0000000000217450
AUTHOR
Reinhild Prange
Deletions in the hepatitis B virus small envelope protein: effect on assembly and secretion of surface antigen particles
The small envelope S protein of hepatitis B virus carrying the surface antigen has the unique property of mobilizing cellular lipids into empty envelope particles which are secreted from mammalian cells. We studied the biogenesis of such particles using site-directed mutagenesis. In this study, we describe the effect of deletions in the N-terminal hydrophobic and hydrophilic domains of the S protein. Whereas short overlapping deletions of hydrophilic sequences flanking the first hydrophobic domain were tolerated, larger deletions of the same sequences were not. Conversely, the hydrophilic region preceding the second hydrophobic domain was not permissive for even short deletions. Deletion of…
Sequence-Specific Repression of Cotranslational Translocation of the Hepatitis B Virus Envelope Proteins Coincides with Binding of Heat Shock Protein Hsc70
AbstractThe large L envelope protein of the hepatitis B virus has the peculiar capacity to adopt two transmembrane topologies. The N-terminal preS domain of L initially remains in the cytosol while the S domain is cotranslationally inserted into the endoplasmic reticulum membrane. The preS region of about half of the L molecules is posttranslationally translocated to the lumenal space. We now demonstrate that the repression of cotranslational translocation of preS is conferred by a preS1-specific sequence. By analysis of L deletion mutants, the cytosolic anchorage determinant was mapped to amino acid sequence 70 to 94 of L. The intrinsic potential of this determinant to suppress cotranslati…
DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response.
In order to design optimized DNA vectors as genetic vaccines against infections with the hepatitis B virus (HBV) we investigated if secretion or retention of the viral antigens has an influence on the quality and quantity of the humoral immune response. Intramuscular injection of plasmid DNA encoding the HBV large L envelope protein, known to be retained within host cells, induced only a weak response in mice whereas a vector expressing the secretion-competent small S envelope protein elicited strong and sustained immunity. Immunization with rearranged envelope genes further demonstrated that secretion affects the magnitude of the immune response. In situ expression of modified small and mi…
γ2-Adaptin, a Ubiquitin-interacting Adaptor, Is a Substrate to Coupled Ubiquitination by the Ubiquitin Ligase Nedd4 and Functions in the Endosomal Pathway
gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-adaptin. By mapping regions of Nedd4 involved in binding to gamma2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of ga…
Hepatitis B Virus Large Envelope Protein Interacts with γ2-Adaptin, a Clathrin Adaptor-Related Protein
ABSTRACT For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified γ2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physic…
Novel transmembrane topology of the hepatitis B virus envelope proteins.
Abstract The small (S), middle (M) and large (L) envelope proteins of the hepatitis B virus (HBV) are initially synthesized as multispanning membrane proteins of the endoplasmic reticulum membrane. We now demonstrate that all envelope proteins synthesized in transfected cells or in a cell-free system adopt more than one transmembrane orientation. The L protein disposes its N-terminal preS domain both to the cytoplasmic and the luminal side of the membrane. This unusual topology does not depend on interaction with the viral nucleocapsid, but is preserved in secreted empty envelope particles. Pulse-chase analysis suggests a novel process of post-translational translocation leading to the non-…
Dual Topology of the Hepatitis B Virus Large Envelope Protein
The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In…
Chaperones Involved in Hepatitis B Virus Morphogenesis
Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum memb…
Possible role of human interleukin-6 and soluble interleukin-6 receptor in hepatitis B virus infection
Human interleukin-6 has been shown to promote hepatitis B virus (HBV) infection. However, it is not clear whether this influence is the result of a direct interaction between interleukin-6 (IL-6) and the HBV envelope proteins or of a rather indirect mechanism. A direct interaction of IL-6 and the preS region of the large envelope protein (L-protein) of HBV has been reported. In this study we assessed the binding of IL-6 and of the IL-6 receptor subunits to the preS region of the L-protein of HBV. Binding of IL-6 and IL-6 receptor subunits sIL-6R and gp130 to preS was assessed by immunoprecipitation with recombinant preS proteins. In patient sera IL-6 and sIL-6R concentrations were analysed …
Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin.
ABSTRACT Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPa…
Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner
Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional …
Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein.
AbstractThe hepatitis B virus L protein forms a dual topology in the endoplasmic reticulum (ER) via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain. Here, we show that preS posttranslocation depends on the action of the ER chaperone BiP. To modulate the in vivo BiP activity, we designed an approach based on overexpressing its positive and negative regulators, ER-localized DnaJ-domain containing protein 4 (ERdj4) and BiP-associated protein (BAP), respectively. The feasibility of this approach was confirmed by demonstrating that BAP, but not ERdj4, destabilizes the L/BiP complex. Overexpressing BAP or ERdj4 inhibits…
γ2-Adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits.
Abstractγ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-adaptin acts by accelerating lysosomal Gag destru…
Rab33B and its autophagic Atg5/12/16L1 effector assist in hepatitis B virus naked capsid formation and release
Hepatitis B virus morphogenesis is accompanied by the production and release of non-enveloped capsids/nucleocapsids. Capsid particles are formed inside the cell cytosol by multimerization of core protein subunits and ultimately exported in an uncommon coatless state. Here, we investigated potential roles of Rab GTPases in capsid formation and trafficking by using RNA interference and overexpression studies. Naked capsid release does not require functions of the endosome-associated Rab5, Rab7 and Rab27 proteins, but depends on functional Rab33B, a GTPase participating in autophagosome formation via interaction with the Atg5-Atg12/Atg16L1 complex. During capsid formation, Rab33B acts in conju…
Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins
The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane …
Posttranslational N-glycosylation of the hepatitis B virus large envelope protein
Abstract Background The addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. Here, we report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation. Results By using an improved detection system, we identified so far unrecognized, novel isoforms of L. Based on mutational analyses, the use of N-glycosylation inhibitors, and pulse-chase studies, we showed that these isoforms are due to posttranslational N-glycan addition to the asparagines 4 and 112 within the preS domain of L. Whi…
Deletion and insertion mutants of HBsAg particles
We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein [1]. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preSl region at the C-terminus have been characterized.
Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins
The envelope of hepatitis B virus contains three related proteins, one of which is myristylated. The nonmyristylated small and middle protein are assembled into empty envelope particles which are secreted from cells, whereas the myristylated large envelope protein is mainly found in complete virions and is not secreted in the absence of the nucleocapsid. The block to secretion can be partially overcome by mutation or deletion of the myristylation site. Creation of a myristyl attachment site in the small protein impairs the secretion of empty envelope particles but not their intracellular assembly. Myristylation may therefore play a crucial role in hepatitis B virus replication by channeling…
Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation
The large L envelope protein of the hepatitis B virus utilizes a new folding pathway to acquire a dual transmembrane topology in the endoplasmic reticulum (ER). The process involves cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain into the ER. Here, we demonstrate that the conformational and functional heterogeneity of L depends on the action of molecular chaperones. Using coimmunoprecipitation, we observed specific interactions between L and the cytosolic Hsc70, in conjunction with Hsp40, and between L and the ER-resident BiP in mammalian cells. Complex formation between L and Hsc70 was abolished when preS translocation was artifici…
Host Cell Rab GTPases in Hepatitis B Virus Infection
Hepatitis B virus (HBV) is a leading cause of liver disease and is presently estimated to infect more than 250 million humans. The extremely successful spread of this virus among the human population is explained by its effective transmission strategies and its manifold particle types, including virions, empty envelopes and naked capsids. Due to its tiny genome, HBV depends on cellular machineries to thrive in infected hepatocytes. To enter, traverse and exit the cell, HBV exploits host membrane trafficking pathways, including intracellular highways directed by Rab GTPases. Here, we review recent discoveries focused on how HBV co-opts and perturbs host Rab GTPase functions with an emphasis …
Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins.
Among the three related L, M, and S envelope proteins of the hepatitis B virus (HBV), the L and S polypeptides are required for virion production. Whereas the pivotal function of the pre-S region of L in nucleocapsid envelopment has been established, the contribution of its S domain and the S protein is less clear. In this study, we evaluated the role of the cytosolic S loop, common to L and S, in HBV assembly by performing mutagenesis experiments. To distinguish between the effect of the mutations on either envelope or virion formation, we investigated the ability of the mutants to assemble into secretable subviral empty envelopes and to replace the wild-type proteins in virion maturation,…
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
Seuls les 100 premiers auteurs dont les auteurs INRA ont été entrés dans la notice. La liste complète des auteurs et de leurs affiliations est accessible sur la publication.; International audience; In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues…
Hepatitis B Virus Exploits ERGIC-53 in Conjunction with COPII to Exit Cells.
Several decades after its discovery, the hepatitis B virus (HBV) still displays one of the most successful pathogens in human populations worldwide. The identification and characterization of interactions between cellular and pathogenic components are essential for the development of antiviral treatments. Due to its small-sized genome, HBV highly depends on cellular functions to produce and export progeny particles. Deploying biochemical-silencing methods and molecular interaction studies in HBV-expressing liver cells, we herein identified the cellular ERGIC-53, a high-mannose-specific lectin, and distinct components of the endoplasmic reticulum (ER) export machinery COPII as crucial factor…
Assessment of determinants affecting the dual topology of hepadnaviral large envelope proteins
For functional diversity, the large (L) envelope protein of hepatitis B virus (HBV) acquires a dual transmembrane topology via co-translational membrane integration of the S region and partial post-translational translocation of the preS subdomain. Because each process requires the second transmembrane segment (TM2), we explored the action of this determinant by using protease protection analysis of mutant L proteins. We demonstrated that neither the disruption of a leucine zipper-like motif by multiple alanine substitutions nor the flanking charges of TM2 affected the topological reorientation of L. The dispensability of both putative subunit interaction modules argues against a link betwe…
Role for calnexin and N-linked glycosylation in the assembly and secretion of hepatitis B virus middle envelope protein particles.
ABSTRACT Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly …
Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry
ABSTRACT The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of chole…
Functional analysis of a rare HBV deletion mutant in chronically infected children.
Liver damage caused by chronic hepatitis B virus (HBV) infection may be enhanced through the selection of deleted HBV preS mutants by intracellular accumulation of viral proteins and subsequent cell death. However, the prevalence and impact of such mutants on the clinical course of infection have not yet been studied in children. Serum samples from 60 children (mean age 9.8 y) were investigated by means of PCR and direct sequencing of the entire preS region. Only one patient (1.5%) was found with a mixed HBV population of a deletion spanning 183 nucleotides and wild-type sequences. This mutation alters the HBV large-surface protein and removes the small-surface promoter. To clarify the sign…
γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation
Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity …
Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing
Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expres…
Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B
Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host-pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly conco…
Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles
AbstractThe envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a…
Development and characterization of a 293 cell line with regulatable expression of the hepatitis B virus large envelope protein
During the life cycle of hepatitis B virus (HBV) the large L envelope protein plays a pivotal role that is related to its peculiar dual transmembrane topology. To study the complex structure and diverse functions of L under regulated conditions of production, a human 293 cell line stably expressing L under the control of the ecdysone-inducible promoter was generated. Cells demonstrated stringent dose- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer. Temporal control of L expression allowed to trace (i) its posttranslational reorientation resulting in the mixed topology; (ii) its spatial redistribution from the endoplasmic reticu…
Erratum
Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; Arozena, AA; Adachi, H; Adams, CM; Adams, PD; Adeli, K; Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghiso, J; Airoldi, EM; Ait-Si-Ali, S; Akematsu, T; Akporiaye, ET; Al-Rubeai, M; Albaiceta, GM; Albanese, C; Albani, D; Albert, ML; Aldudo, J; Algul, H; Alirezaei, M; Alloza, I; Almasan, A; Almonte-Beceril, M; Alnemri, ES; Alonso, C; Altan-Bonnet, N; Altieri, DC; Alvarez, S; Alvarez-Erviti, L; Alves, S; Amadoro, G; Amano, A; Amantini, C; Ambrosio, S; Amelio, I; Amer, AO; Amessou, M; Amon, A; An, Z; Anania, FA; Andersen, SU; Andley, UP; Andreadi, CK; Andrieu-Ab…
ESCRT Requirements for Murine Leukemia Virus Release
The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion releas…
Hepatitis B Virus Subverts the Autophagy Elongation Complex Atg5-12/16L1 and Does Not Require Atg8/LC3 Lipidation for Viral Maturation
ABSTRACT Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended…
Properties of modified hepatitis B virus surface antigen particles carrying preS epitopes
The current hepatitis B virus (HBV) vaccines contain the small (S) and middle (M) viral envelope proteins in particulate form but lack the large (L) protein. Although these particles elicit protective immunity to HBV, inclusion of the immunogenic preS1 region of the L protein may enhance their efficacy. To present preS1-derived epitopes on secretable subviral particles we rearranged the HBV envelope ORF by fusing part or all of the preS1 region to either the N or C terminus of the S protein. Fusion of the first 42 residues of preS1 to either site allowed efficient secretion of the modified particles and rendered the linked sequence accessible at the surface of the particle. Conversely, fusi…