Alpha/beta interferon differentially modulates the clearance of cytoplasmic encapsidated replication intermediates and nuclear covalently closed circular hepatitis B virus (HBV) DNA from the livers of hepatocyte nuclear factor 1alpha-null HBV transgenic mice

Abstract

Treatment with alpha interferon is a standard therapy for patients with chronic hepatitis B virus (HBV) infections. This treatment can reduce virus load and ameliorate disease symptoms. However, in the majority of cases, alpha interferon therapy fails to resolve the chronic HBV infection. The reason alpha interferon therapy is inefficient at resolving chronic HBV infections is assumed to be because it fails to eliminate covalently closed circular (CCC) HBV DNA from the nuclei of infected hepatocytes. In an attempt to address this issue, the stability of HBV CCC DNA in response to alpha/beta interferon induction was examined in HNF1alpha-null HBV transgenic mice. Alpha/beta interferon induction by polyinosinic-polycytidylic acid [poly(I-C)] treatment efficiently eliminated encapsidated cytoplasmic HBV replication intermediates while only modestly reducing nuclear HBV CCC DNA. These observations indicate that nuclear HBV CCC DNA is more stable than cytoplasmic replication intermediates in response to alpha/beta interferon induction. Consequently it appears that for therapies to resolve chronic HBV infection efficiently, they will have to target the elimination of the most stable HBV replication intermediate, nuclear HBV CCC DNA.

FIG. 1.

Effect of poly(I-C) injection on serum HBeAg synthesis in HBV transgenic mice. HNF1(+/−), HNF1α-expressing HBV transgenic mice heterozygous for the wild-type HNF1α allele; HNF1(−/−), HNF1α-null HBV transgenic mice. Mice were injected with phosphate-buffered saline (PBS) or poly(I-C) (pIC). Serum HBeAg levels were measured before (pre) and after (post) injection (OD₄₅₀ nm). The mean HBeAg levels plus standard deviations derived from seven male HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven male HNF1α^+/− HBV transgenic mice injected with poly(I-C), four male HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, eight male HNF1α^−/− HBV transgenic mice injected with poly(I-C), seven female HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven female HNF1α^+/− HBV transgenic mice injected with poly(I-C), eight female HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, and nine female HNF1α^−/− HBV transgenic mice injected with poly(I-C) are shown. The levels of HBeAg in each group of HBV transgenic mice injected with poly(I-C) were decreased in a statistically significantly manner as determined by a paired Student t test (P < 0.05).

FIG. 2.

RNA (Northern) filter hybridization analysis of HBV and 2′,5′-oligoadenylate synthase transcripts in the livers of HBV transgenic mice. Groups of three representative mice of each sex and genotype are shown. (A) The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5- and 2.1-kb RNAs. The probes used were HBVayw genomic DNA plus glyceraldehyde-3-phosphate dehydrogenase cDNA. (B) The synthesis of the 2′,5′-oligoadenylate synthase (2OAS) transcript was used as an indirect measure of the induction of alpha/beta interferon by poly(I-C) injection. The probe used was the 2′,5′-oligoadenylate synthase cDNA. HNF1(+/−), HNF1α-expressing HBV transgenic mice heterozygous for the wild-type HNF1α allele; HNF1(-/-), HNF1α-null HBV transgenic mice. Mice were injected with phosphate-buffered saline (PBS) or poly(I-C) (pIC). (C) Quantitative analysis of the 2OAS transcript in HBV transgenic mice. The mean 2OAS transcript levels plus standard deviations derived from seven male HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven male HNF1α^+/− HBV transgenic mice injected with poly(I-C), four male HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, eight male HNF1α^−/− HBV transgenic mice injected with poly(I-C), seven female HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven female HNF1α^+/− HBV transgenic mice injected with poly(I-C), eight female HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, and nine female HNF1α^−/− HBV transgenic mice injected with poly(I-C) are shown. The levels of the 2OAS transcript in the HNF1α-null HBV transgenic mice are not statistically significantly different from their levels in the control HNF1α-expressing HBV transgenic mice as determined by a Student t test (P > 0.05).

FIG. 3.

RNase protection analysis mapping the transcription initiation sites of the precore (PC) and pregenomic (C) transcripts from the livers of HBV transgenic mice. (A) Groups of three representative mice of each sex and genotype are shown. The 3′ ends of the all the HBV transcripts corresponding to the polyadenylation site (pA) of these RNAs also generated a protected fragment in this analysis. The riboprobes used included the HBVayw sequence spanning nucleotide coordinates 1990 to 1658 and the mouse ribosomal protein L32 gene riboprobe spanning 101 nucleotides of exon 3. The 3.5-kb HBV RNAs protect fragments of 283 (pA), 206 (pC), and 175 (C) nucleotides, respectively. The mouse ribosomal protein L32 RNA protects a fragment of 101 nucleotides, designated L32, when probed with the L32 probe. HNF1(+/−), HNF1α-expressing HBV transgenic mice heterozygous for the wild-type HNF1α allele; HNF1(-/-), HNF1α-null HBV transgenic mice. Mice were injected with phosphate-buffered saline (PBS) or poly(I-C) (pIC). (B) Quantitative analysis of the HBV pregenomic RNA in HBV transgenic mice. The mean HBV pregenomic (C) RNA-to-L32 RNA ratio plus standard deviations derived from seven male HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven male HNF1α^+/− HBV transgenic mice injected with poly(I-C), four male HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, eight male HNF1α^−/− HBV transgenic mice injected with poly(I-C), seven female HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven female HNF1α^+/− HBV transgenic mice injected with poly(I-C), eight female HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, and nine female HNF1α^−/− HBV transgenic mice injected with poly(I-C) are shown. The lower HBV pregenomic RNA to L32 RNA ratios in the poly(I-C)-injected HBV transgenic mice are statistically significantly different from their ratios in the control phosphate-buffered saline injected HBV transgenic mice as determined by a Student t test (P < 0.05).

FIG. 4.

DNA (Southern) filter hybridization analysis of HBV DNA replication intermediates in the livers of HBV transgenic mice. (A) Groups of three representative mice of each sex and genotype are shown. The HBV transgene was used as an internal control for the quantitation of the HBV replication intermediates. The probe used was HBVayw genomic DNA. Tg, HBV transgene; RC, HBV relaxed circular replication intermediates; SS, HBV single-stranded replication intermediates; HNF1(+/−), HNF1α-expressing HBV transgenic mice heterozygous for the wild-type HNF1α allele; HNF1(-/-), HNF1α-null HBV transgenic mice. Mice were injected with phosphate-buffered saline (PBS) or poly(I-C) (pIC). (B) Quantitative analysis of the HBV DNA replicative intermediate (RI) levels in HBV transgenic mice. The mean HBV DNA replicative intermediate levels (values for replicative intermediates per transgene are indicated below the bar graph) plus standard deviations derived from seven male HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven male HNF1α^+/− HBV transgenic mice injected with poly(I-C), four male HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, eight male HNF1α^−/− HBV transgenic mice injected with poly(I-C), seven female HNF1α^+/− HBV transgenic mice injected with phosphate-buffered saline, seven female HNF1α^+/− HBV transgenic mice injected with poly(I-C), eight female HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, and nine female HNF1α^−/− HBV transgenic mice injected with poly(I-C) are shown. The lower levels of the HBV DNA replication intermediates in the poly(I-C)-injected HBV transgenic mice are statistically significantly different from the levels in the control phosphate-buffered saline injected HBV transgenic mice as determined by a Student t test (P < 0.05).

FIG. 5.

DNA (Southern) filter hybridization analysis of HBV CCC DNA replication intermediates and mitochondrial DNA in the livers of HNF1α-null HBV transgenic mice. (A) Groups of three representative mice of each sex are shown. The mitochondrial DNA was used as an internal control for the quantitation of the HBV CCC DNA. The probe used was HBVayw genomic DNA and a mouse mitochondrial DNA fragment. HBV CCC DNA, HBV covalently closed circular DNA; Mito DNA, mouse mitochondrial DNA. Mice were injected with phosphate-buffered saline (PBS) or poly(I-C) (pIC). (B) Quantitative analysis of the HBV CCC DNA replication intermediate levels in HNF1α-null HBV transgenic mice. The mean HBV CCC DNA replication intermediate levels plus standard deviations derived from four male HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, eight male HNF1α^−/− HBV transgenic mice injected with poly(I-C), eight female HNF1α^−/− HBV transgenic mice injected with phosphate-buffered saline, and nine female HNF1α^−/− HBV transgenic mice injected with poly(I-C) are shown. The lower levels of the HBV CCC DNA replication intermediates in the poly(I-C)-injected male but not the female HNF1α-null HBV transgenic mice are statistically significantly different from the levels in the control phosphate-buffered saline-injected HNF1α-null HBV transgenic mice as determined by a Student t test (P < 0.05).