Anneke K. Raney

Nuclear Covalently Closed Circular Viral Genomic DNA in the Liver of Hepatocyte Nuclear Factor 1α-Null Hepatitis B Virus Transgenic Mice

ABSTRACT The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1α-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1α transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1α protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1α-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1α-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.

Replication of the Wild Type and a Natural Hepatitis B Virus Nucleocapsid Promoter Variant Is Differentially Regulated by Nuclear Hormone Receptors in Cell Culture

ABSTRACT A natural hepatitis B virus (HBV) variant associated with seroconversion from HBeAg to anti-HBe antibody contains two nucleotide substitutions (A1764T and G1766A) in the proximal nuclear hormone receptor binding site in the nucleocapsid promoter. These nucleotide substitutions prevent the binding of the retinoid X receptor α (RXRα)–peroxisome proliferator-activated receptor α (PPARα) heterodimer without greatly altering the efficiency of binding of hepatocyte nuclear factor 4 (HNF4) to this recognition sequence. In addition, these nucleotide substitutions create a new binding site for HNF1. Analysis of HBV transcription and replication in nonhepatoma cells indicates that RXRα-PPARα heterodimers support higher levels of pregenomic RNA transcription from the wild-type than from the variant nucleocapsid promoter, producing higher levels of wild-type than of variant replication intermediates. In contrast, HNF4 supports higher levels of pregenomic RNA transcription from the variant than from the wild-type nucleocapsid promoter, producing higher levels of variant than of wild-type replication intermediates. HNF1 can support variant virus replication at a low level but is unable to support replication of the wild-type HBV genome. These observations indicate that the replication of wild-type and variant viruses can be differentially regulated by the liver-specific transcription factors that bind to the proximal nuclear hormone receptor binding site of the nucleocapsid promoter. Differential regulation of viral replication may be important in the selection of specific viral variants as a result of an antiviral immune response.

Characterization of the hepatitis B virus- and nucleocapsid gene transcriptional regulatory elements

The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (-239) and 1238 (-72) increases transcription from the X-gene promoter located between coordinates 1239 (-71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (-193) and 1204 (-106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (-88) and 1238 (-72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (-71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter

It has been demonstrated that the hepatocyte nuclear factor 1 (HNF1) binding site is critical for the majority of the hepatitis B virus (HBV) large surface antigen promoter activity in differentiated hepatoma cell lines. Examination of a series of clustered point mutations in the minimal large surface antigen promoter demonstrated that the HNF1 and TATA box binding sites are the major regulatory elements required for transcription from this promoter. Synthetic promoter constructs containing the large surface antigen promoter HNF1 binding site and TATA box element upstream of the luciferase open reading frame were tested for their transcriptional activities in HepG2. 1 cells in the absence or presence of an HNF1 expression vector. These synthetic promoter constructs displayed a similar level of transcriptional activity and induction by HNF1 in comparison with the full-length large surface antigen promoter, suggesting that additional HBV sequences are dispensable for full transcriptional activity. The distance between the HNF1 binding site and TATA box element in the synthetic promoter constructs appeared to influence the transcriptional activity modestly and in a periodic manner.

Characterization of the hepatitis B virus major surface antigen promoter hepatocyte nuclear factor 3 binding site

Transcription of the HBV 2.1 kb RNAs is regulated by the major surface antigen promoter. Previously, transient transfection analysis identified regulatory sequence elements in this promoter located between -189 and +1 which govern the level of transcription from this promoter and appear to bind only ubiquitous transcription factors including NF1, Sp1 and NF-Y. However, in vivo transcription analysis in transgenic mice has demonstrated that the expression of the HBV 2.1 kb RNAs is largely restricted to hepatocytes. In this study, the presence of a functional HNF3 transcription factor binding site located between -231 and -240 in the major surface antigen promoter suggests that the in vivo liver-restricted expression of the 2.1 kb RNAs may be governed by this liver-enriched transcription factor. The identification of a functional HNF3 binding site upstream of the DNA polymerase open reading frame also supports the contention that transient transfection analysis may fail to detect all of the cis-acting regulatory sequence elements involved in modulating the level of transcription from the viral promoters.