scholarly journals A Putative Amphipathic Alpha Helix in Hepatitis B Virus Small Envelope Protein Plays a Critical Role in the Morphogenesis of Subviral Particles

2021 ◽  
Vol 95 (8) ◽  
Author(s):  
Sisi Yang ◽  
Zhongliang Shen ◽  
Yaoyue Kang ◽  
Liren Sun ◽  
Usha Viswanathan ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Alpha Helix ◽  
Hbv Infection ◽  
20S Proteasome ◽  
S Protein ◽  
B Virus ◽  
Amphipathic Alpha Helix ◽  
Subviral Particles

ABSTRACT Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP-producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent but ubiquitination-independent nonclassic ER-associated degradation pathway. Taken together, the results reported here favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC, whereas the failure of the budding process may result in S protein degradation by 20S proteasome in a ubiquitination-independent manner. IMPORTANCE SVPs are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed those of virion particles by 10,000- to 100,000-fold in the blood of HBV-infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induce immune tolerance and contribute to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by the appearance of anti-HBsAg antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is thus considered to be essential for the restoration of the host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.

scholarly journals Domains of the Hepatitis B Virus Small Surface Protein S Mediating Oligomerization

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Sascha Suffner ◽  
Nadine Gerstenberg ◽  
Maria Patra ◽  
Paula Ruibal ◽  
Ahmed Orabi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Mammalian Cells ◽  
Protein S ◽  
Viral Envelope ◽  
Small Surface ◽  
S Protein ◽  
B Virus

ABSTRACTDuring hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined.IMPORTANCEThe small hepatitis B virus envelope protein S has the intrinsic ability to direct the morphogenesis of spherical 20-nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited, as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic loop, one membrane-embedded domain, and the luminal loop of S independently drive S-S oligomerization.

scholarly journals Expression Level of Small Envelope Protein in Addition to Sequence Divergence inside Its Major Hydrophilic Region Contributes to More Efficient Surface Antigen Secretion by Hepatitis B Virus Subgenotype D2 than Subgenotype A2

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 967
Author(s):  
Qianru Wang ◽  
Shuwen Fu ◽  
Jing Zhang ◽  
Quan Yuan ◽  
Jisu Li ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Envelope Protein ◽  
Hepatitis B Surface Antigen ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Huh7 Cells ◽  
B Virus

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.

scholarly journals Glycine Decarboxylase Mediates a Postbinding Step in Duck Hepatitis B Virus Infection

2004 ◽  
Vol 78 (4) ◽  
pp. 1873-1881 ◽  
Author(s):  
Jisu Li ◽  
Shuping Tong ◽  
Hong Bock Lee ◽  
Ana Luisa Perdigoto ◽  
Hans Christian Spangenberg ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Viral Envelope ◽  
Glycine Decarboxylase ◽  
Virus Family ◽  
Duck Hepatitis B Virus ◽  
S Protein ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.

scholarly journals Drastic Reduction in the Production of Subviral Particles Does Not Impair Hepatitis B Virus Virion Secretion

2009 ◽  
Vol 83 (21) ◽  
pp. 11152-11165 ◽  
Author(s):  
Tamako Garcia ◽  
Jisu Li ◽  
Camille Sureau ◽  
Kiyoaki Ito ◽  
Yanli Qin ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
M Protein ◽  
Dependent Manner ◽  
Drastic Reduction ◽  
S Protein ◽  
L Protein ◽  
B Virus ◽  
S Proteins ◽  
Subviral Particles

ABSTRACT Hepatitis B virus (HBV) contains three coterminal envelope proteins on the virion surface: large (L), middle (M), and small (S). The M and S proteins are also secreted as empty “subviral particles,” which exceed virions by at least 1,000-fold. The S protein serves as the morphogenic factor for both types of particles, while the L protein is required only for virion formation. We found that cotransfecting replication constructs with a small dose of the expression construct for the missing L, M, and S proteins reconstituted efficient virion secretion but only 5 to 10% of subviral particles. The L protein inhibited secretion of subviral particles in a dose-dependent manner, whereas a too-high or too-low L/S protein ratio inhibited virion secretion. Consistent with the results of cotransfection experiments, a point mutation at the −3 position of the S gene AUG codon reduced HBsAg secretion by 60 to 70% but maintained efficient virion secretion. Surprisingly, ablating M protein expression reduced virion secretion but markedly increased the maturity of virion-associated genomes, which could be reversed by providing in trans both L and M proteins but not just M protein. M protein stability was dependent on the coexpression of S protein. Our findings suggest that efficient HBV virion secretion could be maintained despite drastic reduction in subviral particle production, which supports the recent demonstration of separate secretion pathways adopted by the two types of particles. The M protein appears to facilitate core particle envelopment, thus shortening the window of plus strand DNA elongation.

scholarly journals Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1613-1622 ◽  
Author(s):  
Philippe Gripon ◽  
Isabelle Cannie ◽  
Stephan Urban
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Viral Envelope ◽  
Hbv Infection ◽  
Viral Envelope Protein ◽  
Therapeutic Concept ◽  
Heparg Cell ◽  
B Virus

ABSTRACT The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Β.

scholarly journals St, a Truncated Envelope Protein Derived from the S Protein of Duck Hepatitis B Virus, Acts as a Chaperone for the Folding of the Large Envelope Protein

2005 ◽  
Vol 79 (9) ◽  
pp. 5346-5352 ◽  
Author(s):  
Elizabeth V. L. Grgacic ◽  
David A. Anderson
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Cellular Localization ◽  
Transmembrane Domain ◽  
Molar Ratio ◽  
Duck Hepatitis B Virus ◽  
S Protein ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT Envelope proteins of hepadnaviruses undergo a unique folding mechanism which results in the posttranslational translocation of 50% of the large envelope protein (L) chains across the endoplasmic reticulum. This mechanism is essential for the eventual positioning of the receptor-binding domain on the surface of the virus particle and in duck hepatitis B virus (DHBV) is dependent on the small (S) envelope protein as part of the assembly process. In this study, we report the identification of a third envelope protein, St, derived from the S protein and carrying functions previously attributed to S. Antibody mapping and mutagenesis studies indicated St to be C terminally truncated, spanning the N-terminal transmembrane domain (TM1) plus the adjacent cysteine loop. We have previously shown that the mutation of two conserved polar residues in TM1 of S (SAA) eliminates L translocation and assembly. A plasmid expressing a functional equivalent of St was able to rescue assembly, demonstrating that this assembly defect is due to mutations of the corresponding residues in St and not in S per se. Immunofluorescence analysis showed that St directly affects L protein cellular localization. These results indicate that St acts as a viral chaperone for L folding, remaining associated with the DHBV envelope upon secretion. The presence of St at a molar ratio of half that of L suggests that it is St which regulates L translocation to 50%.

scholarly journals Enhancement of Hepatitis B Virus Infection by Noninfectious Subviral Particles

1998 ◽  
Vol 72 (2) ◽  
pp. 1462-1468 ◽  
Author(s):  
Michael Bruns ◽  
Stefan Miska ◽  
Sylvie Chassot ◽  
Hans Will
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Clinical Importance ◽  
Virus Infections ◽  
S Protein ◽  
Duck Hepatitis ◽  
B Virus ◽  
Hepatitis B Virus Infections ◽  
Number Of Particles ◽  
Subviral Particles

ABSTRACT The biological function of the huge excess of subviral particles over virions in hepatitis B virus infections is unknown. Using the duck hepatitis B virus as a model, we unexpectedly found that subviral particles strongly enhance intracellular viral replication and gene expression. This effect is dependent on the multiplicity of infection, the ratio of virions over subviral particles, and the time point of addition of subviral particles. Most importantly, we show that the pre-S protein of the subviral particles triggers enhancement and requires the presence of the binding regions for putative cell-encoded virus receptor proteins. These data suggest that enhancement is due either to the recently described transactivation function of the pre-S protein or to signalling pathways which become activated upon binding of subviral particles to cellular receptors. The findings are of clinical importance, since they imply that infectivity of sera containing hepadnaviruses depends not only on the amount of infectious virions but also decisively on the number of particles devoid of nucleic acids. A similarly dramatic enhancing effect of noninfectious particles in other virus infections is well conceivable.

Preclinical studies on Myrcludex B, a novel Hepatitis B virus (HBV)-envelope protein derived entry inhibitor

2010 ◽  
Vol 48 (01) ◽  
Author(s):  
A Schulze ◽  
A Schieck ◽  
C Gähler ◽  
A Meier ◽  
T Müller ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Entry Inhibitor ◽  
Preclinical Studies ◽  
B Virus
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