Gut Online First, published on June 17, 2015 as 10.1136/gutjnl-2015-309809 Recent advances in basic science HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B Gut: first published as 10.1136/gutjnl-2015-309809 on 5 June 2015. Downloaded from Michael Nassal Correspondence to ABSTRACT frequent viral rebound upon therapy withdrawal Dr Michael Nassal, Department At least 250 million people worldwide are chronically indicates a need for lifelong treatment.6 of Internal Medicine II/ Molecular Biology, University infected with HBV, a small hepatotropic DNA virus that Reactivation can even occur, upon immunosuppres- Hospital Freiburg, Hugstetter replicates through reverse transcription. Chronic infection sion, in patients who resolved an acute HBV infec- Str. 55, Freiburg D-79106, greatly increases the risk for terminal liver disease. tion decades ago,7 indicating that the virus can be Germany; Current therapies rarely achieve a cure due to the immunologically controlled but is not eliminated. [email protected] refractory nature of an intracellular viral replication The virological key to this persistence is an intra- Received 17 April 2015 intermediate termed covalently closed circular (ccc) DNA. cellular HBV replication intermediate, called cova- Revised 12 May 2015 Upon infection, cccDNA is generated as a plasmid-like lently closed circular (ccc) DNA, which resides in Accepted 13 May 2015 episome in the host cell nucleus from the protein-linked the nucleus of infected cells as an episomal (ie, relaxed circular (RC) DNA genome in incoming virions. non-integrated) plasmid-like molecule that gives Its fundamental role is that as template for all viral rise to progeny virus. A cure of chronic hepatitis B RNAs, and in consequence new virions. Biosynthesis of will therefore require elimination of cccDNA. RC-DNA by reverse transcription of the viral pregenomic However, despite >30 years of research, little is RNA is now understood in considerable detail, yet known about the molecular mechanisms of conversion of RC-DNA to cccDNA is still obscure, cccDNA formation and degradation, foremostly foremostly due to the lack of feasible, cccDNA- due to the lack of suitable experimental systems. dependent assay systems. Conceptual and recent Recent discoveries are about to change this situ- experimental data link cccDNA formation to cellular DNA ation, particularly the identification of a liver- repair, which is increasingly appreciated as a critical resident bile acid transporter, sodium taurocholate interface between cells and viruses. Together with new cotransporting polypeptide (NTCP; also known as in vitro HBV infection systems, based on the SLC10A1), as an entry receptor for HBV and hepa- identification of the bile acid transporter sodium titis delta virus (HDV), which usurps HBV’s enve- taurocholate cotransporting polypeptide as an HBV entry lope to enter cells89(box 1). Various aspects of receptor, this offers novel opportunities to decipher, and this finding have recently been reviewed.10 11 eventually interfere with, formation of the HBV A second key for HBV persistence is a flawed http://gut.bmj.com/ persistence reservoir. After a brief overview of the role of immune response, typically including the functional cccDNA in the HBV infectious cycle, this review aims to exhaustion and depletion of cytotoxic T cells, a summarise current knowledge on cccDNA molecular lack of adequate CD4+ T cell help, and failure to biology, to highlight the experimental restrictions that mount neutralising antibodies. While immune res- have hitherto hampered faster progress and to discuss toration will likely be indispensable even if other cccDNA as target for new, potentially curative therapies ways are found to reduce cccDNA,12 for more of chronic hepatitis B. information readers are referred to pertinent on September 27, 2021 by guest. Protected copyright. – reviews.13 16 The focus here will be on a brief history on cccDNA research and its experimental fi INTRODUCTION dif culties, and on recent developments and how At least 250 million people worldwide are chronic- they may translate into new, curative treatments for ally infected with HBV1 and at a greatly increased chronic hepatitis B. risk to develop liver fibrosis, cirrhosis and hepato- cellular carcinoma, causing an estimated 650 000 HBV INFECTION AND REPLICATION: A SHORT deaths per year.2 While an efficient prophylactic OVERVIEW vaccine is available,3 current treatments for chronic HBV is the prototypic member of the hepadnaviri- hepatitis B are limited to type 1 interferons and five dae, a family of small enveloped hepatotropic DNA approved nucleos(t)ide analogues (NAs), which viruses sharing a similar genome organisation and target the viral polymerase, P protein, a multifunc- replication strategy. The mammalian animal viruses tional reverse transcriptase (see below). Due to (orthohepadnaviridae) include, for example, wood- severe side effects, only a fraction of patients are chuck hepatitis virus (WHV) and ground squirrel eligible for interferon therapy, and <10% of them hepatitis virus and, more recently discovered, HBVs show a sustained virological response, measured as of woolly monkeys17 and bats.18 Bird viruses (avi- loss of hepatitis B surface antigen (HBsAg; see hepadnaviridae) include, among others, those of below).4 NAs are much better tolerated, and the Pekin ducks (duck HBV (DHBV)) and heron HBVs. most potent drugs, entecavir and tenofovir, can However, no hepadnavirus has been found in estab- To cite: Nassal M. Gut – Published Online First: reduce viraemia by 5 6 logs, often below detection lished experimental animals such as mice, rats or [please include Day Month limit, and with low rates of viral resistance develop- chicken. Year] doi:10.1136/gutjnl- ment.5 However, HBsAg clearance is very rare As shown in figure 1A, HB virions,19 20 or ‘Dane 2015-309809 (0–5%) even after prolonged treatment,4 and the particles’21, comprise an outer envelope of the Nassal M. Gut 2015;0:1–13. doi:10.1136/gutjnl-2015-309809 1 Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Recent advances in basic science likely further host factors,32 triggers uptake (likely by endocyto- Box 1 Key role of HBV covalently closed circular (ccc) sis) of the virion. Notably, the PreS1 domain is also essential for DNA in viral persistence and chronic hepatitis B nucleocapsid envelopment, which is accounted for by its dual Gut: first published as 10.1136/gutjnl-2015-309809 on 5 June 2015. Downloaded from topology (figure 1A), with one part facing the virion interior and the other the virion surface.33 For the interaction with NTCP, Chronic hepatitis B, caused by persistent infection with HBV, about 50 aa from the PreS1 N terminus and N terminal fatty acyl- puts >250 million people at risk to develop terminal liver ation are sufficient (figure 1C), the basis for entry inhibition by disease. PreS1-derived lipopeptides (‘Myrcludex B’11). Following virion HBV persistence is mediated by an intranuclear, episomal form uptake, the RC-DNA containing nucleocapsids are released into of the viral genome called cccDNA. the host cell cytoplasm. The process is poorly understood but cccDNA is the template for viral RNAs and subsequent likely to yield to new in vitro infection systems based on generation of progeny virions. NTCP-transfected hepatoma cells. Exposure of nuclear localisa- A few copies of cccDNA per liver can (re)initiate full-blown tion signals in the Arg-rich C terminal domains (CTDs)34 of the infection. core protein (figure 1C), possibly regulated by CTD phosphoryl- cccDNA is not targeted by current treatments—but a cure of ation and/or completion of the incomplete (+)-strand in chronic hepatitis B will require elimination of cccDNA. RC-DNA, enables transport of the nucleocapsid to the nuclear Recent advances, including identification of a liver-specific HBV pore where the capsid shell disintegrates,35 releasing the viral receptor and evidence for HBV’s interaction with cellular DNA polymerase-bound RC-DNA into the nucleoplasm; there conver- damage repair, promise to greatly expand the limited sion into cccDNA and formation of a nucleosome-bound mini- knowledge on cccDNA biology. chromosome, likely associated with HBx and core protein, occur.36 As on cellular DNA, this provides numerous options for dynamic epigenetic control of cccDNA transcriptional activity, as lipid-embedded small (S), middle (M) and large (L) surface pro- highly schematically outlined in figure 3. These include DNA teins (HBsAg in serology) and an inner nucleocapsid (core par- modifications such as methylation, repressive and activating post- ticle; hepatitis B core antigen in serology) whose icosahedral translational modifications (PTMs) of the histones such as acetyl- – shell is formed by 120 dimers of the core protein.22 25 Its inter- ation, methylation, phosphorylation and others,37 nucleosome ior harbours the viral genome as a partially double-stranded, cir- spacing and likely more recently discovered mechanisms acting cular but not covalently closed ‘relaxed circular’ (RC) DNA in via non-coding RNAs38 or replacement of normal histones by which the 50 end of the (-)-strand is covalently linked to the specific variants.39 Notably, in the absence of HBx, cccDNA viral P protein26; formation of this unusual structure by protein- appears to be rapidly silenced, whereas HBx promotes a tran- primed reverse transcription is outlined below. scriptionally active state40 that correlates with the presence of The most remarkable features of the genome (figure 1B) are some of the known activating histone PTMs.41 42 However, the its tiny size (∼3 kb) and extremely compact organisation, with mechanism of HBx-mediated de-silencing is unclear, and the full each nucleotide (nt) having coding function in one or even two repertoire of potentially activating versus repressing regulation is http://gut.bmj.com/ (overlapping) open reading frames (ORFs); by necessity, all largely unexplored, as is the question of whether chromatinisa- regulatory elements for gene expression and numerous tion can already be initiated on RC-DNA.