Comparative sequence analysis of duck and human hepatitis B virus genomes

Synthesis of the relaxed-circular (RC) DNA genome of hepadnaviruses requires two template switches during plus-strand DNA synthesis: primer translocation and circularization. Although primer translocation and circularization use different donor and acceptor sequences, and are distinct temporally, they share the common theme of switching from one end of the minusstrand template to the other end. Studies of duck hepatitis B virus have indicated that, in addition to the donor and acceptor sequences, three other cis-acting sequences, named 3E, M, and 5E, are required for the synthesis of RC DNA by contributing to primer translocation and circularization. The mechanism by which 3E, M, and 5E act was not known. We present evidence that these sequences function by base pairing with each other within the minus-strand template. 3E base-pairs with one portion of M (M3) and 5E base-pairs with an adjacent portion of M (M5). We found that disrupting base pairing between 3E and M3 and between 5E and M5 inhibited primer translocation and circularization. More importantly, restoring base pairing with mutant sequences restored the production of RC DNA. These results are consistent with the model that, within duck hepatitis B virus capsids, the ends of the minus-strand template are juxtaposed via base pairing to facilitate the two template switches during plus-strand DNA synthesis.H epadnaviruses are a family of DNA viruses that replicate via reverse transcription of an RNA intermediate (1). Like other reverse transcribing elements, hepadnaviruses use template switches or template exchanges for the synthesis of their genomes. Template switching is the process in which the strand of DNA being synthesized switches from its current template to a new template. For retroviruses, two template switches are required for the synthesis of retroviral DNA: the first and second strong stop template switches (2). These are referred to as replicative template switches. Retroviruses also perform a second type of template switching, called recombinogenic, between the two RNA templates or within a single RNA template during the synthesis of the minus-strand DNA (3, 4). However, these recombinogenic template switches, unlike the replicative template switches, are not obligatory steps in each cycle of reverse transcription. Similar to the replicative template switches in retroviruses, three template switches are needed for the synthesis of the relaxed-circular (RC) genome of hepadnaviruses: one during the synthesis of minus-strand DNA and two for plus-strand RC DNA synthesis. In general, replicative template switches involve translocation of the nascent DNA strand from one end to the other end of the template. Sequence identity at the donor and acceptor sites ensures that the nascent DNA strand elongates from the correct position on the new template.Reverse transcription of hepadnaviruses takes place within the cytoplasmic capsids in hepatocytes ( Fig. 1; for a review, see ref. 5). The first template switch occurs shortly after the initiation of min...

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“…All molecular clones were derived from DHBV strain 3 (18). The plasmids contain 1.5 copies of the genome, and support the synthesis of pgRNA.…”

Section: Methodsmentioning

confidence: 99%

Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription

Liu

Tian

Loeb

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes.…”

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confidence: 99%

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

1998

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Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma. 1 Although a tremendous amount is known about HBV, our knowledge of the virus is by no means complete. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, 2 and the woodchuck hepatitis virus which allows for the in vivo study in an animal model system. 3 The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro. However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. (1) Many HBV expressing cell lines were created using constructs containing strong heterologous promoters proximal to the HBV genome. The effect those promoters have on HBV transcription and replication is unclear but could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters. (2) Cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not integrated routinely but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal covalently closed circular (CCC) DNA molecules. 9 Although the integration of HBV DNA in human liver has been reported, 10 it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found,

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“…HBV may rather employ the ε structure to enhance the efficiency of its preC AUG codon. In fact, among members of hepadnavirus, none has a G at the j4 position of the preC AUG codon (Galibert et al, 1982 ;Seeger et al, 1984 ;Sprengel et al, 1985Sprengel et al, , 1988. However, for a virus with a compact genome organization, such an arrangement may occur fortuitously.…”

Section: Discussionmentioning

confidence: 99%

The encapsidation signal of hepatitis B virus facilitates preC AUG recognition resulting in inefficient translation of the downstream genes.

Hwang¹,

1999

Journal of General Virology

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Hepatitis B virus (HBV) DNA polymerase (P) is translated from a bicistronic pregenomic RNA via a ribosomal leaky scanning mechanism. Another viral transcript, the preC RNA, differs from pregenomic RNA by the presence of some 30 nt at the 5h end that encompass the preC initiation codon. This RNA is used exclusively for expression of the precore protein which is a precursor of secreted HBeAg. Factors leading to inefficient translation of the P and C proteins from the preC RNA were explored using a genetic approach in transient transfection assays. Our data indicate that when translating the precore protein, the elongation arrest that occurs during targeting of nascent polypeptide chains to the endoplasmic reticulum interferes with the scanning of the 40S ribosomal subunits. Such interference seems to hinder initiation of the ribosomes at the downstream genes. Furthermore, the presence of the preC initiator codon in the preC mRNA has resulted in a reduction in the number of scanning ribosomes reaching the C and P initiator codons compared with the case of pregenomic RNA. Finally, although the preC initiator codon is in a suboptimal context for translation initiation, an RNA secondary structure, the encapsidation signal, located downstream to the initiator codon is shown to enhance codon recognition, resulting in a depletion of the number of 40S ribosomal subunits available for scanning of the downstream AUG codons. This study demonstrates that the HBV encapsidation signal plays an additional role in facilitating recognition of the preC initiator codon.

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Comparative sequence analysis of duck and human hepatitis B virus genomes

Cited by 139 publications

References 31 publications

Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription

Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

The encapsidation signal of hepatitis B virus facilitates preC AUG recognition resulting in inefficient translation of the downstream genes.

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