Role of glycosaminoglycans for binding and infection of hepatitis B virus

Institute of Medical Virology, Justus Liebig University, Frankfurter Str. 107, 35392 Giessen, Germany.
Cellular Microbiology (Impact Factor: 4.92). 02/2008; 10(1):122-33. DOI: 10.1111/j.1462-5822.2007.01023.x
Source: PubMed


Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell-surface heparan sulfate proteoglycans as low-affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 microg ml(-1)) or other higher-sulfated polymers, but not by lower-sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1-dependent viral binding at 16 degrees C to the cell surface, subsequent infection could still be inhibited by HBV preS1-lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high-affinity receptor. In summary, we suggest following multistep attachment process: in vivo, HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N-terminal myristic acid to a yet unknown, high-affinity receptor that confers uptake in a yet unknown compartment.

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    • "The exposed part of the " a " determinant comprises three loops (mini-loop aa 121-124 (Qiu et al., 1996), the first-loop aa 124-137 (Dreesman et al., 1982) and the second loop aa 139-147 (Bhatnagar et al., 1982; Brown et al., 1984) held together by disulfide bonds. The HBV surface proteins bear cellular receptor-binding sites located within the aminoterminal preS1 domain of the LHBs interacting with hepatic bile acid transporter, NTCP (Yan et al., 2012) and the HBsAg " a " determinant interacting with heparin sulfate proteoglycans (Leistner et al., 2008; Sureau and Salisse, 2013). The activity of the receptor-binding site within the " a " determinant depends on cysteine residues that are essential for its conformation (Mangold et al., 1995). "
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    ABSTRACT: Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic "a" determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV genotypes. Recombinant scFv consisting of immunoglobulin VH and VL regions joined by a 20 aa-long linker was generated by cloning the respective cDNA sequences from hybridoma HB1. The recombinant scFv generated in E. coli recognized the same epitope as the parental MAb HB1. Cloning of HB1 VH and VL regions allowed determination of their primary structure and subsequent computer modeling of antibody-epitope interaction. The generated molecular models of HB1 variable region with its target peptides were in accordance with experimental data showing the importance of certain aa residues in antibody binding. In conclusion, the current study describes new HBsAg-specific antibodies with HBV-neutralizing potency and a broad cross-reactivity against different HBV strains. The generated MAb HB1 will be of great value in diagnostic and research settings, while the recombinant HB1-derived scFv represents a promising "building block" for producing anti-HBV tools with a potential biopharmaceutical application.
    Virus Research 11/2015; DOI:10.1016/j.virusres.2015.10.024 · 2.32 Impact Factor
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    • "GPC1 is a glycophosphatidylinositol (GPI) linked cell surface protein and member of a large group of heparan sulfate binding proteins that are involved in morphogenesis, adhesion, chemotaxis, and inflammation [Fransson, 2003; Parish, 2006]. Heparan sulfate proteoglycans are key cell surface attachment sites for infectious agents and are thought to play a role in the hepatotropism of hepatitis B virus, hepatitis C virus, and hepatitis D [Barth et al., 2003; Schulze et al., 2007; Leistner et al., 2008; Longarela et al., 2013; Shi et al., 2013]. "
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    ABSTRACT: Acute liver failure is a severe, but rare, outcome of hepatitis A virus infection. Unusual presentations of prevalent infections have often been attributed to pathogen-specific immune deficits that exhibit Mendelian inheritance. Genome-wide resequencing of unrelated cases has proven to be a powerful approach for identifying highly penetrant risk alleles that underlie such syndromes. Rare mutations likely to affect protein expression or function can be identified from sequence data, and their association with a similarly rare phenotype rests on their existence in multiple affected individuals. A rare or novel sequence variant that is enriched to a significant degree in a genetically diverse cohort suggests a candidate susceptibility allele. Whole genome sequencing of ten individuals from ethnically diverse backgrounds with HAV-associated acute liver failure was performed. A set of rational filtering criteria was used to identify genetic variants that are rare in the population, but enriched in this cohort. Single nucleotide polymorphisms, insertions, and deletions were considered and autosomal dominant, autosomal recessive, and polygenic models were applied. Analysis of the protein-coding exome identified no single gene with putatively deleterious mutations shared by multiple individuals, arguing against a simple Mendelian model of inheritance. A number of rare variants were significantly enriched in this cohort, consistent with a complex and genetically heterogeneous trait. Several of the variants identified in this genome-wide study lie within genes important to hepatic pathophysiology and are candidate susceptibility alleles for hepatitis A virus infection. J. Med. Virol. © 2014 Wiley Periodicals, Inc.
    Journal of Medical Virology 10/2014; 86(10). DOI:10.1002/jmv.24007 · 2.35 Impact Factor
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    • "The ubiquitous heparan sulfate proteoglycans (HSPGs), which are widely distributed on mammalian cells, have been identified as potential targets for a number of viruses [82-85], such as herpes virus [86], hepatitis C virus [87], dengue virus [88], human immunodeficiency virus type 1 [55], foot and mouth disease virus [55], human papillomavirus [89], and hepatitis B virus [90]. It has been shown that HSPGs acts as primary binding sites, promoting viral docking and facilitating subsequent interaction of viruses with the specific cellular receptors [91]. "
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    ABSTRACT: Background Hepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain. Methods Lactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry. Results N- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively. Conclusion Generated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.
    BMC Complementary and Alternative Medicine 07/2014; 14(1):219. DOI:10.1186/1472-6882-14-219 · 2.02 Impact Factor
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