Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles

<div><p>Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites <i>via</i> GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.</p></div

Role of GP in the incorporation of Xkr8 in Ebola VLPs.

<p>(A) Characterization of incorporated Xkr8 in Ebola VLPs by means of flow cytometry. FBS- (left), Ebola VLP- (middle), or VLPΔGP- (right) conjugated beads incubated with the rabbit anti-VP40, GP, or Xkr8 polyclonal antibodies. For the binding of antibodies against VP40 and Xkr8, the beads were pre-treated with Triton X-100. 2^nd Ab indicates samples that were not treated with primary antibody. As a control, the rabbit anti-LASV GPC polyclonal antibody was used. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (B) Characterization of incorporated Xkr8 in Ebola VLPs by western blotting. 293T cells were transfected with the expression plasmids of VP40 and NP, with or without GP. At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from the culture medium. Total cell lysates and VLPs were analyzed by western blotting with the rabbit polyclonal antibodies against VP40, GP, Xkr8, or β-actin. (C) Binding of AF-ANX V to the beads. FBS- (left), Ebola VLP- (middle), or VLPΔGP- (right) conjugated beads were incubated with AF-ANX V. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (D) Summary of the binding of the anti-Xkr8 antibody (left) and AF ANX V (right) to the Ebola VLP- or VLPΔGP-conjugated beads. Each experiment was performed in triplicate and the percentages of the positive populations are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test).</p

Viral release from infected cells cultured in the presence of S139/1 IgA or IgG antibodies.

<p>After inoculation with Aichi/H3 (A and B), WSN/H1, Adachi/H2, or Maryland/H13 (C and D), MDCK cells were cultured in the presence of S139/1 IgA or IgG antibodies at the concentrations of 1.0 or 0.1 µg/ml (A and B) and 1.0 µg/ml (C and D). Supernatants were collected 6 and 12 hours after infection, and viral proteins of influenza viruses released into the supernatants were detected by Western blotting (B and D). The relative quantity of the M1 protein was calculated based on the band intensity by using Image Lab version 3.0 (Bio-Rad) (A and C). The intensity of the M1 protein bands detected in the control supernatants collected from infected cells cultured without a MAb (w/o MAb) was set to 100%. Experiments were performed 3 times, and averages and standard deviations are shown (A and C). Statistical significance was analyzed by Student's t-test (**p<0.01, *p<0.05). Asterisks placed directly above the bars indicate significant differences compared to respective controls, and asterisks placed between the bars show significant differences between S139/1 IgA and IgG antibodies.</p

Comparison of neutralizing activities of S139/1 IgA and IgG antibodies.

<p>Appropriately diluted viruses were mixed with S139/1 IgA (continuous lines) or IgG (dashed lines) at the indicated dilutions. Neutralizing activities were evaluated by counting the number of plaques formed on MDCK cells. IC₅₀ values calculated based on the neutralization curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085582#pone-0085582-t003" target="_blank">Table 3</a>.</p

Viral particles deposited on the surface of infected cells cultured with S139/1 IgA.

<p>MDCK cells were infected with Aichi/H3 and incubated for 8 hours in the presence of S139/1 IgA (A and D), IgG (B and E), or in the absence of antibodies (C and F). TEM images of the cell surface are shown at high (A to C) and low (D to F) magnifications. Scale bars represent 1 µm (A to C) and 2 µm (D to F).</p

Comparison of binding activities of S139/1 IgA and IgG antibodies.

<p>Binding activities of S139/1 IgA (continuous lines) and IgG (dashed lines) were tested in ELISA. Disrupted viral particles of Aichi/H3, WSN/H1, Adachi/H2, and Maryland/H13 were used as antigens. Data are mean values of duplicate wells. EC₅₀ values calculated based on the ELISA data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085582#pone-0085582-t001" target="_blank">Table 1</a>.</p

Role of Xkr8 in PS exposure on the surface of virus particles.

<p>Xkr8 is transported to the budding sites together with GP <i>via</i> intracellular vesicles (A). VP40 is independently transported to the PM (B). Transported GP and Xkr8 are incorporated into virus particles (C). Incorporated Xkr8 is activated by a caspase (D), which leads to externalization of PS in the envelope of the EBOV particles (1–3).</p

Effect of a pan-caspase inhibitor on the externalization of PS on the surface of Ebola VLPs and their internalization.

<p>(A) The effect of a pan-caspase inhibitor on Xkr8 in cell lysate and VLPs. 293T cells were transfected with the expression plasmids of EBOV VP40, GP, and NP and incubated for 48 h in the absence or presence of 20 μM Z-VAD-FMK. At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from culture medium. Total cell lysates and VLPs were analyzed by western blotting with rabbit polyclonal antibodies against VP40, Xkr8, or β-actin. % of cleaved Xkr8 was analyzed as the ratio of the intensities of the cleaved bands to total Xkr8 bands. (B) The effect of Z-VAD-FMK treatment on the morphology of VLPs. 293T cells were transfected with the expression plasmids of EBOV VP40, NP, and GP and incubated for 48 h in the absence or presence of 20 μM Z-VAD-FMK. At 48 h.p.t., the culture medium was harvested. Viral particles were purified from the culture medium by ultracentrifugation followed by negative staining. Scale bars; 500 nm. (C–E) The effect of Z-VAD-FMK treatment on the incorporation of viral proteins and Xkr8 into VLPs and on the externalization of PS on the surface of Ebola VLPs. (C) Ebola VLPs obtained from untreated- (left) or Z-VAD-FMK-treated cells (right) were conjugated with latex beads. The beads were then incubated with rabbit polyclonal antibodies against EBOV GP, VP40, or Xkr8 followed by flow cytometric analysis. 2^nd Ab indicates samples that were not treated with primary antibody. As a control, the rabbit anti-LASV GPC polyclonal antibody was used. (D) For detection of externalized PS on the VLPs, the beads were incubated with AF-ANX V and subsequently subjected to flow cytometric analysis. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (E) Summary of the binding of the anti-Xkr8 antibody (left) and AF ANX V (right) to VLPs released from untreated- or Z-VAD-FMK-treated cells. Each experiment was performed in triplicate and the percentages of the positive populations are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test). (F, G) The effect of Z-VAD-FMK treatment on VLP internalization. (F) Purified VLPs were labeled with DiI and adsorbed to Vero-E6 cells for 30 min at room temperature. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsinization for 5 min at 37°C and the internalization of the Ebola VLPs was analyzed by using a confocal laser scanning microscope. (G) The number of internalized DiI-VLPs in 10 individual cells was measured. Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test).</p

Role of Xkr8 in the externalization of PS on Ebola VLPs and PS-dependent VLP internalization.

<p>(A) Downregulation of Xkr8 by shRNA. Individual 293T clones transduced by shRNA plasmids were transfected with the expression plasmids of EBOV VP40, GP, and NP. At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from culture medium by ultracentrifugation. Expression of VP40 and Xkr8 in total cell lysates and VLPs was analyzed by western blotting with rabbit polyclonal antibodies against VP40, Xkr8, or β-actin. (B) Negative staining of VLPs obtained from individual shRNA clones. Individual shRNA cell clones were transfected with the expression plasmids of VP40, GP, and NP. At 48 h.p.t., the culture medium was harvested. Viral particles were purified from the culture medium by ultracentrifugation followed by negative staining. Scale bars; 500 nm. (C–E) The effect of Xkr8 knockdown on the incorporation of viral proteins and Xkr8 into VLPs and on the externalization of PS on the surface of Ebola VLPs. (C) Ebola VLPs obtained from shControl #1 (left) or shXkr8 #1 (right) clones were conjugated with latex beads. The beads were then incubated with rabbit polyclonal antibodies against EBOV GP, VP40, or Xkr8 followed by flow cytometric analysis. 2^nd Ab indicates samples that were not treated with primary antibody. As a control, the rabbit anti-LASV GPC polyclonal antibody was used. (D) For detection of externalized PS on the VLPs, the beads were incubated with AF-ANX V and subsequently subjected to flow cytometric analysis. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (E) Summary of the binding of the anti-Xkr8 antibody (left) and AF ANX V (right) to individual shControl VLP- or shXkr8 VLP-conjugated beads. Each experiment was performed in triplicate and the percentages of the positive populations are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test). (F, G) The effect of Xkr8 knockdown on the internalization of VLPs. (F) Purified VLPs from individual shRNA clones were labeled with DiI and adsorbed to Vero-E6 cells for 30 min at room temperature. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsinization and the internalization of Ebola VLPs was analyzed by using a confocal laser scanning microscope. (G) The number of internalized DiI-VLPs in each of 10 individual clones was measured. Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test).</p

Distribution of extracellular PS in cells expressing EBOV proteins.

<p>Vero-E6 cells grown on 35-mm glass bottom dishes were transfected with the expression plasmids of mCherry-VP40 and wtVP40 at a ratio of 1:5 (a), GP alone (b), mCherry-VP40 and wtVP40 with GP (c), or wtVP40 and GP (d). At 48 h.p.t., the cells were harvested followed by AF-ANX V staining. For detection of GP, the cells were incubated in the medium containing the anti-GP antibody, followed by incubation with Alexa Fluor 647-conjugated secondary antibody. After being washed with medium and ANX V binging buffer, the cells were treated with AF-ANX V. After washing again, the AF-ANX V signal (green) and EBOV proteins were observed by using a confocal microscope. Individual viral proteins are shown in magenta (a, b, and d). In panel (c), mCherry-VP40 and GP are shown in magenta and cyan, respectively. As a control, a backbone plasmid was transfected (e). Panel (f) represents Vero-E6 cells treated with 1 μM STS for 6 h. The nuclei (blue) were counterstained with Hoechst 33342. Insets show the boxed areas. The plots indicate the individual fluorescence intensity along each of the corresponding lines. A.U.; arbitrary unit. Scale bars in the large panels: 10 μm.</p