Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription

Hirsch, Russell C.; Lavine, Joel E.; Chang, Lung‐Ji; Varmus, Harold; Ganem, Don

doi:10.1038/344552a0

Cited by 299 publications

(245 citation statements)

References 17 publications

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“…1B), as reflected by the presence of cytoplasmic capsids (Fig. 2) within which the enzymatic events required for viral replication are known to occur (32). Between days 1 and 4, there was also a decrease in circulating HBsAg and HBeAg (Fig.…”

Section: Discussionmentioning

confidence: 99%

Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection

Yang

Althage²,

Chung³

et al. 2002

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

347

320

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Hepatitis B virus (HBV) is a prototype for liver-specific pathogens in which the failure of the immune system to mount an effective response leads to chronic infection. Our understanding of the immune response to HBV is incomplete, largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models. We have developed a murine model for studying human HBV replication, immunogenicity, and control. After transfection of hepatocytes in vivo with a replicationcompetent, over-length, linear HBV genome, viral antigens and replicative intermediates were synthesized and virus was secreted into the blood. Viral antigens disappeared from the blood as early as 7 days after transfection, coincident with the appearance of antiviral antibodies. HBV transcripts and replicative intermediates disappeared from the liver by day 15, after the appearance of antiviral CD8 ؉ T cells. In contrast, the virus persisted for at least 81 days after transfection of NOD͞Scid mice, which lack functional T cells, B cells, and natural killer (NK) cells. Thus, the outcome of hydrodynamic transfection of HBV depends on the host immune response, as it is during a natural infection. The methods we describe will allow the examination of viral dynamics in a tightly controlled in vivo system, the application of mutagenesis methods to the study of the HBV life cycle in vivo, and the dissection of the immune response to HBV using genetically modified mice whose immunoregulatory and immune effector functions have been deleted or overexpressed. In addition, this methodology represents a prototype for the study of other known and to-be-discovered liver-specific pathogens.H epatitis B virus (HBV) is a human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). Although an effective vaccine has been available for two decades, an estimated 350 million people worldwide are chronically infected. A significant proportion of chronic infections terminate in hepatocellular carcinoma, leading to more than one million deaths annually (2).A reproducible tissue culture model of HBV infection does not exist, nor is HBV infectious for immunologically well-defined laboratory animals. Much of our current understanding of the viral life cycle after HBV infection is derived from studies of duck HBV (DHBV) (3) and woodchuck HBV (WHV) (4) infection in their natural hosts, HBV-infected chimpanzees (5-7), and HBV transgenic mice (8-10). For various reasons, however, none of these models is ideal. DHBV (11) and WHV (12, 13) are genetically divergent from HBV, and immunological studies in genetically outbred and immunologically uncharacterized ducks and woodchucks are difficult. Chimpanzee experiments are limited by cost, availability, and ethical considerations, whereas transgenic mice are immunologically tolerant to the virus, thereby compromising the greatest potential strength of a mouse model of HBV infection. We present here work describing a recently developed mouse model that alleviates many of thes...

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Section: Discussionmentioning

confidence: 99%

Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection

Yang

Althage²,

Chung³

et al. 2002

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

347

320

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“…The plasmids contain 1.5 copies of the genome, and support the synthesis of pgRNA. The molecular clone used to express the WT reference virus (p503-3) is null for P protein due to a frameshift mutation in the P gene (19). The molecular clone used to express WT P protein has been described previously (14).…”

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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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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“…This pregenomic RNA functions additionally as mRNA for the synthesis of two viral proteins, core protein and pol, which in turn interact with the package signal (termed ⑀) that appeared at the 5Ј-end of the pre-genomic RNA to initiate the RNA encapsidation process (49,50). Encapsidation of the RNA template is under stringent control, because only the pre-genomic RNA is selectively encapsidated.…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Mechanisms for Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatitis C Virus Core Protein

Chen¹,

Kao²,

Chen³

et al. 2003

Journal of Biological Chemistry

144

132

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We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg 39 -Arg 40 ) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.

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Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription

Cited by 299 publications

References 17 publications

Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection

Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection

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

Mechanisms for Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatitis C Virus Core Protein

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