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

Externalization of PS on the surface of Ebola VLPs.

<p>(A) Representative dot plots of unconjugated (top), FBS- (middle), or Ebola VLP- (bottom) conjugated beads. Purified Ebola VLPs or FBS were incubated with 4-μm latex beads and then analyzed by means of flow cytometry. Single beads (circle) were gated for further analysis. X-axis: forward scatter corner signals, Y-axis: side scatter corner signals. (B) Electron micrograph of Ebola VLP-conjugated beads. Latex beads conjugated with Ebola VLPs were subjected to negative staining. Scale bar: 1 μm. (C) Binding of antibodies to the beads. FBS- (left) or Ebola VLP-conjugated (right) beads were incubated with rabbit polyclonal antibodies against EBOV GP, or VP40, followed by incubation with Alexa Fluor 488-labeled secondary antibody. To analyze the binding of the anti-VP40 antibody, we treated Ebola VLP-conjugated beads with 0.05% Triton X-100 for 10 min at room temperature. Antibody binding to the beads was analyzed by flow cytometry. 2^nd Ab indicates beads 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: fluorescent intensity, Y-axis: forward scatter corner signals showing the size of the gated events. The results are representative of three individual experiments. (D) Binding of fluorescently labeled-ANX V to the beads. FBS- (left) or Ebola VLP-conjugated (right) beads were incubated with or without Alexa Fluor 488 (AF)-ANX V. The percentages of the positive populations are indicated. X-axis: fluorescent intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (E) The effect of unlabeled ANX V on the binding of AF-ANX V to the beads. Ebola VLP-conjugated beads were pretreated with unlabeled ANX-V at the indicated concentrations. After being washed, the beads were incubated with 1.2 μg/ml AF-ANX V for 15 min at room temperature. Binding of AF-ANX V to the beads was analyzed by flow cytometry. Relative binding of AF-ANX V to the beads is shown. Each experiment was performed in triplicate and the results are presented as the mean ± SD.</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

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

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

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

Differences among epitopes recognized by neutralizing antibodies induced by SARS-CoV-2 infection or COVID-19 vaccination

Summary: SARS-CoV-2 has gradually acquired amino acid substitutions in its S protein that reduce the potency of neutralizing antibodies, leading to decreased vaccine efficacy. Here, we attempted to obtain mutant viruses by passaging SARS-CoV-2 in the presence of plasma samples from convalescent patients or vaccinees to determine which amino acid substitutions affect the antigenicity of SARS-CoV-2. Several amino acid substitutions in the S2 region, as well as the N-terminal domain (NTD) and receptor-binding domain (RBD), affected the neutralization potency of plasma samples collected from vaccinees, indicating that amino acid substitutions in the S2 region as well as those in the NTD and RBD affect neutralization by vaccine-induced antibodies. Furthermore, the neutralizing potency of vaccinee plasma samples against mutant viruses we obtained or circulating viruses differed among individuals. These findings suggest that genetic backgrounds of vaccinees influence the recognition of neutralizing epitopes