the identified VDRE cluster (capitalized) labeled with the 6-FAM fluorophore at the 5′ end of the probe. B, EMSA was performed using in vitro translated (IVT) VDR and RXR as detailed in the Experimental procedures section. Briefly, the 6-FAM-labeled probe was incubated with VDR and/or RXR and unlabeled competitor oligonucleotides when required and resolved on a native 8% polyacrylamide gel. VDR interacts with the VDREs in the HBV core promoter in the absence of RXR, as observed by the presence of the indicated band (see arrow) in lane 4. The addition of unlabeled competitor oligonucleotides reduced the complex formed in a concentration-dependent manner, demonstrating the specificity of the binding reaction (see lanes 6 and 7). Representative gel image from one of three separate experiments is shown. C, ChIP-quantitative PCR (qPCR) using anti-VDR antibody confirmed the increased binding of VDR at the HBV core promoter in HepG2 cells transfected with 1.3× HBV-genome construct in the presence of 10 nM calcitriol for 24 h. D, whereas, no enrichment of RXR was observed at the core promoter when ChIP-qPCR was performed with anti-RXR antibody in the presence of the ligand in similar conditions. The CYP24A1 promoter having established VDREs was used as a positive control, whereas the HBV PreS1 promoter having no putative VDREs was used as a negative control. All data are means ± SD for three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and NS (analyzed by paired Student's t test). 6-FAM, 6-carboxyfluorescein; HBV, hepatitis B virus; NS, not significant; RXR, retinoid X receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.

Figure 5

Calcitriol inhibits HBV core promoter transcripts, HBV secretory proteins, but not HBV virion secretion. The 1.3×-HBV genome construct was transfected in HepG2 and Huh7 cells, whereas HepG2.2.15 stably expresses HBV genome. In the HBV infection model, HBV particles derived from HepG2.2.15 cells were used to infect HepG2-NTCP cells as described in the Experimental procedures section. Vehicle control (without calcitriol) or 10 nM calcitriol was added immediately after transfection or infection, and samples were processed as described in the Experimental procedures section. RNA isolation and quantitation by quantitative PCR (qPCR) shows that (A) HBV pcRNA and (B) HBV pgRNA are suppressed in the presence of calcitriol. Quantitation of HBV secretory proteins by ELISA demonstrates that calcitriol negatively regulates (C) HBeAg and (D) HBsAg. E, finally, HBV virion secreted in supernatant was captured 72 h after ligand treatment, and its DNA was quantitated by qPCR. The virion secretion in the presence of calcitriol was marginally reduced in all HBV cell culture models (up to 12%) in vitro, though the observation was not statistically significant in any model. All data are means ± SD for three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and NS (analyzed by paired Student's t test). HBeAg, hepatitis B “e” antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; NS, not significant; NTCP, sodium taurocholate cotransporting polypeptide; pcRNA, precore RNA; pgRNA, pregenomic RNA.

Figure 6

Calcitriol inhibits HBV activity directlythroughthe vitamin D signaling pathway. The HBV core promoter (CP) transcribes the pgRNA and pcRNA. The pgRNA serves as the genetic template for the virus and is packaged into virus particles before secretion. The pcRNA is translated to the secretory HBeAg and then secreted via the endoplasmic reticulum (ER) (upper panel, see left to right). Calcitriol (metabolically activated vitamin D) binds and activates its nuclear receptor, VDR (see lower panel). We identified a VDRE cluster in the HBV core promoter. Calcitriol-activated VDR interacts with the identified VDREs in the HBV core promoter independent of RXR, suppressing its activity. This inhibits transcription of pgRNA and pcRNA. HBeAg and HBsAg secretion is also inhibited in the presence of the ligand. Numerous studies have highlighted the pathogenic function of HBeAg and HBsAg; hence, the vitamin D-mediated inhibition of these secretory proteins may be of clinical relevance. We did not observe significant inhibition in HBV virion secretion in the presence of vitamin D. HBV, hepatitis B virus; HBeAg, hepatitis B “e” antigen; HBsAg, hepatitis B surface antigen; pcRNA, precore RNA; pgRNA, pregenomic RNA; VDR, vitamin D receptor; VDRE, vitamin D response element.