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. 2001 Feb 13;98(4):1841-6.
doi: 10.1073/pnas.98.4.1841. Epub 2001 Feb 6.

Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism

H Tang  1 , A McLachlan
Affiliations

Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism

H Tang et al. Proc Natl Acad Sci U S A. .

Abstract

Hepatotropism is a prominent feature of hepatitis B virus (HBV) infection. Cell lines of nonhepatic origin do not independently support HBV replication. Here, we show that the nuclear hormone receptors, hepatocyte nuclear factor 4 and retinoid X receptor alpha plus peroxisome proliferator-activated receptor alpha, support HBV replication in nonhepatic cells by controlling pregenomic RNA synthesis, indicating these liver-enriched transcription factors control a unique molecular switch restricting viral tropism. In contrast, hepatocyte nuclear factor 3 antagonizes nuclear hormone receptor-mediated viral replication, demonstrating distinct regulatory roles for these liver-enriched transcription factors.

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Figures

Figure 1
Figure 1
Nuclear hormone receptors activate HBV replication in a nonhepatoma cell line. (A) Structure of the HBV DNA (4.1 kbp) construct used in transient transfection analysis. The 4.1-kbp greater-than-genome length HBV DNA sequence in this construct spans coordinates 1072–3182/1–1990 of the HBV genome (subtype ayw). The locations of the HBV 3.5-, 2.4-, 2.1-, and 0.7-kb transcripts are indicated. EnhI/Xp, enhancer I/X-gene promoter region; Cp, nucleocapsid or core promoter; pA, polyadenylation site; PS1p, presurface antigen promoter; Sp, major surface antigen promoter; X, X-gene; S, surface antigen gene; C, core gene; P, polymerase gene. (B–E) Cells were transiently transfected with the HBV DNA (4.1 kbp) construct and liver-enriched transcription factors. Mouse NIH 3T3 fibroblasts (3T3), human differentiated hepatoma cells (Huh7 and HepG2), and human dedifferentiated hepatoma cells (HepG2.1) were used for this analysis. (B and D) 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. (C and E) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. All-trans retinoic acid and clofibric acid at 1 μM and 1 mM, respectively, were used to activate the nuclear hormone receptors RXRα and PPARα (+lig).
Figure 2
Figure 2
HNF3 inhibits nuclear hormone receptor-activated HBV replication. (A–D) Mouse NIH 3T3 fibroblasts were transiently transfected with the HBV DNA (4.1 kbp) construct and liver-enriched transcription factors. (A and C) RNA (Northern) filter hybridization analysis of HBV transcripts. (B and D) DNA (Southern) filter hybridization analysis of HBV replication intermediates.
Figure 3
Figure 3
The proximal nucleocapsid HNF4 binding site is a major determinant of nuclear hormone receptor-mediated HBV replication in mouse fibroblasts. (A) Sequence of the HBV core promoter region. The 4-nt HNF4 site mutation indicated above the wild-type sequence inhibits the binding of nuclear hormone receptors to the proximal HNF4 binding site (results not shown). The nucleotide substitutions do not alter the X-gene polypeptide sequence. The HNF3 and Sp1 binding sites are also indicated. (B and C) Mouse NIH 3T3 fibroblasts were transiently transfected with HBV DNA (4.1 kbp) constructs and liver-enriched transcription factors. The HBV HNF4mut DNA (4.1 kbp) construct (lanes 6–10) contained the 4-nt mutation (A) in the proximal HNF4 binding site of the core promoter. Both core promoter regions in this terminally redundant HBV construct (Fig. 1A) were mutated for this analysis, but similar results were obtained when only the upstream core promoter region was mutated (results not shown). (B) RNA (Northern) filter hybridization analysis of HBV transcripts. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates.
Figure 4
Figure 4
Effect of HNF3 and mutation of the proximal nuclear hormone binding site in the core promoter on the transcription initiation site of the 3.5-kb HBV RNA. Mouse NIH 3T3 fibroblasts were transiently transfected with HBV DNA (4.1 kbp) constructs and liver-enriched transcription factors. The HBV HNF4mut DNA (4.1 kbp) construct (lanes 6–10) contained the 4-nt mutation (Fig. 3A) in the proximal HNF4 binding site of the core promoter. Both core promoter regions in this terminally redundant HBV construct (Fig. 1A) were mutated for this analysis. RNase protection analysis was performed to map the transcription initiation sites of the HBV precore (PC) and pregenomic or core (C) transcripts. The HBV probe also protected a fragment (pA) derived from the 3′ end of all of the HBV RNAs that terminated at the HBV polyadenylation site. The protected fragment indicated with an asterisk is generated as a result of the cleavage of the pA-protected fragment at the site of the discontinuity between the wild-type probe and the HNF4mut containing HBV RNA. A riboprobe detecting the ribosomal gene L32 transcripts was included as an internal control.
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