Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.

It is demonstrated that nucleoprotein likely serves as a core for the nucleocapsid (NC) complex, which is critical for replication and transcription of viral genome. The interaction of nucleoprotein with matrix protein VP40 is found to be essential for transport of NCs to the cell surface and for the incorporation of NCs into virions, resulting in the formation of mature virus particles. Unique among all viruses to our knowledge, Ebolavirus particles containing NCs mainly emerge horizontally from the cell surface, whereas the other viruses bud vertically. These findings form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.

Ebolavirus is a causative agent of a severe, mostly fatal hemorrhagic fever in humans. Like all viruses, it must reproduce its various components and assemble them to reproduce progeny virus particles. However, because Ebolavirus needs special containment for biosafety, progress in understanding its morphogenesis has been slow, resulting in no effective therapies currently available for humans. To further our understanding of the Ebolavirus life cycle, the authors examined the specific interactions among viral proteins and the processes of Ebolavirus morphogenesis.

Funding: This work was supported by Core Research for Evolutional Science and Technology (CREST) grants from the Japan Science and Technology Corporation Agency (JST), Japan; by grants from the National Institutes of Health, NIAID; by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Health, Labor, and Welfare, Japan; and by the NIH/NIAID Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE) Program. The authors also acknowledge membership within and support from the Region V “Great Lakes” RCE (NIH award 1-U54-AI-057153). TN was the recipient of Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.

Copyright: © 2006 Noda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In Ebolavirus infection, newly synthesized viral proteins and genomic RNA in the form of NCs are transported to the budding site where the viral components assemble to form virions [ 7 , 10 – 13 ]. However, many questions regarding assembly of Ebolavirus particles have yet to be answered. How are NCs formed in the cytoplasm? How are NCs transported to the cell surface and incorporated into virions? How do the filamentous virions bud from the cell surface? In an attempt to answer these questions, we performed structural and biochemical assessments of cells transfected with plasmids expressing various combinations of Ebola viral proteins and of cells infected with Ebolavirus.

Ebolavirus, together with Marburgvirus, comprise the family Filoviridae in the order Mononegavirales [ 1 , 2 ]. It has a nonsegmented, negative-sense RNA genome that encodes at least seven structural proteins [ 1 , 3 ]. These proteins form filamentous particles 80 nm in diameter with lipid membrane derived from host cells. The viral glycoprotein (GP) protrudes from the surface of the viral envelope, while the matrix protein VP40 plays a central role in the morphogenesis of the filamentous virions [ 4 – 7 ]. Along the central axis of the filamentous virion resides a nucleocapsid (NC) of approximately 50 nm in diameter. This viral genomic RNA-protein complex has an axial channel at its center [ 8 ]. The NCs of Ebolavirus, which represent the principal units of transcription and replication of the viral genome, are thought to consist of four proteins: the L polymerase protein, VP35, nucleoprotein (NP), and VP30. Huang et al. [ 9 ] showed that expression of NP, the membrane-associated VP24 protein, and VP35 results in the formation of structures morphologically indistinguishable from the NCs observed in Ebolavirus-infected cells, demonstrating the involvement of VP24 in the formation of such structures.

Results/Discussion

Formation of NC-Like Structures To confirm whether the expression of NP, VP24, and VP35 are sufficient for the formation of NC-like structures [9,14], we cotransfected 293T cells with various combinations of plasmids for the expression of the viral proteins (i.e., NP, VP24, VP30, VP35, and L) and the minigenome RNA, which consists of a green fluorescent protein gene flanked by 3′ leader and 5′ trailer sequences. As previously described [9,14], coexpression of NP, VP24, and VP35 was indispensable for the formation of NC-like structures (Figures 1A and S1A) that are morphologically indistinguishable from NCs in virus-infected cells (Figure 1B), whereas the viral minigenome RNA was not essential for this process. To understand the roles of the individual proteins in the formation of the NC-like structures, we transfected cells with plasmids expressing NP, VP24, or VP35. When NP was expressed alone, helical tubes, the diameter of which was almost the same as that of a central portion of the NCs (approximately 20–25 nm in diameter), possessing a central channel (approximately 15–20 nm in diameter), were arranged in a bundle in the cytoplasm (Figure S1B–S1D). In the cytoplasm of VP35-expressing cells, large (approximately 8-μm × 3-μm) electron-dense structures with small opaque areas (less than 200 nm in diameter) were observed near the nucleus (Figure S1E). By contrast, in cells transfected with the VP24 plasmid, numerous, small electron-dense pleiomorphic aggregates, identified as VP24 by immunoelectron microscopy with an anti-VP24 antibody (unpublished data), were seen scattered throughout the cytoplasm (Figure S1F). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Formation of NC-Like Structures upon the Expression of NP, VP24, and VP35 (A) Expression of NP, VP24, and VP35 produced filamentous and tubular NC-like structures approximately 50 nm in diameter. (B) In Ebolavirus-infected cells, a large number of NCs newly synthesized in the cytoplasm were observed. (C) When NP, VP24, and VP35 were expressed simultaneously, NC-like structures were found at the edge of the NP tubes. Bars, 1 μm (A, B, and C). https://doi.org/10.1371/journal.ppat.0020099.g001 To understand how NC-like structures are formed, we coexpressed NP, VP24, and VP35 in different combinations. On coexpression of VP35 with VP24, we saw a large number of pleiomorphic particles near the nucleus that were different from the VP35- or VP24-induced structures (Figure S1G), although an interaction between VP35 and VP24 was not detected by a coimmunoprecipitation assay (unpublished data). When VP35 was expressed with NP, small pleiomorphic structures, whose electron density was similar to that of the VP35-induced structures, were observed at the periphery of the clusters formed with NP tubes (Figure S1H). By contrast, when VP24 was expressed with NP, the morphologies of the small pleiomorphic structures formed by VP24 and of the NP tubes did not change (unpublished data). Finally, when the three proteins were expressed simultaneously, NC-like structures approximately 50 nm in diameter were found at the edge of the clusters of NP tubes (Figure 1C). These results, together with the previous biochemical studies demonstrating interactions between NP and VP24 (Ebolavirus) or NP and VP35 (Ebolavirus and Marburgvirus) [9,15], suggest that NP helical structures likely serve as the core for the formation of the NC-like structures, and that VP35 and VP24 contribute to this process by interacting with NP at the periphery of the NP clusters.

VP40 Is Critical for the Transport of NC-Like Structures and for Virion Incorporation Upon expression of NP, VP35, and VP24, NC-like structures were not found at the plasma membrane where Ebolavirus buds off, indicating that the formation and transportation of NCs to the cell surface are separate events and likely independently regulated. To determine which viral proteins are required for NC transport to the cell surface, we expressed the viral proteins involved in the formation of NC-like structures, other viral proteins (i.e., L, VP30, VP40, and GP), and the minigenome RNA in 293T cells. When VP40 was coexpressed with the proteins required for the NC-like structures (i.e., NP, VP24, and VP35), these structures were found immediately beneath the plasma membrane (Figure 2A) in an orderly arrangement, even in the absence of other viral proteins (i.e., L, VP30, and GP) and the minigenome viral RNA (unpublished data). Furthermore, VP40 proteins were detected by immunoelectron microscopy near the NC-like structures underneath the plasma membrane (Figure S2A). However, without VP40, NC-like structures at the plasma membrane could not be detected (Figure S2B). These results indicate that VP40 plays an important role in the transport of NC-like structures to the cell surface. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. VP40 Expression Is Essential for the Transport of NC-Like Structures to the Plasma Membrane (A) A micrograph of the transport of NC-like structures in cells expressing NP, VP24, VP35, and VP40. (A, inset) Enlargement of the boxed area depicting the cross section of six NC-like structures beneath the plasma membrane. (B) NC-like structures were incorporated into VLPs. Inside the VLPs, tubular NC-like structures are observed. NC-like structures released from VLPs broken during sample preparation can also be seen in the same field (arrow). (C and D) An NC-like structure (indicated by broken lines in D) residing along the central axis of a filamentous VLP. Bars, 2μm (A), 500 nm (B), or 100 nm (C). https://doi.org/10.1371/journal.ppat.0020099.g002 Expression of VP40 results in the formation of virus-like particles (VLPs), which are released from plasmid-transfected cells [4,5]. To determine whether NC-like structures are incorporated into VLPs, we examined the VLPs released from cells expressing NP, VP24, VP35, and VP40 by negative-staining electron microscopy. Smooth-surfaced, filamentous VLPs were found in the supernatants (Figure 2B). In most of the VLPs, NC-like structures were present along the central axis (Figure 2C and 2D), as is seen in NCs detected in authentic Ebola virions [8]. These observations indicate that VP40 alone is sufficient for NC incorporation into virions and that the surface membrane GP is not required for this event, unlike influenza virus glycoproteins [16].

VLP Budding Is Dependent on Microtubules To determine which cellular components are involved in the transport of the NC-like structures to the plasma membrane, we expressed NP, VP24, VP35, and VP40 in cells treated with an intracellular vesicular trafficking inhibitor (monensin), an actin polymerization inhibitor (cytochalasin D), a microtubule polymerization inhibitor (nocodazole), or a microtubule depolymerization inhibitor (taxol). Even when actin polymerization was disturbed by 10 μg/ml cytochalasin D (the effect of the drug was confirmed by a change in enhanced yellow fluorescent protein [EYFP-β] actin distribution visualized by fluorescence microscopy; not shown), the amounts of VP40 and NP detected in the supernatants of the drug-treated cells did not differ from those of untreated cells (Figure 3A), despite a previous report suggesting the involvement of actin in virion formation [17]. When vesicular transport was inhibited by 5 μM monensin (a condition under which the transport of the vesicular stomatitis virus glycoprotein was confirmed to be inhibited [18]; unpublished data), a significant effect on release of VP40 and NP was not observed (Figure 3A). By contrast, perturbation of the microtubule structures by either 10 μM nocodazole or 1 μM taxol (as indicated by changes in EYFP-tubulin distribution; not shown) led to a reduction in the levels of NP and VP40 detected in the culture supernatants by more than 56% for NP and more than 57% for VP40 (Figure 3A). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. VLP Budding Is Dependent on Microtubules 10 μM nocodazole (noc), 1 μM taxol (tax), 10 μg/ml cytochalasin D (cytD), or 5 μM monensin (mon) was added to cells 3 h after they were transfected with plasmids expressing (A) NP, VP24, VP35, and VP40, or (B) VP40 alone. At 16 h post-transfection, proteins in the cell lysates and supernatants were separated by SDS-PAGE and examined by Western blotting with anti-NP and anti-VP40 antibodies. Following nocodazol or taxol treatment, the amounts of both VP40 and NP (A) or VP40 (B) in the supernatants (i.e., efficiency of VLPs budding) were reduced. cont, mock-treated control. https://doi.org/10.1371/journal.ppat.0020099.g003 Using electron microscopy we next examined whether microtubule-perturbating drugs inhibit the transport of NC-like structures to plasma membrane. In the presence of nocodazole or taxol, we did not detect an appreciable difference in the intracellular localization of NC-like structures, by comparison to localization in the absence of these drugs (unpublished data). We therefore tested the effect of these drugs on VLPs produced by VP40 alone. We found that perturbation of microtubules with these drugs reduced VP40-induced VLP release into culture media by 62% for nocodazole and 64% for taxol (Figure 3B). This finding is consistent with a recent report that Ebolavirus VP40 directly associates with microtubules [19]. These data suggest that a microtubule-dependent pathway may be involved in the release of VLPs from cells, which likely explains the concomitant reduction of NC-like structures in the culture supernatant.

Interaction between NP and VP40 during the Formation of VP40-Induced VLPs When VP40 and NP are coexpressed, NP migrates beneath the plasma membrane [20], and large amounts of NP are detectable in the supernatant [21]. To make certain that the NP detected in the supernatant was indeed incorporated into VLPs and not released as free NP, we performed a floatation analysis with the supernatants of cells expressing either NP alone or NP and VP40. When NP was coexpressed with VP40 (Figure S3A), it was detected in fractions of low sucrose concentration (i.e., approximately 1.12 g/cm3, fraction number 3) and as free protein in fractions of higher sucrose concentration. However, when NP was expressed alone, it was not detected in the supernatant of the transfected cells (Figure S3B). These results suggest that NP exists in membrane-bound form in the supernatant of cells that express both NP and VP40. To obtain direct morphologic evidence that NP is incorporated into VLPs, we used transmission electron microscopy (TEM) to examine cells coexpressing NP and VP40. Following coexpression of NP and VP40, we observed filamentous VLPs containing NP helical structures (Figure 4A), which were not observed in VLPs produced by the expression of VP40 alone (Figure 4C). Indeed, helical NP structures were found even in VLPs that were just about to bud (Figure 4B, arrows). To demonstrate a direct interaction between VP40 and NP, we transfected 293T cells with a VP40-expressing plasmid alone or together with an NP-expressing plasmid and subjected the cell lysates and supernatants to immunoprecipitation with appropriate antibodies. VP40 coimmunoprecipitated with NP, and conversely, NP with VP40, demonstrating the direct interaction between these two proteins (Figure S4A and S4B). These results strongly suggest that the interaction between NP and VP40 is responsible for the incorporation of NCs into virions. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. NP Helices Are Incorporated into VLPs (A and B) In cells coexpressing NP and VP40, NP helices can be seen in newly released VP40-induced VLPs (A, arrow), as well as in VLPs just about to bud (B, arrows). (C) Empty VLPs released from cells expressing VP40 alone. Bars, 100 nm (A and C) or 500 nm (B). https://doi.org/10.1371/journal.ppat.0020099.g004