cells Review Intracellular Trafficking of HBV Particles Bingfu Jiang 1 and Eberhard Hildt 1,2,* 1 Department of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; [email protected] 2 German Center for Infection Research (DZIF), TTU Hepatitis, Marburg-Gießen-Langen, D63225 Langen, Germany * Correspondence: [email protected]; Tel.: +49-61-0377-2140 Received: 12 August 2020; Accepted: 2 September 2020; Published: 2 September 2020 Abstract: The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles. Keywords: hepatitis B virus; trafficking; entry; morphogenesis; release 1. Introduction The human hepatitis B virus (HBV) belongs to the family of Hepadnaviridae, a group of small hepatotropic DNA viruses. HBV is an enveloped virus. The three viral surface proteins are LHB (large hepatitis B virus surface protein), MHB (middle hepatitis B virus surface protein) and SHB (small hepatitis B virus surface protein) that are integral membrane proteins embedded into the membrane. The membrane surrounds the nucleocapsid. The nucleocapsid is assembled by the core protein (HBc) and harbors the viral DNA genome. The viral genome is a partially double-stranded DNA with a size of about 3.2 kb. This very compact genome encompasses four overlapping open reading frames (ORFs) encoding the viral polymerase (P), the capsid forming core protein (HBc) and its secretory variant HBeAg, the regulatory protein HBx, and the three envelope proteins—LHBs, MHBs and SHBs [1–3]. In addition to being part of the infectious viral particle, the surface proteins can also form non-infectious subviral particles (SVPs) that lack capsid and genome. The SVPs exist as spheres with a diameter of 22 nm and filaments with a variable length between 80 and 400 nm (Figure1). Cells 2020, 9, 2023; doi:10.3390/cells9092023 www.mdpi.com/journal/cells Cells 2020, 9, 2023 2 of 21 Cells 2020, 9, x FOR PEER REVIEW 2 of 21 Figure 1. Schematic presentation of HBV viral particle, subviral filament and sphere. Figure 1. Schematic presentation of HBV viral particle, subviral filament and sphere. TheThe HBV HBV envelope envelope proteins, proteins, that that are are also also term termeded HBVHBV surface surface antigens antigens (HBsAg), (HBsAg), are are encoded encoded by aby single a single open open reading reading frame frame that that is is divided divided by by three inin frameframe standing standing start start codons codons in thein the PreS1, PreS1, the thePreS2 PreS2 and and the the S region. S region. The The three three surface surface protei proteinsns share thethe S-domain S-domain with with 226 226 amino amino acids acids (aa), (aa), which forms SHBs. MHBs contains an additional PreS2 domain with 55 aa, while LHBs encompasses which forms SHBs. MHBs contains an additional PreS2 domain with 55 aa, while LHBs encompasses the PreS2 domain and the PreS1 domain with a length of 108, 118 or 119 aa depending on the the PreS2 domain and the PreS1 domain with a length of 108, 118 or 119 aa depending on the genotype. The HBV surface proteins are integral membrane proteins anchored by the S-domain genotype.in the membrane.The HBV surface SHBs is proteins inserted byare various integral transmembrane membrane proteins (TM) regions anchored in the by ER the membrane. S-domain in the Themembrane. first ER transmembraneSHBs is inserted region by various (TM1) encompasses transmembrane aa 8–22 (TM) [4], regions which is in followed the ER bymembrane. a cytosolic The firstloop ER transmembrane aa 23–79. The second region transmembrane (TM1) encompasses domain (TM2)aa 8–22 (aa [4], 80–98) which directs is followed the luminal by a exposure cytosolic of loop aa 23–79.aa 99–169 The containingsecond transmembrane the major conformational domain (TM2) epitope (aa of80–98) HBsAg directs (a-determinant the luminal aa exposure 124–147) andof aa a 99– 169 N-glycosylationcontaining the sitemajor [5]. Theconformational detailed structure epitope of the of following HBsAg C-terminal(a-determinant sequence aa 124–147) has not yet and been a N- glycosylationstudied experimentally, site [5]. The but detailed it has beenstructure considered of the as following one or two C-terminal transmembrane sequence regions has (TM3 not/4) yet [5,6 been]. studiedMHBs experimentally, shows no difference but it in has the topologybeen consider of its S-domained as one as or compared two transmembrane to SHBs. The PreS2regions domain (TM3/4) [5,6].of MHBs MHBs shows directs inno thedifference ER lumen in andthe istopology N-glycosylated of its S-domain at Asn-4 [as7]. compared The N-terminus to SHBs. of LHBs The PreS2 is domainsubjected of MHBs to myristoylation. directs in the Integrity ER lumen of the and myristoylation is N-glycosylated site is crucialat Asn-4 for [7]. the infectivityThe N-terminus of HBV of [8, LHBs9]. is subjectedWith respect to myristoylation. to the PreS1PreS2 Integrity domain, of LHBs the ismyristoylation characterized by site a dual is crucial topology: for the The infectivity TM1 of nascent of HBV [8,9].LHBs With does respect not functionto the PreS1PreS2 as a co-translational domain, LHBs translocation is characterized signal sequence, by a dual leading topology: to the cytosolicThe TM1 of exposure of the PreS1PreS2 domain and S-domain up to aa 79. However, due to a posttranslational nascent LHBs does not function as a co-translational translocation signal sequence, leading to the translocation, about 50% of TM1 are integrated in the ER membrane. In this case, the PreS1PreS2 cytosolic exposure of the PreS1PreS2 domain and S-domain up to aa 79. However, due to a domain and the first 7 aa of the S-domain face the lumen of the ER, [10–12]. The PreS-domain directing posttranslationalin the ER lumen translocation, (e-PreS) is exposed about on50% the of viral TM1 surface are integrated and mediates in the the virus–cellER membrane. interaction In this [13 case,], the whilePreS1PreS2 the one domain facing the and cytosol the first (i-PreS) 7 aa hasof the been S-do postulatedmain face to the interact lumen with of thethe nucleocapsid ER, [10–12]. duringThe PreS- domainviral directing envelopment in the [14 ].ER Moreover, lumen (e-PreS) the PreS2 is domainexposed facing on the the viral cytoplasm surface acts and as mediates a regulatory the proteinvirus–cell interactionthat binds [13], and while activates the classic one isoformsfacing the of PKCcytosol that (i leads-PreS) to anhas activation been postulated of c-Raf-dependent to interact signaling with the nucleocapsidcascades. For during more viral information, envelopment readers [14]. are referredMoreover, to pertinent the PreS2 reports domain [15, 16facing]. the cytoplasm acts as a regulatoryThe HBV protein core protein that (HBc,binds capsid and activates protein) contains classic genotype-dependentisoforms of PKC that 183 leads or 185 to aa. an The activation primary of c-Raf-dependentstructure of HBc signaling can be divided cascades. into anFor N-terminal more inform domaination, (NTD, readers also designatedare referred as assemblyto pertinent domain) reports [15,16].and a C-terminal domain (CTD), which are connected by a linker region. The NTD consists of the firstThe N-terminal HBV core 149 protein or 151 (HBc, aa (depending capsid protein) on the genotype), contains whichgenotype-dependent are sufficient for the183 self-assembly or 185 aa. The α primaryof viral structure capsids [17of –HBc19]. Thecan structure be divided of the into core an polypeptide N-terminal is characterizeddomain (NTD, by aalso highly designated-helical as fraction. Two α-helices (α3, α4) connected by a loop constitute the central domain of a monomer. It is assembly domain) and a C-terminal domain (CTD), which are connected by a linker region. The NTD assumed to be linked by a disulfide bridge, the stem constitutes the dimer interface and exposes the consists of the first N-terminal 149 or 151 aa (depending on the genotype), which are sufficient for spikes on the outer surface of the icosahedral capsid [20,21]. However, there is evidence that fully the self-assembly of viral capsids [17–19]. The structure of the core polypeptide is characterized by a highly α-helical fraction. Two α-helices (α3, α4) connected by a loop constitute the central domain of a monomer. It is assumed to be linked by a disulfide bridge, the stem constitutes the dimer interface and exposes the spikes on the outer surface of the icosahedral capsid [20,21]. However, there is evidence that fully oxidized dimers assemble slower into capsids as compared to the reduced dimers. Apart from the velocity of capsid formation, the stability of the formed capsid is affected by the Cells 2020, 9, 2023 3 of 21 oxidized dimers assemble slower into capsids as compared to the reduced dimers.