scholarly journals Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site

2010 ◽  
Vol 84 (24) ◽  
pp. 12850-12861 ◽  
Author(s):  
Kiyoaki Ito ◽  
Yanli Qin ◽  
Michael Guarnieri ◽  
Tamako Garcia ◽  
Karen Kwei ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Immune Escape ◽  
Envelope Proteins ◽  
Glycosylation Site ◽  
Single Amino Acid ◽  
Dominant Negative Effect ◽  
Escape Mutation ◽  
B Virus

ABSTRACT Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site (131NST133). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.

scholarly journals The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

2009 ◽  
Vol 83 (23) ◽  
pp. 12443-12451 ◽  
Author(s):  
Yann Le Duff ◽  
Matthieu Blanchet ◽  
Camille Sureau
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Envelope Protein ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Receptor Binding Site ◽  
B Virus

ABSTRACT The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides—estimated at three to four per virion—is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S- and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.

scholarly journals A Short N-Proximal Region in the Large Envelope Protein Harbors a Determinant That Contributes to the Species Specificity of Human Hepatitis B Virus

2001 ◽  
Vol 75 (23) ◽  
pp. 11565-11572 ◽  
Author(s):  
P. Chouteau ◽  
J. Le Seyec ◽  
I. Cannie ◽  
M. Nassal ◽  
C. Guguen-Guillouzo ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Host Range ◽  
Envelope Protein ◽  
Envelope Proteins ◽  
Species Specificity ◽  
Wild Type ◽  
Woolly Monkey ◽  
L Protein ◽  
B Virus

ABSTRACT Infection by hepatitis B virus (HBV) is mainly restricted to humans. This species specificity is likely determined at the early phase of the viral life cycle. Since the envelope proteins are the first viral factors to interact with the cell, they represent attractive candidates for controlling the HBV host range. To investigate this assumption, we took advantage of the recent discovery of a second virus belonging to the primateOrthohepadnavirus genus, the woolly monkey HBV (WMHBV). A recombinant plasmid was constructed for the expression of all WMHBV envelope proteins. In additional constructs, N-terminal sequences of the WMHBV large envelope protein were substituted for their homologous HBV counterparts. All wild-type and chimeric WMHBV surface proteins were properly synthesized by transfected human hepatoma cells, and they were competent to replace the original HBV proteins for the production of complete viral particles. The resulting pseudotyped virions were evaluated for their infectious capacity on human hepatocytes in primary culture. Virions pseudotyped with wild-type WMHBV envelope proteins showed a significant loss of infectivity. By contrast, infectivity was completely restored when the first 30 residues of the large protein originated from HBV. Analysis of smaller substitutions within this domain limited the most important region to a stretch of only nine amino acids. Reciprocally, replacement of this motif by WMHBV residues in the context of the HBV L protein significantly reduced infectivity of HBV. Hence this short region of the L protein contributes to the host range of HBV.

scholarly journals Impaired Virion Secretion by Hepatitis B Virus Immune Escape Mutants and Its Rescue by Wild-Type Envelope Proteins or a Second-Site Mutation

2012 ◽  
Vol 87 (4) ◽  
pp. 2352-2357 ◽  
Author(s):  
K. Kwei ◽  
X. Tang ◽  
A. S. Lok ◽  
C. Sureau ◽  
T. Garcia ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Immune Escape ◽  
Envelope Proteins ◽  
Wild Type ◽  
Site Mutation ◽  
B Virus ◽  
Escape Mutants

scholarly journals A Metastable Form of the Large Envelope Protein of Duck Hepatitis B Virus: Low-pH Release Results in a Transition to a Hydrophobic, Potentially Fusogenic Conformation

2000 ◽  
Vol 74 (11) ◽  
pp. 5116-5122 ◽  
Author(s):  
E. V. L. Grgacic ◽  
H. Schaller
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Envelope Proteins ◽  
Low Ph ◽  
Active State ◽  
Hydrophobic Domain ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.

scholarly journals Role of the Antigenic Loop of the Hepatitis B Virus Envelope Proteins in Infectivity of Hepatitis Delta Virus

2005 ◽  
Vol 79 (16) ◽  
pp. 10460-10466 ◽  
Author(s):  
Georges Abou Jaoudé ◽  
Camille Sureau
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Hepatitis Delta Virus ◽  
Envelope Proteins ◽  
Single Amino Acid ◽  
Hepatitis Delta ◽  
B Virus ◽  
Virion Surface ◽  
Delta Virus

ABSTRACT The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.

scholarly journals Entry of Hepatitis Delta Virus Requires the Conserved Cysteine Residues of the Hepatitis B Virus Envelope Protein Antigenic Loop and Is Blocked by Inhibitors of Thiol-Disulfide Exchange

2007 ◽  
Vol 81 (23) ◽  
pp. 13057-13066 ◽  
Author(s):  
Georges Abou-Jaoudé ◽  
Camille Sureau
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Hepatitis Delta Virus ◽  
Envelope Proteins ◽  
Single Amino Acid ◽  
Hepatitis Delta ◽  
Disulfide Exchange ◽  
B Virus ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) particles are coated with the envelope proteins (large, middle, and small) of the hepatitis B virus (HBV). The large protein bears an infectivity determinant in its pre-S1 domain, whereas a second determinant has been proposed to map to the cysteine-rich antigenic loop (AGL) within the S domain of all three envelope proteins (G. Abou Jaoudé and C. Sureau, J. Virol. 79:10460-10466, 2006). In this study, the AGL cysteines were substituted by serine or alanine, and the mutants were evaluated for their function at viral entry using HDV particles and susceptible HepaRG cells. Mutations of cysteines 121 to 149 were tolerant of the production of HDV virions. The mutations altered the structure and antigenicity of the conserved “a” determinant of the AGL, as measured by conformation-sensitive antibodies, and they created a block to infectivity. Substitution of Cys-90 or Cys-221, located outside of the AGL, had no impact on the “a” determinant or viral entry. Furthermore, infectivity was maintained when the AGL CxxC motif at position 121 to 124 was modified by single-amino-acid deletion or insertion, suggesting that cysteines 121 and 124 are not catalyzers of thiol/disulfide exchange. However, membrane-impermeable inhibitors of thiol/disulfide isomerazation demonstrated a dose-dependent inhibition of infection in an in vitro assay when applied to the virus prior to inoculation or during the virus-cell interaction period. Overall, the results demonstrate the essential role of the AGL cysteines at viral entry, and they establish a correlation between the cysteine disulfide network, the conformation of the “a” determinant, and infectivity.

Regulation of Hepatitis B Virus Virion Release and Envelopment Timing by Nucleocapsid and Envelope Interactions

2021 ◽  
Author(s):  
Ji Xi ◽  
Haitao Liu ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Capsid Protein ◽  
Envelope Protein ◽  
Critical Role ◽  
Envelope Proteins ◽  
Linker Sequence ◽  
Hbv Replication ◽  
Virion Release ◽  
B Virus

Interactions between the N-terminal (assembly) domain (NTD) and the linker region of the hepatitis B virus (HBV) capsid protein and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulates covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA was further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment, and highlights the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. Importance Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases including cancer. The interactions between the HBV capsid and the large (L) envelope protein is required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.

Sign in / Sign up

Export Citation Format

Share Document