Combination of sotrastaurin with calcineurin inhibitors.

<p>(A, B) Effect of different concentrations of sotrastaurin plus (A) CsA or (B) tacrolimus on HCV Jc1-Luc replication at 48 h after electroporation.</p

Effect on early HCV replication cycle stages.

<p>(A) Infection of Huh-7.5 cells with Jc1-Luc produced in the absence of sotrastaurin. The drug was added for at least 4 h during infection. (B) Strain H77 HCVpp infection of Huh-7.5 cells in the presence or absence of sotrastaurin. Pseudoparticles bearing the glycoprotein of the vesicular stomatitis virus (VSVG) served as controls. (C) Pseudoparticle infection of Huh-7.5 cells in the presence of increasing concentrations of BIM-I. pcDNA represents pseudoparticles devoid of viral glycoproteins.</p

Sotrastaurin does not affect cell-to-cell spread of HCV.

<p>(A) HCV-positive donor cells were mixed with HCV-negative target cells under an agarose overlay preventing cell-free virus spread in the presence or absence of sotrastaurin. Target cells contained a tagRFP-reporter that assumes a cytosolic or nuclear localization depending whether the cells are uninfected or infected with HCV. After 96 hours of co-culture nuclei were co-stained with DAPI. (B) Percentage of infected target cells after 96 hours of co-culture.</p

Effects of sotrastaurin on the HBV lifecycle.

<p>(A) Differentiated HepaRG cells were infected with HBV virions. Sotrastaurin 5 or 10 µg/ml was present either during inoculation (initial 16 h), during inoculation and infection (throughout), or after inoculation (from 16 h onwards). An inhibitory fragment from the HBV L-protein (HBV pre-S/2-48my) applied during inoculation served as a positive control. All reactions were adjusted to contain the same final concentration of DMSO. HBeAg levels secreted into the cell culture supernatant were determined at day 9 after infection. (B) Cytotoxic effect of sotrastaurin on differentiated HepaRG cells as measured by and LDH release assay. (C) HBV antigens released from AD38 harboring a replicating HBV genome after 72 hours of sotrastaurin treatment. IU – international units; S/CO - signal to cutoff ratio. (D) AD38 cells were treated with increasing amounts of sotrastaurin. After 72 h cells were stained with a live/dead cell stain kit and analyzed by FACS.</p

Effect of short-term administration of chenodeoxycholic acid on serum HDV RNA.

<p>Three individuals with chronic HBV/HDV coinfection (circle, triangle and square) were administered chenodeoxycholic acid 15 mg/kg daily orally in three doses. (A) Serum HDV RNA, (B) serum HBV DNA and (C) HBsAg were determined before the first dose and at the indicated time points thereafter. The last symbol in each line indicates serum levels after the final dose of chenodeoxycholic acid. HBV DNA for one patient (square) was not detectable on day 8.</p

Quantification of inhibition of HDV cell entry by BA.

<p>HuH-7/hNTCP cells were seeded in 96-well plates and infected with HDV in the presence of <b>(A)</b> 184 or 368 nM or preS1 peptide or <b>(B-D)</b> 100 or 200 µM of BA. Cells were exposed to virus +/- BA for 6 h, washed and then kept in regular media for another 5 days. TCID₅₀ values were determined as described in the method section. Each data point is based on 48 wells and a representative of two independent experiments is shown.</p

Inhibition-toxicity relationship for chenodeoxycholic acid and sodium taurocholate.

<p>To assay HDV infectivity (filled symbols) HuH-7/hNTCP cells were infected as described above in a 96-well plate and TCID₅₀ was performed in the presence of increasing concentrations of chenodeoxycholic acid (CDCA; triangles) or sodium taurocholate (STCA; circles). In parallel, using identical concentrations and duration of exposure HuH-7/hNTCP cells stably expressing firefly luciferase were treated with CDCA or STCA and luciferase activity after drug treatment was measured as an aggregate measure of cytotoxic and anti-proliferative effects (open symbols). A representative of three independent experiments (only two for cytotoxicity) is shown. Each data point is based on analysis of 48 wells in a TCID₅₀ setup.</p

Effect of BA on HDV infection of HuH-7/hNTCP cells.

<p>HuH-7/hNTCP cells were exposed to HDV containing supernatant in the presence or absence of 200 µM of different BA. For glycocholic acid 50 µM results are shown because the BA is toxic at 200 µM. preS1 peptide (368 nM) and cyclosporine A (25 µg/mL) served as positive controls. After 6 h cells were washed and repleted with regular media without virus, BA or drugs. After another 5 d cells were fixed and stained with a polyclonal antibody against HDV antigen (right hand images in each panel). Nuclei were counterstained with DAPI (left hand images). A representative of at least three independent experiments is shown.</p

VOC Alpha and B.1 efficiently dampen induction of innate immunity in hBAECs.

hBAECs were infected with B.1 or VOC Alpha (MOI of 0.5) and cell lysates were generated at the indicated time points followed by total RNA extraction. The experiment was performed with cells derived from 1–5 adult donors and that were infected in duplicates. (A) Cell-associated expression of envelope in hBAECs during the early phase of infection determined by Q-RT-PCR. TBP was used for normalization. (B) Cell-associated expression of sgN in hBAECs during an early phase of infection determined by Q-RT-PCR. (C–I) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold change +/− SD. (J) Relative change (to preinfection) of cytokines and chemokines concentration in the basal medium of infected hBAECs (MOI 0.5). Concentration of cytokines and chemokines was determined by MagPix Luminex technology. Paired t tests were conducted between B.1 and VOC Alpha-infected groups and scored negative. AEC, airway epithelial cells; hBAEC, human bronchial airway epithelial cell; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein; VOC, variant of concern. See S1 Data.</p

Relative sgRNA level normalized to total RNA reads and infection efficiency in B.1- and VOC Alpha-infected Calu-3 cells.

(A) RNA-seq analysis was conducted from total cell lysates that were obtained 24 hours postinfection to quantify sgRNA proportions in SARS-CoV-2-infected cells (MOI of 2). Canonical, as well as ORF9b and N* sgRNAs were quantified from the RNA-seq dataset. Data were normalized to total RNA reads. (B, C) Number of SARS-CoV-2 nucleocapsid (N)-positive Calu-3 cells was determined by flow cytometry. Calu-3 were left either UI or were infected with B.1 and VOC Alpha (MOI of 2) for 24 hours, permeabilized and immunostained with rabbit-anti-SARS-CoV-2 nucleocapsid antibody, followed by goat anti-rabbit Alexa 488 secondary antibody. (B) Percentage of SARS-CoV-2 N-positive cells. (C) Gating strategy of living-, single-, and N-positive cells is depicted for UI, B.1-, and VOC Alpha-infected cells. MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, subgenomic RNA; UI, uninfected; VOC, variant of concern. See S1 Data. (TIF)</p