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

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

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

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

BAG3 inhibits egress of infectious recombinant virus VSV-M40.

<p>HEK293T cells were first transfected with vector alone, BAG3-WT or BAG3-ΔN for 24 hours, and then infected with recombinant virus VSV-M40 <b>(A)</b> or VSV-M40-P2728A <b>(C)</b> at a MOI of 0.1 for 8 hours. Supernatants were harvested and virus titers were determined by standard plaque assay on BHK-21 cells. Each bar represents the average of three independent experiments performed in duplicate. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. The indicated proteins from VSV-M40 <b>(B)</b> or VSV-M40(P2728A) <b>(D)</b> infected cell extracts were detected by Western blotting.</p

BAG3 inhibits budding of eVP40 and mVP40 VLPs in a WW-domain dependent manner.

<p><b>A)</b> HEK293T cells were transfected with the indicated plasmid combinations, and proteins were detected in cell extracts and VLPs by Western blotting. <b>B)</b> Quantification of the relative budding ratios of eVP40 VLPs from three independent experiments. eVP40 VLPs from control cells was set to 1.0. Statistical significance was analyzed by a one-way ANOVA. ns: not significant, *** = p<0.001. <b>C)</b> HEK293T cells were transfected with the indicated plasmid combinations, and proteins were detected in cell extracts and VLPs by Western blotting. <b>D)</b> Quantification of the relative budding ratios of mVP40 VLPs from three independent experiments. mVP40 VLPs from control cells was set to 1.0. Statistical significance was analyzed by a one-way ANOVA. * = p<0.05, *** = p<0.001.</p

BAG3 sequesters VP40 away from the plasma membrane.

<p>HEK293T cells were mock-transfected or transfected with eVP40 <b>(A)</b> or mVP40 <b>(C)</b> plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 <b>(B)</b> or mVP40 <b>(D)</b> from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. <b>E)</b> HEK293T cells were transfected with eVP40 plus vector, BAG3-WT, or BAG3-ΔN as indicated. Cells were fixed at 24 hrs post-transfection, and then incubated with rabbit anti-eVP40 antiserum and mouse anti-myc antiserum (to detecting BAG3-WT and BAG3-ΔN). Cells were then stained with Alexa Fluor 488 goat anti-rabbit and 594 goat anti-mouse secondary antibodies. Microscopy was performed using a Leica SP5 FLIM inverted confocal microscope and XZY scanning. Representative images displaying eVP40 (green) and BAG3-WT (red) or BAG3-ΔN (red) localized at the PM are shown. Cell nuclei were stained with NucBlue. Scale bars = 10μm.</p

BAG3 inhibits eVP40 and mVP40 VLP egress in a dose-dependent manner.

<p>HEK293T cells were transfected with a constant amount of eVP40 plus vector (-), or increasing amounts of BAG3-WT <b>(A)</b>, BAG3-ΔC <b>(B)</b>, or BAG3-ΔN <b>(C)</b>. The indicated proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control. HEK293T cells were transfected with a constant amount of mVP40 plus vector (-), or increasing amounts of BAG3-WT <b>(D)</b>, BAG3-ΔC <b>(E)</b>, or BAG3-ΔN <b>(F)</b>. The indicated proteins were detected in cell extracts and VLPs by Western blotting. mVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control.</p

BAG3 interacts with eVP40 and mVP40 in a WW-domain dependent manner.

<p><b>A)</b> Schematic diagram of BAG3-WT and mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains including the single N-terminal WW-domain (blue), two IPV domains (orange), the PxxP region (yellow), and the BAG domain (green). All three proteins contain both His and <i>cmyc</i> epitope tags. <b>B)</b> Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated (IP) with either rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot (WB) using mouse anti-c<i>myc</i> antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown. <b>C)</b> Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with either mouse IgG or anti-flag (mVP40) antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot using polyclonal anti-His antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown.</p

Proline-rich reading array identifies BAG3 as an eVP40 interactor.

<p><b>A)</b> Schematic diagram of the “proline-rich” reading array chip. Each lettered square contains 12 numbered WW- and/or SH3 GST fusion domains in duplicate. A mock (M) GST sample is in the center of each square. <b>B)</b> The fluorescent pattern following binding of the EBOV VP40-WT biotinylated peptide to the array. The fluorescent spots indicate a positive peptide/WW-domain interaction. EBOV VP40 peptide interactions with WW1 of Rsp5 (square A, green boxes), WW3 of Nedd4 (square B, purple boxes), WW1 of ITCH (square C, yellow boxes), and the BAG3 WW domain (square G, red oval) are highlighted.</p