Elsevier

Virus Research

Volume 134, Issues 1–2, June 2008, Pages 235-249
Virus Research

Hepatitis B viruses: Reverse transcription a different way

https://doi.org/10.1016/j.virusres.2007.12.024Get rights and content

Abstract

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is the type member of the Hepadnaviridae, hepatotropic DNA viruses that replicate via reverse transcription. Beyond long-established differences to retroviruses in gene expression and overall replication strategy newer work has uncovered additional distinctions in the mechanism of reverse transcription per se. These include protein-priming by the unique extra terminal protein domain of the reverse transcriptase (RT) utilizing an RNA hairpin for de novo initiation of first strand DNA synthesis, and the strict dependence of this process on cellular chaperones. Recent in vitro reconstitution systems enabled first biochemical insights into this multifactorial reaction, complemented by high resolution structural information on the RNA, though not yet the protein, level. Genetic approaches have revealed long-distance interactions in the nucleic acid templates as an important factor enabling the puzzling template switches required to produce the relaxed circular (RC) DNA found in infectious virions. Finally, the failure of even potent HBV RT inhibitors to eliminate nuclear covalently closed circular (ccc) DNA, the functional equivalent of integrated proviral DNA, has spurred a renewed interest in the mechanism of cccDNA generation. These new developments are in the focus of this review.

Introduction

Hepatitis B virus (HBV) is the prototypic member of the Hepadnaviridae, a family of hepatotropic DNA viruses infecting selected mammalian (orthohepadnaviruses) and avian (avihepadnaviruses) hosts. Despite the tiny size of its genome (about 3 kb) HBV is one of the most successful human pathogens, with an estimated 2 billion people having contracted the virus and 350 million or more being chronic carriers (WHO Fact sheet no. 204); chronic infection is correlated with a strongly increased risk for the development of severe, potentially lethal, liver disease (Ganem and Prince, 2004).

Soon after the discovery of HBV as causative agent of B-type hepatitis in humans (Blumberg, 1997), and of a related virus (DHBV) in ducks (Mason et al., 1980), it was recognized that the DNA in infectious hepadnavirions is generated by reverse transcription of an RNA intermediate (Summers and Mason, 1982).

Despite this kinship to retroviruses, there are various principal differences between hepadnaviral and retroviral replication (Nassal and Schaller, 1993, Nassal and Schaller, 1996). The most obvious are DNA synthesis in the initially infected producer cell and intracellular persistence of the hepadnavirus genome as an episomal covalently closed circular DNA (cccDNA) rather than as an integrated provirus; therefore hepadnaviruses, together with some plant viruses such as Cauliflower Mosaic Virus (CaMV), have been classified as pararetroviruses (Rothnie et al., 1994). Other distinctions include, beyond the much smaller genome size, generation of separate mRNAs by internal promoters rather than by splicing of a single LTR driven transcript; translation of the individual gene products as separate entities rather than as polyprotein precursors; consequently, the lack of virus encoded protease and integrase functions; and autonomously budding-competent envelope proteins, giving rise to massive amounts of secreted empty envelopes, or subviral particles, which constitute the bulk of the serologically important marker hepatitis B surface antigen (HBsAg) whereas, in contrast to Gag-particles (Demirov and Freed, 2004), membrane-enwrapped nucleocapsids cannot be released without envelope proteins.

However, the differences also extend into the actual mechanism of reverse transcription. Selective encapsidation of the pregenomic RNA (pgRNA) template into the icosahedral capsid (Wynne et al., 1999) is mediated by specific interactions with the viral reverse transcriptase (RT), called P protein, rather than by the capsid, or core, protein (Hirsch et al., 1990, Junker-Niepmann et al., 1990, Hirsch et al., 1991, Knaus and Nassal, 1993, Pollack and Ganem, 1993). In all likelihood, a single pgRNA plus a single P protein are packaged per particle (Bartenschlager and Schaller, 1992), hence hepadnaviruses lack a copy-choice mechanism as a means to increase genome diversity or to repair lethal defects from the previous replication round. Reverse transcription occurs inside intact nucleocapsids rather than in discernable preintegration complexes; although the HBV capsid shell displays high structural plasticity (Böttcher et al., 2006), only subtle differences have been detected between recombinant capsids from E. coli and virion-derived DNA genome containing nucleocapsids (Roseman et al., 2005).

Several of these established distinctions have previously been reviewed and the major conclusions are still valid (Nassal and Schaller, 1993, Nassal and Schaller, 1996, Nassal, 2000, Ganem and Schneider, 2001). Readers interested in other aspects of HBV biology are referred to a series of recent reviews covering the as yet poorly understood attachment and entry process (Glebe and Urban, 2007), intracellular transport (Kann et al., 2007), virus morphogenesis (Bruss, 2007), pathogenesis (Baumert et al., 2007) and therapy (Tillmann, 2007), as well as the two most pertinent animal model viruses, namely woodchuck hepatitis virus WHV (Menne and Cote, 2007) and in particular DHBV (Schultz et al., 2004) which has helped to unravel many of the mechanisms underlying hepadnavirus replication that could not, or only inadequately, be addressed with the human virus due to various experimental restrictions, including the lack of an experimental in vivo infection system other than chimpanzees.

At the heart of reverse transcription, recent research has uncovered major additional differences between retroviruses and hepadnaviruses. The first is initiation of DNA synthesis which in hepadnaviruses occurs by a unique protein-priming mechanism (Wang and Seeger, 1992, Wang and Seeger, 1993, Pollack and Ganem, 1994), reflected in the presence in P proteins of an extra terminal protein (TP) domain not found in any other RT. A second is the absolute chaperone dependence of P proteins for gaining template RNA binding and DNA synthesis competence. A third, possibly shared with retroviruses, is the emerging importance of long-distance interactions in the template nucleic acids that facilitate the puzzling template switches required to generate the mature relaxed circular (RC) DNA genome (Liu et al., 2003). Finally, there is a renewed interest in the mechanism of HBV cccDNA formation, spurred by the extreme longevity of this molecule even under treatment with effective RT inhibitors (Zhu et al., 2001, Michalak et al., 2004, Mulrooney-Cousins and Michalak, 2007). These issues are in the focus of the present article. As a necessary framework, we provide a very condensed and simplified outline of the hepadnaviral replication cycle in general; an in-depth treatment can be found in a recent review (Beck and Nassal, 2007) which also includes a comprehensive list of primary literature references.

Section snippets

A short outline of the hepadnaviral replication cycle

Basic features of the genome organization and infectious cycle of HBV are shown in Fig. 1; with some modifications they also apply to the avihepadnaviruses. Virions consist of a lipid envelope into which the three surface glycoproteins L (large), M (middle), and S (small) are embedded, and an inner nucleocapsid, or core, which harbors the 3 kb genome in the form of a partially double-stranded, non-covalently closed circular DNA, referred to as relaxed circular (RC) DNA. The long (−)-strand DNA

Hepadnaviral reverse transcription: the main actors

The principal viral actors in hepadnavirus replication are the pgRNA and its two translations products, P protein and core protein; in fact, artificial introduction of DHBV pgRNA into duck hepatocytes is sufficient to initiate infection (Huang and Summers, 1991). For HBV, the ɛ stem-loop alone can mediate encapsidation, including packaging of heterologous RNAs (Junker-Niepmann et al., 1990, Knaus and Nassal, 1993, Rieger and Nassal, 1995). In DHBV, the situation is more complex in that a second

Cis-elements for (−)-DNA completion, (+)-DNA synthesis and RC-DNA formation

Generation of the final product of hepadnaviral reverse transcription, RC-DNA, requires several template switches from one to the other end of the corresponding template. Intriguingly, all these events occur inside the restricted volume of intact nucleocapsids, with P protein continuously attached via its TP domain to the 5′ end of the (−)-DNA strand; hence after the first template switch from 5′ ɛ to 3′ DR*, about 3 kb of DNA are present in between the RT and the TP domain (Fig. 6A) when

First glimpses into RC to cccDNA conversion

Generation of cccDNA, structurally organized as a nucleosomal minichromosome (Bock et al., 2001), strictly requires RC-DNA as precursor. Hence it was hoped with the advent of potent RT inhibitors (see above) that blocking reverse transcription would also lead to cccDNA elimination. However, drops in viral load by several orders of magnitude are accompanied by an only extremely slow decline in cccDNA levels, and cccDNA may even persist, in a small percentage of hepatocytes, after apparent

Conclusions

The recent work summarized above has confirmed that, beyond long-established differences to retroviruses, hepadnaviruses have evolved their own genuine strategy of performing reverse transcription, with probably some mechanistic variation even between different members of the hepadnavirus family; examples are the activity of DHBV but not HBV P protein in in vitro reconstitution systems, and the differential use of cis-elements that control specific RNA encapsidation and conversion into RC-DNA.

Acknowledgements

Work cited from the author's lab has been or is supported by grants from the Deutsche Forschungsgemeinschaft (Na 154/4-3 and Na 154/7-2), and by the European Commission through the Virgil network (LSHM-CT-2004-503359) and the FSG-V-RNA (LSHG-CT-2004-503455) project. I am grateful to past and present lab members for their contributions, and to Dan Loeb and Megan Maguire for a private seminar on hepadnavirus cis-elements.

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