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Virus entry into host cells is the first step of infection and a crucial determinant of pathogenicity. Here we show that Ebola virus-like particles (EBOV-VLPs) composed of the glycoprotein GP(1,2) and the matrix protein VP40 use macropinocytosis and clathrin-mediated endocytosis to enter cells. EBOV-VLPs applied to host cells induced actin-driven ruffling and enhanced FITC-dextran uptake, which indicated macropinocytosis as the main entry mechanism. This was further supported by inhibition of entry through inhibitors of actin polymerization (latrunculin A), Na(+)/H(+)-exchanger (EIPA), and PI3-kinase (wortmannin). A fraction of EBOV-VLPs, however, colocalized with clathrin heavy chain (CHC), and VLP uptake was reduced by CHC small interfering RNA transfection and expression of the dominant negative dynamin II-K44A mutant. In contrast, we found no evidence that EBOV-VLPs enter cells via caveolae. This work identifies macropinocytosis as the major, and clathrin-dependent endocytosis as an alternative, entry route for EBOV particles. Therefore, EBOV seems to utilize different entry pathways depending on both cell type and virus particle size.

The Zaire Ebola virus (EBOV) protein VP35 is multifunctional; it inhibits IFN-α/β production and functions as a cofactor of the viral RNA polymerase. Mass spectrometry identified the double stranded RNA binding protein 76 (DRBP76/NFAR-1/NF90) as a cellular factor that associates with the VP35 C-terminal interferon inhibitory domain (IID). DRBP76 is described to regulate host cell protein synthesis and play an important role in host defense. The VP35-IID-DRBP76 interaction required the addition of exogenous dsRNA, but full-length VP35 associated with DRBP76 in the absence of exogenous dsRNA. Cells infected with a Newcastle disease virus (NDV)–expressing VP35 redistributed DRBP76 from the nucleus to the cytoplasm, the compartment in which EBOV replicates. Overexpression of DRBP76 did not alter the ability of VP35 to inhibit type I IFN production but did impair the function of the EBOV transcription/replication complex. These data suggest that DRBP76, via its association with VP35, exerts an anti-EBOV function.

VP24, the smallest of the 7 structural proteins of Ebola virus (EBOV) [1], is membrane associated [2]. Although the function of VP24 is not fully understood, it is multifunctional, because (1) together with NP and VP35, it is necessary for the formation of nucleocapsid-like structures [3]; (2) it is necessary for the formation of a functional viral ribonucleoprotein (vRNP) complex [4]; (3) it has an inhibitory effect on the transcription and replication of the EBOV genome through its association with the vRNP complex [5]; and (4) it functions as an interferon antagonist by binding to karyopherin α, which is required for nuclear import of phosphorylated STAT1, a component of the Jak-STAT signal transduction pathway [6, 7]. In the present study, to better understand the function of VP24, we first identified host proteins that interact with VP24 by using coimmunoprecipitation and mass spectrometry. Of the 68 candidate host proteins identified, we selected and investigated the role in EBOV replication of Sec61α, a component of the heterotrimeric Sec61 complex that plays a critical role in promoting protein translocation at the ER membrane [8–10]. Sec61α was chosen because its intracellular distribution is similar to that of VP24 (ie at the perinuclear region) and because of its involvement in various biological functions [11] and its roles in the pathogenesis of type 2 diabetes and hepatosteatosis [12–14].

The matrix protein VP40 of Marburg virus promotes the formation and release of virus-like particles (VLPs). Marburg virus VP40 interacts with cellular Tsg101 via its L domain motif; however, mutation of this motif does not affect VLP budding or the accumulation of VP40 in multivesicular bodies (MVBs), which are platforms for virus particle formation. To identify regions of Marburg virus VP40 that are important for VLP budding, we examined deletion mutants and alanine-scanning mutants at the N- and C-terminus of VP40 for their involvement in VLP budding. VLPs were not detected in the presence of alanine-replacement mutants at Ile39 and Thr40, and the level of VLP budding for the alanine mutant at Asn297 was decreased. Moreover, these mutants did not accumulate in MVBs. Our results suggest the involvement of a novel host factor(s) in VLP budding and VP40 transport to MVBs.

Marburg virus (MARV) and the closely related Ebola virus (EBOV) belong to the filovirus family and cause a severe hemorrhagic fever in humans, with mortality rates up to 90%. Currently, there is no approved vaccine or antiviral treatment. Filoviruses have a nonsegmented negative-sense RNA genome encoding 7 structural proteins. Four of these proteins constitute the nucleocapsid complex, containing the nucleoprotein (NP), the viral polymerase L, the polymerase cofactor VP35, and the viral protein VP30 in close association with the viral genome (for review, see [1]). Cytoplasmic inclusions, which are thought to represent active sites of viral replication, are present as large aggregates in filovirus-infected cells. These inclusions are formed by all 4 nucleocapsid proteins, with NP being the driving force for aggregation due to self-assembly of NP [2, 3]. NP interacts with VP35, VP30, and L, either directly or via a linker protein, thereby redirecting the nucleocapsid proteins into cytoplasmic aggregates [4–8]. Rescue systems to recover infectious virus from full-length complementary DNA (cDNA) clones have been established for both MARV and EBOV [9–13]. These techniques were used to generate recombinant forms of EBOV, derived from isolates of the Zaire ebolavirus (ZEBOV) species, containing the enhanced green fluorescent protein (EGFP) gene within an additional transcription unit (ATU), which provide a sensitive and quantitative readout for antiviral drug screening assays and virus spread studies [14–21]. EGFP was efficiently expressed over 10 passages, confirming the stability of the EBOV constructs [15]. In this study, we rescued a recombinant MARV from a clone containing an ATU encoding EGFP. This clone allows for the visualization of MARV spread in infected cells and was used to assess the localization of EGFP and nucleocapsid proteins in infected cells.

Ebola virus VP35 is a cofactor of the viral RNA polymerase complex and, together with NP and VP24, is an essential component for nucleocapsid formation. In the present study, we examined the interactions between VP35 and NP and found that VP35 interacts with helical NP-RNA complexes through the C-terminus of NP. We also found that coexpression of excess VP35 with NP reduced the yields of NP-RNA complexes purified by CsCl gradient ultracentrifugation and inhibited the formation of the NP-induced inclusion bodies that typically form in Ebola virus-infected cells. These findings suggest that the NP to VP35 ratio is important in the Ebola virus replication cycle and advance our knowledge of nucleocapsid morphogenesis.

Background. sGP, which is perceived as nonstructural, secretory glycoprotein, shares 295 amino acids at its N-terminal with GP1,2, which include the specific residue necessary to interact with GP2. In the present study, we tested whether the sGP protein of Zaire ebolavirus (ZEBOV) could substitute for GP1 and form a complex with GP2, thus serving as a structural protein. Methods. We expressed ZEBOV GP1,2, VP40, and NP proteins, together with sGP protein, from expression plasmids and examined the resultant virus-like particles by using Western blot. Cells expressing GP2 in combination with either GP1 or sGP were analyzed by using flow cytometry with the KZ52 antibody, which recognizes a GP1,2 conformational epitope. A VSV pseudotype, VSVΔG*, which expresses a GFP reporter gene instead of the G protein, was used to produce pseudotyped viruses encoding sGP and variants of GP to test the contribution of sGP to infectivity. Results. Western blot and flow cytometric analyses suggested the existence of a covalently linked sGP-GP2 molecule. VSVΔG*(sGP + GP2) and VSVΔG*(GP1,2) infected Vero E6 cells and were neutralized by the KZ52 antibody. Overexpression of sGP reduced the titer of VSVΔG*(GP1,2). Conclusions. ZEBOV sGP can substitute for GP1, forming a sGP-GP2 complex and conferring infectivity. Our studies suggest a novel role for sGP as a structural protein.

Type I interferon (IFN) signaling is mediated through several signaling pathways, including the Janus kinase and signal transducer and activator (JAK-STAT) and p38 mitogen-activated protein (MAP) kinase pathways. The VP24 protein of Ebolavirus is an IFN antagonist, blocking type I IFN signaling through the JAK-STAT pathway. Here, we show that, in 293 cells, VP24 also interferes with the p38 MAP kinase pathway by blocking IFN-β–stimulated phosphorylation of p38-α. Similar inhibition was not observed in HeLa cells, suggesting cell type–specific differences in signal transduction.

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean–Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

Infection with Ebola virus (EBOV) causes hemorrhagic fever in humans with high case-fatality rates. The EBOV-glycoprotein (EBOV-GP) facilitates viral entry and promotes viral release from human cells. African fruit bats are believed not to develop disease upon EBOV infection and have been proposed as a natural reservoir of EBOV. We compared EBOV-GP interactions with human cells and cells from African fruit bats. We found that susceptibility to EBOV-GP-dependent infection was not limited to bat cells from potential reservoir species, and we observed that GP displayed similar biological properties in human and bat cells. The only exception was GP localization, which was to a greater extent intracellular in bat cells as compared to human cells. Collectively, our results suggest that GP interactions with fruit bat and human cells are similar and do not limit EBOV tropism for certain bat species.

Background. The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) augments Ebola virus (EBOV) infection. However, it its unclear whether DC-SIGN promotes only EBOV attachment (attachment factor function, nonessential) or actively facilitates EBOV entry (receptor function, essential). Methods. We investigated whether DC-SIGN on B cell lines and dendritic cells acts as an EBOV attachment factor or receptor. Results. Engineered DC-SIGN expression rendered some B cell lines susceptible to EBOV glycoprotein (EBOV GP)-driven infection, whereas others remained refractory, suggesting that cellular factors other than DC-SIGN are also required for susceptibility to EBOV infection. Augmentation of entry was independent of efficient DCSIGN internalization and might not involve lectin-mediated endocytic uptake of virions. Therefore, DC-SIGN is unlikely to function as an EBOV receptor on B cell lines; instead, it might concentrate virions onto cells, thereby allowing entry into cell lines expressing low levels of endogenous receptor(s). Indeed, artificial concentration of virions onto cells mirrored DC-SIGN expression, confirming that optimization of viral attachment is sufficient for EBOV GP-driven entry into some B cell lines. Finally, EBOV infection of dendritic cells was only partially dependent on mannose-specific lectins, such as DC-SIGN, suggesting an important contribution of other factors. Conclusions. Our results indicate that DC-SIGN is not an EBOV receptor but, rather, is an attachmentpromoting factor that boosts entry into B cell lines susceptible to low levels of EBOV GP-mediated infection.

International audience; The Ebola virus matrix protein VP40 plays an essential role in virus assembly and budding. In this study we reveal that transient VP40 expression results in the release into the culture medium of substantial amounts of soluble monomeric VP40 in addition to the release of virus-like particles containing an oligomeric form of this protein as previously described. We show that VP40 secretion is endoplasmic reticulum/Golgi-independent and is not associated with cell death. Soluble VP40 was observed during Ebola virus infection of cells and was also found in the serum of virus-infected animals albeit in lower amounts. Unconventional secretion of VP40 may therefore play a role in Ebola virus pathogenicity.

The virion protein 40 (VP40) and nucleoprotein (NP) of Ebola (EBOV) and Marburg viruses (MARV) play key roles during virion assembly and egress. The ability to detect interactions between VP40-VP40, VP40-NP, and NP-NP and follow these complexes as they traffic through mammalian cells would enhance our understanding of the molecular events leading to filovirus assembly and budding, and provide new insights into filovirus replication and pathogenesis. Here, we successfully employed a bimolecular complementation (BiMC) approach to visualize interactions between EBOV and MARV VP40-VP40, NP-NP, and VP40-NP proteins and localize these protein complexes in mammalian cells using confocal microscopy. We demonstrate that VP40-VP40 complexes localized predominantly at the plasma membrane, whereas VP40-NP and NP-NP complexes displayed a more dispersed pattern throughout the cytoplasm. As expected based on previous findings, efficient interactions between EBOV or MARV VP40-VP40 proteins were independent of L-domains PTAPPEY and PPPY, respectively. In contrast, the formation of EBOV or MARV VP40-VP40 complexes was dependent on the previously characterized LPLGVA and LPLGIM motifs of EBOV and MARV VP40 proteins, respectively, indicating that these motifs are important for VP40 oligomerization and subsequent budding. These results highlight the feasibility and usefulness of the BiMC approach as a strategy to further characterize both filovirus protein interactions as well as filovirus-host interactions in real time in the natural environment of the cell.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.