Cellular factors controlling the activity of woodchuck hepatitis virus enhancer II

Ueda, Keiji; Yu, Wei; Ganem, Don

doi:10.1128/jvi.70.7.4714-4723.1996

Cited by 13 publications

(15 citation statements)

References 71 publications

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“…Mapping studies have confirmed that WHV contains promoters analogous to the major HBV promoters (2,29). Recent studies have shown that WHV enhancer II is a strongly liver-specific enhancer that regulates the production of pregenomic RNAs, which is an important ratelimiting step of hepadnavirus replication (6,32). Surprisingly, the WHV region homologous to HBV enhancer I lacks enhancer activity in the three human liver cell lines tested (2,6,31).…”

mentioning

confidence: 95%

Woodchuck Hepatitis Virus Contains a Tripartite Posttranscriptional Regulatory Element

Donello

Loeb

Hope

1998

J Virol

325

126

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The hepatitis B virus posttranscriptional regulatory element (HBVPRE) is a cis-acting RNA element that partially overlaps with enhancer I and is required for the cytoplasmic accumulation of HBV surface RNAs. We find that the closely related woodchuck hepatitis virus (WHV), which has been shown to lack a functional enhancer I, also contains a posttranscriptional regulatory element (WPRE). Deletion analysis suggests that the WPRE consists of three independent subelements. Comparison of the bipartite HBVPRE and tripartite WPRE activities reveals that the tripartite WPRE is two to three times more active than the bipartite HBVPRE. Mutation of a single WPRE subelement decreases WPRE activity to the level of the HBVPRE. Bipartite and tripartite chimeras of the WPRE and HBVPRE possess activities which suggest that elements containing three subelements are posttranscriptionally stronger than those containing two. These data demonstrate that the posttranscriptional regulatory element is conserved within the mammalian hepadnaviruses and that its strength is determined by the number of subelements within the RNA.

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confidence: 95%

Woodchuck Hepatitis Virus Contains a Tripartite Posttranscriptional Regulatory Element

Donello

Loeb

Hope

1998

J Virol

325

126

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“…Our studies of viral DNA sequences in N-myc2 activation suggested that the region corresponding to HBV EnII played a major role in the activation (22). Accordingly, we and others have mapped and characterized EnII in WHV (3,23). The activity of WHV EnII maps to an 88-nucleotide DNA fragment (nucleotides 1772 to 1859) upstream from the transcription initiation site of pgRNA.…”

mentioning

confidence: 79%

“…, and EnIIABC(Ϫ)E1bCAT were constructed by cloning the insert fragments of pW1697-1919WT, pW1697-1919IIA(Ϫ), pW1697-1919IIB(Ϫ), pW1697-1919IIC (Ϫ), pW1697-1919IIAB(Ϫ), pW1697-1919IIAC(Ϫ), pW1697-1919IIBC(Ϫ) and pW1697-1919IIABC(Ϫ) into the XhoI and XbaI sites in E1bCAT, which has been described elsewhere (23).…”

Section: Methodsmentioning

confidence: 99%

“…The fragments used as probes for electrophoretic mobility shift assay (EMSA) were generated by PCRs in which pW1697-1919WT, -IIA(Ϫ), -IIB(Ϫ), -IIC(Ϫ), -IIAB(Ϫ), IIAC(Ϫ), -IIBC(Ϫ), and -IIABC(Ϫ) were amplified with Taq polymerase and primers with the sequences 5Ј-CTTTGTCTCCTGGC-3Ј (nucleotides 1760 to 1773) and 5Ј-GCCCTCCTCCCATT-3Ј (nucleotides 1859 to 1846). The fragments (spanning nucleotides 1760 to 1859) were then labeled with T4 polynucleotide kinase and [ 32 P]ATP and incubated with nuclear extracts from HepG2 cells as previously described (23).…”

Section: Methodsmentioning

confidence: 99%

“…The activity of WHV EnII maps to an 88-nucleotide DNA fragment (nucleotides 1772 to 1859) upstream from the transcription initiation site of pgRNA. Biochemical and genetic studies have identified three host transcription factors that recognize elements within this region: the liver-enriched factors HNF-1 and HNF-4 and the ubiquitous factor Oct-1 (23). Deletion analyses suggested that HNF-1 and HNF-4 are the main contributors to the EnII activity in transient assays and together account for its strong liver specificity.…”

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confidence: 99%

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In Vivo Effects of Mutations in Woodchuck Hepatitis Virus Enhancer II

Yu¹,

Tennant

Ganem

1998

J Virol

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Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-myc2gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.

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