scholarly journals The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

2009 ◽  
Vol 83 (22) ◽  
pp. 11819-11829 ◽  
Author(s):  
Charlotte Lepère-Douard ◽  
Maud Trotard ◽  
Jacques Le Seyec ◽  
Philippe Gripon
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Fusion Peptide ◽  
S Protein ◽  
Pest Sequence ◽  
B Virus ◽  
Infected Cells ◽  
Infection Processes

ABSTRACT The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.

scholarly journals Phospholipid interactions of the putative fusion peptide of hepatitis B virus surface antigen S protein

1995 ◽  
Vol 76 (2) ◽  
pp. 301-308 ◽  
Author(s):  
I. Rodriguez-Crespo ◽  
E. Nunez ◽  
J. Gomez-Gutierrez ◽  
B. Yelamos ◽  
J. P. Albar ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Fusion Peptide ◽  
Virus Surface ◽  
S Protein ◽  
Virus Surface Antigen ◽  
B Virus

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 Prediction of a putative fusion peptide in the S protein of hepatitis B virus

1994 ◽  
Vol 75 (3) ◽  
pp. 637-639 ◽  
Author(s):  
I. Rodriguez-Crespo ◽  
J. Gomez-Gutierrez ◽  
M. Nieto ◽  
D. L. Peterson ◽  
F. Gavilanes
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Fusion Peptide ◽  
S Protein ◽  
B Virus

Fine Mapping of Neutralization Epitopes on Duck Hepatitis B Virus (DHBV) pre-S Protein Using Monoclonal Antibodies and Overlapping Peptides

Virology ◽  
1993 ◽  
Vol 192 (1) ◽  
pp. 217-223 ◽  
Author(s):  
Sylvie Chassot ◽  
Véronique Lambert ◽  
Alan Kay ◽  
Catherine Godinot ◽  
Bernard Roux ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Fine Mapping ◽  
Duck Hepatitis B Virus ◽  
S Protein ◽  
Duck Hepatitis ◽  
B Virus

Hepatitis B Virus DNA in Serum and Infected Cells

1987 ◽  
pp. 113-130
Author(s):  
Ch. Bréchot ◽  
A. Dejean ◽  
C. Pasquinelli ◽  
F. Laure ◽  
P. Tiollais
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepatitis B Virus Dna ◽  
B Virus ◽  
Infected Cells

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 N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

2018 ◽  
Vol 115 (35) ◽  
pp. 8829-8834 ◽  
Author(s):  
Hasan Imam ◽  
Mohsin Khan ◽  
Nandan S. Gokhale ◽  
Alexa B. R. McIntyre ◽  
Geon-Woo Kim ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Mutational Analysis ◽  
Regulatory Function ◽  
Messenger Rnas ◽  
Stem Loop ◽  
B Virus ◽  
A Site

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

scholarly journals Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes

2019 ◽  
Vol 93 (16) ◽  
Author(s):  
Dmytro Kornyeyev ◽  
Dhivya Ramakrishnan ◽  
Christian Voitenleitner ◽  
Christine M. Livingston ◽  
Weimei Xing ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Half Life ◽  
Natural Infection ◽  
Spatiotemporal Analysis ◽  
Human Hepatocytes ◽  
X Protein ◽  
B Virus ◽  
Infected Cells ◽  
Structural Maintenance Of Chromosomes

ABSTRACTThe structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blotting and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding-deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (∼3 h) in HBV-infected PHH. In addition, we found that treatment with small interfering RNAs (siRNAs) that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.IMPORTANCEHepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection, and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscore its potential as a novel target for the treatment of chronic HBV infection.

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 Complete Genome Sequence of a Precore-Defective Hepatitis B Virus Genotype D2 Strain Isolated in Bangladesh

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Md Golzar Hossain ◽  
Md Muket Mahmud ◽  
Md Arifur Rahman ◽  
Sharmin Akter ◽  
K. H. M. Nazmul Hussain Nazir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Nucleotide Sequence ◽  
Hepatitis B ◽  
Complete Genome Sequence ◽  
Complete Nucleotide Sequence ◽  
Mutational Analysis ◽  
Limited Information ◽  
Virus Genotype ◽  
Hbv Genotype ◽  
B Virus

Hepatitis B virus (HBV) genomic mutations affect viral replication, disease progression, and diagnostic and vaccination efficiency. There is limited information regarding characterization and mutational analysis of HBV isolated in Bangladesh. Here, we report the complete nucleotide sequence of a precore-defective HBV genotype D2 strain isolated in Bangladesh.

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