Budding of Ebola Virus Particles Requires the Rab11-Dependent Endocytic Recycling Pathway

Abstract

The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.

Figure 1.

Characterization of incorporation of Rab11 in Ebola virus-like particles (VLPs). HEK293T cells were transfected with expression plasmids for Ebola virus VP40, glycoproteins, and nucleoproteins. At 48 hours posttransfection, culture medium was harvested. Viral particles were purified from the culture medium and treated with or without trypsin in the presence or absence of Triton X-100. Incorporation of VP40 and Rab11 in Ebola VLPs was analyzed by Western blot analysis. The experiment was performed 3 times independently, and a representative blot is shown (A). The intensity of the bands was quantified, and the average of the relative expression values and its standard deviation are shown (B). *, P < .05; **, P < .01 vs respective control (Student’s t test).

Figure 2.

The subcellular distribution of Rab11 in Vero-E6 cells transiently expressing VP40 derivatives. Vero-E6 cells grown on coverslips were transfected with the expression plasmids for wild-type VP40 (B), VP40 L117R (C), or VP40 I370R (D). At 48 hours posttransfection, the cells were harvested and the subcellular distribution of VP40 and Rab11 was analyzed by immunofluorescence staining. As a control, a backbone plasmid was transfected (A). In merged images, VP40 and Rab11 are shown in green and magenta, respectively. The nuclei (blue) were counterstained with Hoechst 33342. Insets show the boxed areas. Scale bars = 10 µm.

Figure 3.

The effect of a dominant-negative form of Rab11 on the distribution of VP40. Vero-E6 cells grown on coverslips were transfected with the expression plasmids for green fluorescent protein (GFP)-wtRab11 (B) or -dnRab11 (D). At 48 hours posttransfection (h.p.t.), the cells were transfected with the expression plasmids for VP40. At 48 h.p.t., the cells were harvested and the subcellular distribution of VP40 was analyzed by immunofluorescence staining. As a control, GFP-wtRab11 (A) or -dnRab11 (C) was expressed alone. In merged images, VP40 and GFP-fused Rab11 derivatives are shown in magenta and green, respectively. The nuclei (blue) were counterstained with Hoechst 33342. Scale bars = 10 µm. The plots indicate the individual fluorescence intensity along each of the corresponding lines. Abbreviation: A.U., arbitrary unit.

Figure 4.

The role of Rab11 in budding of Ebola virus-like particles (VLPs). HEK293T cells were transfected with small interfering ribonucleic acid (siRNA) against Rab11a, Rab11b alone, or Rab11a and Rab11b. At 72 hours posttransfection (h.p.t.), the cells were transfected with the expression plasmids for VP40, nucleoproteins (NP), and glycoproteins (GP). At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from the culture medium. Total cell lysates (TCL) (A) and VLPs (B) were analyzed by Western blotting with the antibodies against VP40, NP, GP, Rab11, or β-actin. The experiment was performed 3 times independently, and the representative blot is shown. The intensity of the bands correspondence to each protein was quantified. The intensity of corresponding bands to VP40, NP, GP, or Rab11 was normalized with that of corresponding bands to β-actin in the same sample. The average of relative expression values and its its standard deviation are shown. *, P < .05; **, P < .01 vs respective control (Student’s t test).

Figure 5.

The role of Rab11 in VP40-induced clusters in the plasma membrane (PM). Vero-E6 cells were transfected with control small interfering ribonucleic acids (siRNAs) (A) or siRNAs against Rab11a and Rab11b (B). At 72 hours posttransfection (h.p.t.), the cells were transfected with the expression plasmid of VP40. At 48 h.p.t., the subcellular distribution of VP40 and Rab11 was analyzed by immunofluorescence staining. In merged images, VP40 and Rab11 are shown in green and magenta, respectively. The nuclei (blue) were counterstained with Hoechst 33342. White arrows represent the remaining VP40 clusters in the PM. Scale bars = 10 µm.