Duck Hepatitis B Virus Expresses a Regulatory HBx-Like Protein from a Hidden Open Reading Frame

Abstract

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho-and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.Since the identification of hepatitis B virus (HBV) in humans, several related viruses have been isolated from mammalian and avian species (9, 53, 68). These viruses, known as hepadnaviruses, display high liver tropism, have a narrow host range, and cause acute and chronic hepatitis. Chronic infection with mammalian hepadnaviruses (orthohepadnaviruses) is associated with the development of liver cancer.The hepadnaviruses are small enveloped DNA viruses with a unique virion ultrastructure. The viral genome is a partially duplexed, relaxed circular DNA molecule (rcDNA) with a size of about 3 kb which upon entry of the cell is converted into a covalently closed circular episome. This covalently closed circular DNA is then transcribed by the host RNA polymerase II, synthesizing subgenomic mRNAs and a greater-than-genomelength RNA known as pregenomic RNA or C-mRNA. Distinctive for all hepadnaviruses is the method of replication by reverse transcription of the pregenomic RNA into rcDNA. Transcripts encoding the envelope proteins, the nucleocapsid protein, the polymerase protein (P protein), and, as exclusively described so far in orthohepadnaviruses, the X protein have been identified. The organization of the viral genome is very compact, with overlapping reading frames and promoters and enhancer elements located within coding regions (20).The X proteins of orthohepadnaviruses affect signal transduction pathways, transcription, cell transformation, and proliferation (1,8,46,70). The HBV-specific X protein (HBx) is expressed in vivo, as shown by immunohistology and indirectly by identification of an HBx-specific immune response in infected individuals (64). In vivo expressio...

Avian hepatitis B viruses: Molecular and cellular biology, phylogenesis, and host tropism

“…DHBV lacks an apparent X-ORF. But a cryptic X-like ORF has recently been reported [22] . In vivo experiments have suggested no functional role for this gene product in DHBV short term infection [79] .…”

“…Regulatory sequences such as enhancers and promoters, as well as several cis-acting elements, overlap with coding regions. The first ORF encodes the surface proteins L and S, the second codes for the capsid protein as well as the e-antigen, the third for the polymerase and a cryptic fourth for the X protein [22] . Two different types of spherical viral particles can be detected in the serum of infected ducks: infectious virus particles (virions) with a diameter between 40 and 60 nm and subviral particles (SVPs) with a diameter between 30 and 60 nm, which lack the 27 nm nucleocapsid, including the viral genome [23] .…”

Hepatitis B virus-induced oncogenesis

“…HBx is a small polypeptide (17 kDa) that is produced at very low levels during chronic and acute hepatitis. Recently, a HBxlike regulatory protein was identified for duck hepatitis B virus (DHBV) [42] . Since the time when HBx initially was described to act as a transcriptional activator [43,44] , a variety of functions have been ascribed to the still enigmatic HBx [45,46] .…”

Establishment of mice model with human viral hepatitis B

“…At present, the animal models for HBV research included chimpanzee [11] , Tupaia Glis [12] and other hepadnaviridae virus infected animals [13] . Although duck hepatitis B virus (DHBV) has been found to be similar to HBV in mechanism of pathogenesis, ducks infected with DHBV did not develop hepatic cirrhosis and hepatocellular carcinoma because DHBV lacks X gene [14] . Mice Figure 6 Liver structure of mice by HE-staining (×200).…”