CPP sequences and IC50 of DHBV release.

<p>Sequences are shown in uppercase letters for peptides. The amino acids are <b>D</b> amino acids isomers. The carbohydrates chains are shown in lowercase letters. IC₅₀ represent the concentration of CPP resulting in 50% inhibition of DHBV release in PDH and LMH-D2 cells.</p

Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

<div><p>Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)₈ having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)₈ peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)₈ caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)₈-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis <em>via</em> interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)₈ may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.</p> </div

Separation of DHBV DNA-containing particles and SVPs by sucrose-gradient ultracentrifugation.

<p>Supernatants of LMH-D2 cells were concentrated and separated using a 0%–60% a sucrose-based linear density gradient and 17 fractions were recovered from top to bottom. Aliquots from untreated cells (<b>A</b>) or Deca-(Arg)₈ treated cells (<b>C</b>) of each fraction were separated by 4%–20% gradient SDS-PAGE and analyzed for viral envelope. Images of immunoblot were juxtaposed. DHBV capsids were subjected to electrophoresis through a non-denaturing agarose gel and detected by immunostaining of the viral core protein and viral DNA within nucleocapsids was detected by probing the blots with radiolabeled viral DNA. Total viral DNA in the same fractions was analyzed by Dot blot hybridization. In the bottom, the deduced gradient positions and quantification of the viral markers from untreated cells (<b>B</b>) or Deca-(Arg)₈ treated cells (<b>D</b>) are indicated together with the density profile of the gradient.</p

Dose dependent inhibition of hepadnaviral release by Deca-(Arg)₈ in different cell culture systems.

<p>DHBV-infected PDHs (<b>A</b>), and stable transfected LMH-D2 cells (<b>B</b>) were treated with increasing amounts of Deca-(Arg)₈ ranking from 0.25 µM to 2 µM in duplicates for 6 and 4 days, respectively. Cell culture supernatants were collected daily during treatment. The kinetics of viral release in cell culture supernatants, monitored by dot-blot hybridization and quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics) is represented on the upper panel. Relative areas under curves determined by the means of duplicate, compared to untreated cells, are represented in the lower panel. The curves are representative of at least two independent experiments.</p

Analysis of subcellular fractions from LMH-D2 for viral and cellular markers.

<p>Homogenates of cells were subfractionated using a 0%–30% iodixanol-based linear density gradient and 17 fractions were recovered from top to bottom. Aliquots from untreated cells (<b>A</b>) or Deca-(Arg)₈ treated cells (<b>C</b>) of each fraction were separated by 4%–20% gradient SDS-PAGE and analyzed for viral envelope, core protein, and organelle marker proteins PDI (ER), Rab5B (early endosomes). Images of immunoblot were juxtaposed. Viral DNA in the same fractions was analyzed by Dot blot hybridization. In the bottom, the deduced gradient positions and quantification of the viral markers from untreated cells (<b>B</b>) or Deca-(Arg)₈ treated cells (<b>D</b>) are represented together with the density profile of the gradient.</p

Effect of Deca-(Arg)₈ on PDHs and DHBV.

<p>(<b>A</b>); PDHs were plated and treated with (Arg)₈ (left panel) or Deca-(Arg)₈ (right panel) coupled to FITC at different concentration ranking from 0.5 µM to 2 µM and cells were incubated at 37°c, 5% of CO₂ for 24 hours. Cells were observed by fluorescence microscopy (Magnificationx20). (<b>B</b>); PDH were plated and infected with DHBV. PDHs were either treated with 2 µM of Deca-(Arg)₈, washed 2 hours later and then were infected with DHBV or before infection the DHBV inoculum was treated overnight with 2 µM of Deca-(Arg)₈ and was use for infection of PDHs. The release of viral particles in cell culture supernatants was monitored by dot-blot hybridization and was quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics).</p

Effect of Deca-(Arg)₈ on the intracellular hepadnavirus intermediate replicative forms.

<p>Infected PDH (<b>A</b>), and stably DHBV-transfected LMH-D2 (<b>B</b>) cells were treated with different amounts of Deca-(Arg)₈ ranking from 0.5 µM to 2 µM for 5 days. At the end of treatment, the cells were harvested and lysed for DNA and RNA extraction. Intracellular intermediate replicative forms was subjected to Southern blot and Northern blot analysis with an DHBV probe ³²P labeled, and was quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics). For the Northern blot the level of DHBV RNA was normalized on the GAPDH RNA amount. The top panel shows the autoradiography. The bottom panel shows histograms of autoradiography quantifications. Bands corresponding to the expected size of relaxed circular (RC), linear (L), single-stranded (SS) and covalently closed circular (ccc) DHBV DNA are indicated. Bands corresponding to the expected size of pregenomic (pg), preS (preS), and S (S) DHBV RNA are indicated.</p

Inhibition of DHBV release in PDHs and LMH-D2 cells by modified cationic peptides and effect of Deca-(Arg)₈ on cell viability.

<p>Infected PDH and LMH-D2 cells were treated with 2 µM of several modified cationic peptides for 5 days. Cells supernatants were collected daily and were spotted onto positively charged nylon membrane and DHBV DNA was detected by hybridization with a DHBV DNA probe labeled with ³²P, and then was quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics) to monitor DHBV release. Relative areas under curves determined by the means of duplicate, compared to untreated cells set at 100% are represented. The two representations show the effect of modified cationic peptides derived from (Arg)₈ sequence with increasing number of arginine in the peptide (<b>A</b>) or with increasing number of carbon in the fatty acid chain (<b>B</b>). Dose-dependent effects of Deca-(Arg)₈ transduction on cell viability (<b>C</b>). Cell viability was determined by enzymatic activity MTT assay after daily incubation with different concentrations of Deca-(Arg)₈ ranking from 1 µM to 4 µM during six days. The error bars display the standard deviation of duplicates in three independent experiments.</p

Effect of Deca-(Arg)₈ peptide treatment on viral structural proteins expression and on intracellular replication-competent core particles formation.

<p>Detection of viral structural proteins expression (DHBV-preS/S envelope, DHBV-S envelope and DHBV-core protein) of PDHs (<b>A</b>), LMH-D2 cells (<b>B</b>) by immunoblotting. After 5 days of treatment by different amount of Deca-(Arg)₈, cells were harvested and total protein recovered from 12 wells plates was loaded in each lane for SDS-polyacrylamide gel electrophoresis. Following transfer to a PVDF membrane envelope, core protein was detected with specific antibodies. β-actin, detected with mouse anti-human actin, was assayed as a loading control. The next two panels show the results of capsid gel analysis of the native nucleocapsids of PDHs (<b>C</b>), LMH-D2 cells (<b>D</b>). Core protein was detected by western blotting analysis using a rabbit antiserum reactive to purified DHBV nucleocapsids and the viral DNA was denatured with NaOH and the membrane was probed to detect viral DNA.</p

Deca-(Arg)₈ induces an abnormal distribution and an accumulation of viral envelope proteins in large clusters.

<p>LMH-D2 cells (<b>A</b>) and DHBV infected PDHs (<b>B</b>) were treated with increased concentrations (1–4 µM) of Deca-(Arg)₈ peptide. Four days after treatment the cells were fixed and immunostained for DHBV-core (rows 2) and DHBV-PreS/S (row 3) using rabbit anti-DHBV-core and mouse anti-DHBV-PreS/S antibodies, respectively. The primary antibodies were followed by staining with AlexaFluor 488-conjugated goat anti-rabbit or AlexaFluor 555-conjugated goat anti-mouse, and the fluorescent signals of DHBV-core (green) and DHBV-PreS/S (red) are shown in corresponding row in absence or presence of Deca-(Arg)₈ treatment (<b>A</b>; columns 1–4) (<b>B</b>; columns 1–2). The overlays of the fluorescences are shown in the bottom row (<b>A–B</b>). (<b>A</b>) Magnificationx100; (<b>B</b>) Magnificationx40.</p