viruses Review Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation João Diogo Dias, Nazim Sarica and Christine Neuveut * Laboratoire de Virologie Moléculaire, Institut de Génétique Humaine, CNRS, Université de Montpellier, UMR9002 Montpellier, France; [email protected] (J.D.D.); [email protected] (N.S.) * Correspondence: [email protected] Abstract: Hepatitis B virus (HBV) remains a major public health concern, with more than 250 million chronically infected people who are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma. Although antiviral treatments efficiently control virus replication and improve liver function, they cannot cure HBV infection. Viral persistence is due to the maintenance of the viral circular episomal DNA, called covalently closed circular DNA (cccDNA), in the nuclei of infected cells. cccDNA not only resists antiviral therapies, but also escapes innate antiviral surveillance. This viral DNA intermediate plays a central role in HBV replication, as cccDNA is the template for the transcription of all viral RNAs, including pregenomic RNA (pgRNA), which in turn feeds the formation of cccDNA through a step of reverse transcription. The establishment and/or expression of cccDNA is thus a prime target for the eradication of HBV. In this review, we provide an update on the current knowledge on the initial steps of HBV infection, from the nuclear import of the nucleocapsid to the formation of the cccDNA. Keywords: HBV; HBVcccDNA; nuclear import; nuclear pore; DNA repair; DNA synthesis; HBV Citation: Diogo Dias, J.; Sarica, N.; cure; HBc Neuveut, C. Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation. Viruses 2021, 13, 757. https://doi.org/ 1. Introduction 10.3390/v13050757 The human hepatitis B virus (HBV) remains a major health threat with an estimated 250 million people chronically infected worldwide [1]. Epidemiological studies have Academic Editor: Francesca Di established that persistent HBV infection is a major risk factor for the development of Nunzio hepatocellular carcinoma (HCC), making HBV one of the most important environmental carcinogens for humans [2,3]. HBV is the prototype member of a family of small, en- Received: 29 March 2021 veloped DNA viruses called hepadnaviruses that preferentially infect hepatocytes. All Accepted: 23 April 2021 Published: 26 April 2021 hepadnaviruses have the ability to replicate their genomes in the cytoplasm through re- verse transcription of the encapsidated pregenomic RNA (pgRNA) by the viral reverse Publisher’s Note: MDPI stays neutral transcriptase (Pol; Figure1). This enzyme has DNA-dependent DNA polymerase and with regard to jurisdictional claims in RNA-dependent DNA polymerase activities and RNase H activity that generate a partially published maps and institutional affil- double-stranded DNA genome called relaxed circular DNA (rcDNA) [4]. iations. The 3.2 kb rcDNA is enclosed in an icosahedral nucleocapsid that is enveloped to form infectious particles. Upon infection, the rcDNA intermediate form is repaired so as to give rise to a complete double stranded covalently closed circular DNA (cccDNA) that is located in the nucleus and serves as a template for the transcription of all viral RNAs, including pgRNA [5,6]. In the cytoplasm, after reverse transcription, mature capsids containing Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. rcDNA assemble with the viral envelope at the cellular multivesicular body, allowing This article is an open access article for the formation and secretion of viral particles (Figure1)[ 7–9]. Alternatively, capsids distributed under the terms and can return directly to the nucleus, resulting in intracellular cccDNA amplification [10,11]. conditions of the Creative Commons The mechanism that directs the viral capsids towards either of these two pathways is still Attribution (CC BY) license (https:// unknown. Intracellular recycling appears early after infection, before virus secretion, for creativecommons.org/licenses/by/ the related virus duck hepatitis B virus [12]. It has thus been suggested that the fate of 4.0/). the capsids is controlled by the level of viral envelope proteins [13]. The establishment Viruses 2021, 13, 757. https://doi.org/10.3390/v13050757 https://www.mdpi.com/journal/viruses VirusesViruses2021 ,202113, 757, 13, x FOR PEER REVIEW 2 of 142 of 14 offate cccDNA of the capsids in the nucleusis controlled requires by the the level completion of viral envelope of different proteins steps, [13]. startingThe establish- with the attachmentment of cccDNA of the in viral the particlenucleus requires to the cell the surface,completion followed of different by the steps, delivery starting of with the nu- cleocapsidthe attachment into theof the cytoplasm, viral particle the to translocation the cell surface, of followed the rcDNA by the into delivery the nucleus, of the nu- and its repaircleocapsid to form into the the cccDNA. cytoplasm, The the exact translocation chronology of the of rcDNA the two into last the steps nucleus, is not and yet its fully understoodrepair to form and the may cccDNA. overlap, The as exact will bechronology discussed of below. the two Of last note, steps besides is not theyet secretionfully ofunderstood viral particles and thatmay consistoverlap, of as rcDNA-containingwill be discussed below. virions, Of note, different besides types the secretion of incomplete of particlesviral particles are released, that consist particularly of rcDNA-containing empty capsids virions, consisting different oftypes genome-free of incomplete enveloped par- capsidsticles are that released, are found particularly in infected empty humans capsids in consisting 100-fold orof moregenome-free excess enveloped [14]. These cap- empty sids that are found in infected humans in 100-fold or more excess [14]. These empty viral viral particles are currently the subject of research by different groups, as they represent a particles are currently the subject of research by different groups, as they represent a po- potential biomarker to monitor intrahepatic HBV activities and the response to antiviral tential biomarker to monitor intrahepatic HBV activities and the response to antiviral ther- therapies [15–17]. HBV RNA-containing capsids are also secreted as both non-enveloped apies [15–17]. HBV RNA-containing capsids are also secreted as both non-enveloped and andenveloped enveloped particles particles [18,19]. [18 ,19]. FigureFigure 1. Schematic 1. Schematic representation representation of theof the human human hepatitis hepatitis B virusB virus (HBV) (HBV) life life cycle. cycle. After After binding binding to to the the high high affinity affinity receptor re- ceptor sodium taurocholate co-transporting polypeptide (NTCP), HBV is internalized via endocytosis, and the viral capsid sodium taurocholate co-transporting polypeptide (NTCP), HBV is internalized via endocytosis, and the viral capsid is is released into the cytoplasm. The capsid travels to the nuclear pore using the microtubule network. The rcDNA is re- releasedleased into in the the nucleus cytoplasm. and is The converted capsid travelsinto covalently to the nuclear closed circular pore using DNA the (cccDNA), microtubule which network. becomes Thechromatinized, rcDNA is releasedand in theis nucleusthe template and for is convertedthe transcription into covalently of all HBV closedRNAs. circularIn the cytoplasm, DNA (cccDNA), pgRNA is which encapsidated becomes and chromatinized, retrotranscribed and into is the templatercDNA. for The the transcriptioncapsids are then of alleither HBV enve RNAs.loped In at the MVB cytoplasm, and secreted pgRNA as new is encapsidated virions, or transported and retrotranscribed back to the intonuclear rcDNA. Thepore capsids to increase are then the either pool of enveloped cccDNA. The at MVBsteps andof the secreted viral life ascycle new discussed virions, in or the transported review are delineated back to the in nuclearthe red box. pore to increase the pool of cccDNA. The steps of the viral life cycle discussed in the review are delineated in the red box. Current treatments for chronic hepatitis B efficiently control virus replication and improveCurrent liver treatments function, but for do chronic not lead hepatitis to complete B efficiently viral clearance, control and virus lifelong replication treatment and improveis required liver for function, these patients but do [20]. not Therapeutic lead to complete failure viralis due, clearance, in part, to and the lifelonginability treatmentof the isdrugs required to eliminate for these the patients cccDNA, [20 ].making Therapeutic this the failure key target is due, in order in part, to achieve to the inabilityHBV cure. of the Understanding HBV cccDNA biology from the entry of viral particles to the establishment drugs to eliminate the cccDNA, making this the key target in order to achieve HBV cure. of cccDNA, as well as its transcriptional regulation and persistence will allow for the iden- Understanding HBV cccDNA biology from the entry of viral particles to the establishment tification of new therapeutic targets to clear the viral reservoir, thus representing a very of cccDNA, as well as its transcriptional regulation and persistence will allow for the active field of investigation. In this review, we summarize and provide an update on early identification of new therapeutic targets to clear the viral reservoir, thus representing