The spatio-temporal distribution dynamics of Ebola virus proteins and RNA in infected cells

Abstract

Here, we used a biologically contained Ebola virus system to characterize the spatio-temporal distribution of Ebola virus proteins and RNA during virus replication. We found that viral nucleoprotein (NP), the polymerase cofactor VP35, the major matrix protein VP40, the transcription activator VP30, and the minor matrix protein VP24 were distributed in cytoplasmic inclusions. These inclusions enlarged near the nucleus, became smaller pieces, and subsequently localized near the plasma membrane. GP was distributed in the cytoplasm and transported to the plasma membrane independent of the other viral proteins. We also found that viral RNA synthesis occurred within the inclusions. Newly synthesized negative-sense RNA was distributed inside the inclusions, whereas positive-sense RNA was distributed both inside and outside. These findings provide useful insights into Ebola virus replication.

Figure 1. Spatio-temporal distribution dynamics of EBOV proteins in EbolaΔVP30-infected cells.

Vero-VP30 cells were infected with EbolaΔVP30 at an m.o.i. of 0.1. The cells were fixed with 4% PFA at the indicated times after infection. The distribution of EBOV NP (A), VP35 (B), VP40 (C), GP (D), VP30 (E), and VP24 (F) was analyzed by use of immunofluorescence staining. The nuclei were counterstained with DAPI. Yellow arrow (C, top) shows nuclear localization of VP40. Scale bars: 20 μm. White arrows indicate distribution patterns as described in the text.

Figure 2. Co-localization analysis of various combinations of EBOV proteins in EbolaΔVP30-infected cells.

Vero-VP30 cells were infected with EbolaΔVP30 at an m.o.i. of 0.1. The cells were harvested at the indicated times after infection. The co-localization of EBOV proteins was analyzed by use of double immunofluorescence staining in the following combinations: VP40 (red) and NP (green) (A), VP40 (red) and VP35 (green) (B), GP (red) and VP40 (green) (C), NP (red) and VP30 (green) (D), NP (red) and VP24 (green) (E). The nuclei were counterstained with DAPI. Scale bars: 20 μm. The percentage of co-localization (proportion of co-localized protein to individual proteins) is shown in the individual panels.

Figure 3. Intracellular localization of EBOV RNA.

Vero-VP30 cells were infected with EbolaΔVP30 at an m.o.i. of 0.1. Vero-VP30 cells were pretreated for 30 min with actinomycin D at 24 h.p.i., transfected with 10 mM BrUTP, and incubated for 2 h (A) or 12 h (B) in the presence of actinomycin D. The co-localization of BrUTP-labeled viral RNA (green) and VP40 (red) was analyzed by using immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bars: 20 μm. The percentage of co-localization is shown in the individual panels.

Figure 4. Spatio-temporal distribution dynamics of negative-sense EBOV RNA in EbolaΔVP30-infected cells.

Vero-VP30 cells were infected with EbolaΔVP30 at an m.o.i. of 0.1. The cells were fixed with 4% PFA at the indicated times after infection. The co-localization of negative-sense viral RNA (green) and NP protein (red) was detected by using a combination of FISH and immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bars: 20 μm. The percentage of co-localization is shown in the individual panels.

Figure 5. Spatio-temporal distribution dynamics of positive-sense EBOV RNA in EbolaΔVP30-infected cells.

Vero-VP30 cells were infected with EbolaΔVP30 at an m.o.i. of 0.1. The cells were fixed with 4% PFA at the indicated times after infection. The co-localization of positive-sense viral RNA (green) and NP protein (red) was detected by using a combination of FISH and immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bars: 20 μm. The percentage of co-localization is shown in the individual panels.

Figure 6. A model of EBOV assembly.

Viral mRNA is transcribed from genomic negative-sense RNA, is released into the cytoplasm, where viral proteins are translated. NP, together with VP35, VP40, VP30, and VP24 forms small inclusions (A), which become larger near the nucleus (B). At the edge of the inclusion bodies, the NC is formed. VP40 associates with the NC, contributing to its transport to the plasma membrane (C1). Alternatively, NC initially associates with a few VP40 molecules and then moves to plasma membrane, where it is enveloped with membrane-associated VP40 (C2). Synthesized GP is independently transported to the plasma membrane (D). The viral components then assemble, and the progeny virions bud (E).