Inhibition of HDV infection by suramin, PPADS and BBG.

<p>PHH were exposed to HDV in HGM for 3 h. From −1 to +3 h the inhibitors were also present, at the concentrations indicated. At +3 h both inhibitors and virus were removed and replaced by media containing preS1 peptide (50 nM) for the next 16 h, after which the cells were incubated in HGM out to 6 days, at which time total RNA was extracted and assayed by qPCR for HDV antigenomic RNA. As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015784#s4" target="_blank">Methods</a>, the mean values obtained are expressed relative to untreated control cultures. Error bars represent calculated standard error of the mean.</p

Inhibition of HDV and HBV infections by inhibitors of purinergic receptors.

<p>PHH in HGM were exposed to HDV (red) or HBV (blue) for 16 h in the absence (panel A) or presence (panel B) of 5% PEG, along with the indicated inhibitors and their concentrations. After 16 h media was changed to HGM and the infections allowed to continue for 6 or 12 days, for HDV and HBV, respectively, at which times total RNA was extracted and assayed by qPCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015784#s4" target="_blank">Methods</a>. Evaluation was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015784#pone-0015784-g001" target="_blank">Fig. 1</a>.</p

HDV replication is inhibited by application of interferons alpha or gamma.

<p>Times of addition of inhibitors are measured relative to 3-h of virus exposure. The preS1 peptide was present at 50 nM while the interferons were at 600 U/ml. All assays were performed in at least triplicate, and the data expressed relative to control hepatocytes that were not treated with inhibitors. The indicated standard error of the mean includes the errors in both the control and the treated cultures.</p

Time-dependent induction by interferons alpha and gamma of innate immune response proteins in primary hepatocytes.

<p>Hepatocytes were exposed to either interferons alpha or gamma at 600 units/ml. At time points out to 24 h, as indicated, total cell protein was extracted and analyzed by immunoblot to detect the indicated host proteins. Note that after 2 hours of treatment with interferon gamma, the pSTAT1 increases but not the total STAT1; this is because only a small fraction of the total STAT1 undergoes phosphorylation.</p

Representation of time-lines for exposure of primary hepatocytes to HDV and potential inhibitors.

<p>For the five time-lines shown the open box indicates the period of HDV exposure and the shaded box the exposure to inhibitor. The effects on HDV replication of such treatments with three different inhibitors are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022415#pone-0022415-t001" target="_blank">Table 1</a>.</p

Dose response of HDV and VSV infections to interferon-alpha and preS1 peptide.

<p>For HDV infections the inhibitors were present from 24 h prior to infection and during the 16 h of exposure to virus. Replication was assayed at 6 days post-infection by realtime PCR, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022415#pone-0022415-g001" target="_blank">Fig. 1C</a>. The assays were performed in triplicate, and average values are expressed as a percentage relative to the untreated controls, with the indicated standard error of the mean. For the VSV infections, the interferon was only present during the 16 h of virus exposure. VSV replication was assayed at 16 h by counting GFP positive cells. The values are expressed relative to untreated controls and the errors indicate the standard deviation as the square root of the mean. Interferon-alpha concentrations are expressed here as molarities, with 600 units/ml = 0.12 nM. The preS1 peptide, like interferon-alpha, can inhibit HDV infection, but the needed molarity is 300-times more. Interferon-alpha can inhibit VSV infection but the concentration is 1,000-time less than that needed to inhibit HDV.</p

Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

<p>Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress</p

VP40/BAG3 GST pulldown assay.

<p><b>A)</b> Extracts from HEK293T cells transfected with eVP40-WT or eVP40-ΔPT/PY plasmids were incubated with GSH beads conjugated with GST-BAG3WW or GST alone. Input and pulled-down proteins were detected by Western blotting using anti-eVP40 or anti-GST antisera. <b>B)</b> Extracts from HEK293T cells expressing flag-tagged mVP40-WT were incubated with GSH beads conjugated with GST-BAG3WW or GST alone. Input and pulled-down proteins were detected by Western blotting using anti-flag or anti-GST antisera.</p

BAG3 alters the intracellular localization of eVP40 in live cells.

<p><b>A)</b> HEK293T cells were transfected with GFP-eVP40 (green) plus either vector, or BAG3-mCherry (red), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with arrows highlighting the typical localization pattern of GFP-eVP40 at the plasma membrane and in PM projections, while arrowheads highlight the altered diffuse cytoplasmic localization pattern of eVP40 observed in BAG3 expressing cells. Cell nuclei were stained with NucBlue. Scale bars = 10μm. <b>B)</b> HeLa cells were transfected with GFP-eVP40 (green) plus mCherry-LC3 (red) and vector alone (top row), or BAG3 (bottom two rows), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with white arrows highlighting the colocalization of GFP-eVP40 and mCherry-LC3 in aggresomes. Cell nuclei were stained with NucBlue. Scale bars = 10μm.</p

siRNA knockdown of BAG3 enhances eVP40 VLP egress.

<p><b>A)</b> HEK293T cells were transfected with eVP40 plus either random (control) or BAG3-specific siRNA as indicated. Proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLPs from control cells (lane 1) was set at 1.0. <b>B)</b> Quantification of the relative budding ratio of eVP40 VLPs from four independent experiments. Statistical significance was analyzed by a student t test, *** = p<0.001.</p