Proteoglycans act as cellular hepatitis delta virus attachment receptors.

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved

Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms.

Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5' splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection

Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms.

Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5' splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection

Immunotherapy With the PreS-based Grass Pollen Allergy Vaccine BM32 Induces Antibody Responses Protecting Against Hepatitis B Infection

Background: We have constructed and clinically evaluated a hypoallergenic vaccine for grass pollen allergy, BM32, which is based on fusion proteins consisting of peptides from the IgE binding sites of the major grass pollen allergens fused to preS (preS1 + preS2), a domain of the hepatitis B virus (HBV) large envelope protein which mediates the viral attachment and entry. Aim of this study was the characterization of the HBV-specific immune response induced by vaccination of allergic patients with BM32 and the investigation of the vaccines' potential to protect against infection with HBV. Methods: Hepatitis B-specific antibody and T cell responses of patients vaccinated with BM32 were studied using recombinant preS and synthetic overlapping peptides spanning the preS sequence. The specificities of the antibody responses were compared with those of patients with chronic HBV infection. Furthermore, the capacity of BM32-induced antibodies, to inhibit HBV infection was investigated using HepG2-hNTCP cell-based in vitro virus neutralization assays. Findings: IgG antibodies from BM32-vaccinated but not of HBV-infected individuals recognized the sequence motif implicated in NTCP (sodium-taurocholate co-transporting polypeptide)-receptor interaction of the hepatitis B virus and inhibited HBV infection. Interpretation: Our study demonstrates that the recombinant hypoallergenic grass pollen allergy vaccine BM32 induces hepatitis B-specific immune responses which protect against hepatitis B virus infection in vitro

Inhibition of HDV and HBV infection by P2XR antagonists and agonists is dependent on their charge. A

<p>) HepaRG cells were infected with serum of an HDV-positive patient (GTA7) in presence of P2XR antagonists (PPADS, suramin and AZ11645373) and agonists (ivermectin) for 16 h at 37°C. PreS/2-48^myr peptide (200 nM) was used in parallel as control. HDV-specific IF and nuclei staining was done 5 days p.i. The number of total and HDV-infected cells was quantified using ImageJ software. The results are depicted as percentage of the untreated control. In total, 290368 cells were counted for the analysis. Cell lysates of in parallel infected HepaRG cells were used for an HDAg-specific Western Blot. <b>B</b>) HepaRG cells were infected with HBV in presence of P2XR antagonists (PPADS, suramin and AZ11645373) and agonists (ivermectin) for 16 h at 37°C. Supernatants from days 8–11 p.i. were collected and secreted HBeAg was quantified by ELISA. Experiments were performed in duplicate and repeated at least two times independently. The values presented are mean ± SD. <b>C)</b> HepaRG cells were infected with serum of an HDV-positive patient (GTA7). Ivermectin (1 µM) was added at different time points during or after virus inoculation. HDV-specific IF and nuclei staining was done 6 days p.i. The number of total and HDV-infected cells was quantified using the ImageJ software. The results are depicted as percentage of the untreated control. In total, 128450 cells were counted for the analysis.</p

Chemical structures, charge and function of the compounds used in the study.

<p>Listed are the structures of the repeating units (disaccharide or lysine) or the individual molecules. Charged groups are depicted in red. Possible acetylations are marked in blue. The number of charged groups per repeating unit or molecule and the function of the substance are listed. Not included are derivatives of the compounds.</p

The obstruction or removal of negatively charged cellular interaction sites inhibit the HDV infection.

<p><b>A</b>) HepaRG cells were pre-incubated with increasing concentrations of poly-L-lysine for 30 min at 37°C and subsequently infected in presence of the polycation for 16 h at 37°C. <b>B</b>) HDV was pre-incubated for 1 h at 37°C with heparin (100 µg/ml and 500 µg/ml), suramin (100 µg/ml) and dextran sulfate (100 µg/ml). Unbound polyanions were removed by ultrafiltration. HepaRG cells were incubated with the pre-treated viruses for 8 h at 37°C. <b>C</b>) HepaRG cells were pre-incubated with heparin, suramin, dextran sulfate, pentosan polysulfate or poly-L-lysine for 1 h at 37°C. Cells were washed and incubated with HDV for 8 h at 37°C in absence of the compounds. <b>D</b>) Sulfation of cellular proteoglycans was inhibited by treatment of HepaRG cells with increasing concentrations of sodium chlorate for 48 h prior to HDV infection. <b>E</b>) HepaRG cells were incubated for 1 h at 37°C with the indicated concentrations of heparinase III or 1 µM preS/2-48^myr. The cells were washed and inoculated with HDV for 16 h at 37°C in presence of the substances. HDV-specific IF was performed for all experiments on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cells in comparison to the untreated control. In total, 822302 cells were counted for the analysis.</p

Optimization-by-design of hepatotropic lipid nanoparticles targeting the sodium-taurocholate cotransporting polypeptide

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP;; SLC10A1; ) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed; in vitro; . The pharmacokinetics and targeting capacity of selected lead formulations was investigated; in vivo; using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide,; i.e.; Myr‑HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP‑mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by; 111; In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles