Cellular localization of KSHV particles following inhibition of proteasome activity.

<p>(A, B and C) HUVEC treated with DMSO (A), MG132 (B) or EPOX (C) were incubated with KSHV for 4 hr, stained for viral particles (red), cell membrane (white) and nuclei (blue). Z-stack images were acquired with confocal laser-scanning microscopy. Three-dimensional software was used to generate z-projection images (left column). Three-dimensional contoured images (middle and right columns) were generated using Imaris image analysis software to determine the localizations of KSHV particles in relation to the cell membrane, the cell interior, and the cell nucleus (upper panels). The 3-D images in the upper panels were rotated on the x-axis to visualize viral particles attached at the cell membrane (small green arrows), internalized into the cytoplasmic space (orange triangles), or docked at the nucleus (green triangles) (lower panels). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s005" target="_blank">Videos S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s006" target="_blank">S2</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s007" target="_blank">S3</a> correspond to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat-1002703-g003" target="_blank">Figures 3A, B, and C</a> respectively. Video S4 depicts viral particles that have been engulfed, and are enclosed within a membrane-bound vesicle. (D–F) Quantification of cellular localization of cell-associated KSHV particles as illustrated in A, B, and C, respectively. Box and whisker plots depict the statistical analyses of the cellular localization of KSHV particles. Open boxes indicate the 75th and 25th percentiles. Top and bottom short lines indicate the 90th and 10th percentiles, respectively. Middle thick lines in the boxes indicate the medians. Outliers outside the 90th and 10th percentiles are represented as black dots. To ensure representation, only cells with at least five particles per cell were counted. The percentages of KSHV particles retained at the membrane for each sample are illustrated in (D), the percentages of internalized KSHV particles in the cytoplasmic space are depicted in (E), and the percentages of KSHV particles docked at the nuclei are illustrated in (F). “n” represents total number of cells analyzed per sample. <i>p</i>-values <0.05 are statistically significant.</p

Colocalization of KSHV particles with E3 ligases during KSHV entry and intracellular trafficking in endothelial cells.

<p>(A) Most KSHV particles are colocalized with E3 ligase c-Cbl or its activated phosphorylated forms but, to a less extent, with Rabex5. HUVEC infected with KSHV for 4 hr were stained for KSHV particles (red), E3 ligase Rabex5, c-Cbl or its phosphorylated forms (pY700 or pY774) (green), and cell nuclei (blue). Z-stack images were acquired and used for colocalization analysis with the Imaris software. Regions of 3-D colocalization are depicted in yellow. (B) Quantification of colocalization of KSHV particles with Rabex5, c-Cbl, phospho-Y700 c-Cbl and phospho-Y774 c-Cbl.</p

Ubiquitination mediates internalization of integrin β1 and KSHV entry and intracellular trafficking.

<p>(A) Colocalization of KSHV particles with integrin β1 during entry into endothelial cells. HUVEC infected with KSHV were stained for viral particles (red) and integrin β1 (white). Images were subjected to 3-D colocalization analysis. Regions of colocalization are depicted in green. (B) Increase of ubiquitination of integrin β1 following KSHV infection, which is blocked by UBEI-41. HUVEC pretreated with DMSO or UBEI-41 for 1 hr were infected with KSHV. At 1 hpi, integrin β1 was immunoprecipitated and the ubiquitinated protein detected by Western-blotting. (C and D) Ubiquitination mediates internalization of integrin β1. HUVEC pretreated with DMSO (C) or UBEI-41 (D) for 1 hr were labeled with a monoclonal antibody to integrin β1 for 1 hr, fixed and labeled with an AlexaFluor488 goat anti-mouse antibody. Z-stack images were acquired to visualize the cellular location of the labeled integrin β1. (E) Inhibition of E1 activating enzyme reduces KSHV entry and intracellular trafficking in endothelial cells. HUVEC were pretreated for 1 hr with UBEI-41 before infection with KSHV for 4 hr, and stained for viral particles (red) and nuclei (blue). (F) Quantification of the numbers of KSHV particles docked at nuclei following inhibition with UBEI-41 as described in E.</p

Inhibition of phosphorylation of E3 ligases reduces KSHV entry and intracellular trafficking in endothelial cells.

<p>(A) Treatment with PP1 analog inhibits KSHV entry and intracellular trafficking in a dose-dependent manner. HUVEC pretreated with PP1 analog for 1 hr, infected with KSHV for 4 hr, and stained for KSHV particles (red) and nuclei (blue). (B) Quantification of the total number of Orf65+ particles docked at each nucleus following treatment with PP1 analog. (C) Treatment with PP1 analog prevents phosphorylation of c-Cbl during KSHV infection. HUVEC pretreated with Src kinase inhibitor PP1 analog for 1 hr were infected with KSHV for 30 min. E3 ligase c-Cbl, phospho-Y700 c-Cbl, and phospho-Y774 c-Cbl were detected using β-actin as a loading control. EGF was used as a positive control for c-Cbl activation.</p

Inhibition of proteasome activity reduced KSHV infectivity in endothelial cells.

<p>(A) Proteasome inhibitors MG132 and EPOX reduced the numbers of LANA-positive cells. HUVEC grown on coverslips were pretreated with proteasome inhibitors for 1 hr and infected with KSHV for 4 hr in the presence of the inhibitors. Cells were fixed and stained for LANA (red) and nuclei (blue) at 48 hpi. (B) Analyses of LANA-positive cells following treatment with proteasome inhibitors depicted as box and whisker plots. Open boxes indicate the 75th and 25th percentiles. Top and bottom short lines indicate the 90th and 10th percentiles, respectively. Middle lines in the boxes indicate the medians, also shown in “%” on tops. Outliers outside the 90th and 10th percentiles are represented as black dots. Some elements of the box and whisker plots are missing because of overrepresentation at lower or higher percentiles. “n” represents total number of fields analyzed per sample. <i>p</i>-values <0.05 are statistically significant.</p

Proteasome inhibitors increase KSHV particles associated with early endosomal marker EEA1.

<p>(A) HUVEC treated with DMSO, MG132 or EPOX were incubated with KSHV for 4 hr, stained for viral particles (red), EEA1 (green), membrane (white), and nuclei (blue). Z-stacks were deconvolved, and Imaris image analysis software was used to generate 3-D contoured images. Videos S5, S6 and S7 correspond to cells treated with DMSO, MG132 or EPOX, respectively, labeled for KSHV particles and EEA1 rotated around the x-axis. Video S8 depicts KSHV particles enclosed in an EEA1+ vesicle. (B–D) Imaris 3-D colocalization analyses determine the total numbers of KSHV particles in a cell that are colocalized with EEA1 (B), the numbers of viral particles localized at cell membranes that are EEA1+ (C), and the numbers of viral particles localized within the cytoplasmic spaces that are EEA1+ (D). Box and whisker plots depict the statistical analyses of the cellular localization of KSHV particles as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat-1002703-g003" target="_blank">Figure 3</a>.</p

Inhibition of proteasome function reduces KSHV particles associated with late endosomal/lysosomal marker LAMP1.

<p>(A) HUVEC treated with DMSO, MG132 or EPOX were incubated with KSHV for 4 hr, stained for KSHV particles (red), LAMP1 (green), membrane (white), and nuclei (blue). Z-stacks were deconvolved and, Imaris image analysis software was used to generate 3-D contoured images. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s013" target="_blank">Videos S9</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s014" target="_blank">S10</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s015" target="_blank">S11</a> correspond to cells treated with DMSO, MG132 or EPOX, respectively, labeled for KSHV particles and LAMP1 rotated around the x-axis. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s016" target="_blank">Video S12</a> depicts KSHV particles enclosed in an LAMP1+ vesicle. (B–D) Imaris 3-D colocalization analyses determine the total numbers of KSHV particles in a cell that are colocalized with LAMP1 (B), the numbers of viral particles localized at cell membranes that are LAMP1+ (C), and the numbers of viral particles localized within the cytoplasmic spaces that are LAMP1+ (D). Box and whisker plots depict the statistical analyses of the cellular localization of KSHV particles as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat-1002703-g003" target="_blank">Figure 3</a>.</p

Association of KSHV particles with ubiquitin-interacting proteins during entry into endothelial cells.

<p>(A) Colocalization of KSHV particles with Epsin and Eps15 during entry into HUVEC. HUVEC infected with KSHV for 1 hr were stained for viral particles (red) and ubiquitin-interacting proteins (green) Epsin (top) or Eps15 (bottom). Images were analyzed in 3-D for colocalization of KSHV particles with Epsin or Eps15. Regions of colocalization are depicted in yellow (second column). (B) Inhibition of proteasome function depletes the cellular pool of free ubiquitin. HUVEC were treated with DMSO, MG132, or EPOX for 1 hr, and then infected with KSHV for 3 hr. Cell lysates were collected and analyzed by immunoblotting to detect both low molecular weight free ubiquitin as well as ubiquitinated proteins (top panel). β-actin was used as a loading control (bottom panel). Longer exposure shown on the left was used to detect the low molecular weight band corresponding to monomeric ubiquitin at 7.5 kD. The shorter exposure shown at the right panel was used to illustrate relative levels of ubiquitinated proteins. (C) Quantification of relative levels of ubiquitinated proteins detected in B.</p

Knock-down of c-Cbl but not Rabex-5 inhibits KSHV entry and intracellular trafficking, and prevents KSHV infection of endothelial cells.

<p>(A) HUVEC mock-treated or infected with c-Cbl, Rabex-5 or control shRNA lentivirus particles for 4 days were examined for the expression of c-Cbl, Rabex-5 and β-tubulin by Western-blotting. (B) HUVEC grown on coverslips were mock treated or infected with c-Cbl, Rabex-5 or control lentivirus particles for 4 days, and infected by KSHV for 4 hr. Cells were stained for Orf65+ viral particles (red) and nuclei (blue). Knock-down with c-Cbl shRNA but not Rabex-5 shRNA or control shRNA decreased the numbers of KSHV particles successfully docked at perinuclear regions. (C) Analyses of numbers of KSHV particles docked on each nucleus following shRNA lentivirus infection depicted as box and whisker plots as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat-1002703-g003" target="_blank">Figure 3</a>. (D) Quantification of LANA-positive cells following KSHV infection for 48 hr in cells with prior knock-down with c-Cbl, Rabex-5, or control shRNA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat.1002703.s004" target="_blank">Figure S4</a>). HUVEC grown on coverslips were mock treated or infected with lentivirus particles for 4 days, and infected by KSHV. At 48 hpi, cells were stained for LANA (red) and nuclei (blue). LANA-positive cells were analyzed and depicted as box and whisker plots as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002703#ppat-1002703-g002" target="_blank">Figure 2B</a>. c-Cbl shRNA but not Rabex-5 shRNA or control shRNA decreased the numbers of LANA-positive cells.</p

Inhibition of proteasome activity prevents KSHV entry into endothelial cells and intracellular trafficking to nuclei.

<p>(A and C) HUVEC were treated with proteasome inhibitors MG132 (A) and EPOX (C) at indicated doses for 1 hr. Cells were then infected with KSHV in the presence of the inhibitors, fixed at 4 hpi, and stained for Orf65+ viral particles (red) and nuclei (blue). (B and D) Quantification of the total number of Orf65+ particles docked at each nucleus following treatment with proteasome inhibitors MG132 (B) and EPOX (D).</p