Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol

Abstract

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP_1,2 or the subunit GP₂ depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

ARTICLE

Received 1 May 2015 | Accepted 29 May 2015 | Published 9 Jul 2015

DOI: 10.1038/ncomms8688

Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol

Moritz Hacke1, Patrik Bjrkholm2, Andrea Hellwig3, Patricia Himmels1, Carmen Ruiz de Almodvar1, Britta Brgger1, Felix Wieland1 & Andreas M. Ernst1

The high pathogenicity of the Ebola virus reects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufcient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

1 Heidelberg University Biochemistry Center (BZH), 69120 Heidelberg, Germany. 2 Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, 75124 Uppsala, Sweden. 3 Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg 69120, Germany. Correspondence and requests for materials should be addressed to A.M.E. (email: mailto:[email protected]

Web End [email protected] ).

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The current Ebola virus outbreak is the largest and most severe in the history of this virus. Ebola virus poses a threat to thousands of people in West Africa, with outbreaks

reported in Guinea, Liberia, Sierra Leone and Nigeria1,2. Despite the devastating consequences of Ebola virus infection, the treatment options remain limited and experimental. Recently, the use of cholesterol-reducing agents as an adjuvant therapy to reduce the sepsis-like side effects of Ebola virus disease has been suggested3,4. The dysregulation of the immune response and a heavy inammatory reaction, described as a cytokine storm, are induced on EBOV infection5. Whether the detachment of infected endothelial cells and the concomitant leakage of the endothelial barrier are important factors in the pathogenesis of Ebola virus disease remains controversial611. Among other factors, the cytotoxicity of EBOV has been attributed to the envelope glycoprotein (GP)6; however, the molecular mechanism underlying the pathogenicity of this virus is unclear. GP is an essential virulence factor that plays a key role in viral entry, membrane fusion, epitope shielding and EBOV assembly1217. Virulence has been attributed not only to the membrane-anchored GP but also to the soluble form of this protein, sGP, which results from RNA editing18. sGP increases endothelial permeability by acting as an activator of neutrophils and macrophages, which regulates the host immune response1922.

Recently, GP shedding was shown to impact endothelial permeabilization23. In addition, the interaction between b1-integrin and GP was shown to contribute to the loss of adhesion of GP-expressing cells7,24. Furthermore, a role for the GP2 subunit in counteracting interferon-induced antiviral response via the protein tetherin (BST-2) has been proposed25.

GP is a type I membrane protein that exhibits high N- and O-glycosylation, and undergoes proteolytical cleavage through furin in the trans-Golgi network, which yields the large amino-terminal GP1 subunit and the small carboxy-terminal

GP2 subunit26. The GP2 subunit contains a putative fusion peptide and a transmembrane domain (TMD) and is palmitoylated twice on the short cytoplasmic tail27,28. Owing to an intramolecular disulde bond, GP1 and GP2 do not dissociate on furin cleavage29. The mature GP forms a trimer that comprises of disulde-bridged GP1,2 subunits30,31. The ectopic expression of

GP is sufcient to induce the detachment of adherent cells32, and a mucin-like domain within subunit GP1 has been suggested to strongly impact this process6. Cell detachment accounts for the loss of the endothelial barrier of infected blood vessels, which putatively leads to vascular leakage and contributes to the high pathogenicity of the virus6,9. Ebola virions are lamentous in shape, and the matrix protein VP40 and GP are sufcient to produce protrusions at the plasma membrane of adherent cells33,34. The protrusions resulting from VP40 are virus like, whereas the structures resulting from GP alone appear pleiomorphic35,36. The combination of VP40 and GP is sufcient to produce lamentous virus-like particles that exhibit the morphology of Ebola virions37,38.

Here we report that GP-induced cell detachment might be attributed to the GP2 subunit and that cytotoxicity is strongly impacted through cholesterol and a GXXXA motif in the TMD. Furthermore, we show that the GP2 subunit induces lamentous protrusions at the plasma membrane that also depend on cholesterol and the GXXXA motif, which implies a role for GP in the assembly of the lamentous Ebola virus.

ResultsA mucin-like domain within GP has been attributed to the induction of cell detachment6. The isolated GP2 subunit has not been analysed in this context, and notably, the contribution of

GP2 to cell detachment or the production of virus-like laments was not observed using yellow uorescent protein (YFP)-GP2 fusions25. These results left a possibility that the mucin-like domain, rather than being involved in mediating detachment, acts as a spacer to maintain an appropriate distance between GP1 and

GP2 and thereby allow the interaction of GP2 subunits (Fig. 1a). In this case, the deletion of the mucin-like domain then would reduce the relative distance of GP1 to the membrane, causing GP1 to interfere with the oligomerization of GP2. Accordingly, YFP-GP2 might not induce cell detachment, as the bulky YFP moiety may have taken the role of GP1 in the GP1,2-Dmucin construct, potentially disturbing GP2 oligomerization and masking the contribution of this protein to cell detachment.

FLAG-GP2 induces protrusions at the plasma membrane. We observed the presence of a seam-like arrangement of Gly and Ala residues with a central G/A/S-XXX-G/A/S motif in the TMD of GP2. These motifs are well-established hubs for

TMDTMD interactions39; thus, these motifs likely contribute to the oligomerization of GP. To challenge the hypothesis that TMDTMD interactions might be disrupted due to the steric hindrance resulting from the presence of a bulky protein (unduly close GP1 in the mucin-like domain deletions or YFP in

YFP-GP2), we designed constructs based on the GP from Ebola Zaire (ZEBOV) by using a small FLAG tag directly fused to the GP2 domain (as depicted in Fig. 1a; Supplementary Table 2). This tag, which contained eight amino acids (with a mass of B2 kDa, including the linker peptide), was not expected to exert strong steric hindrance.

Surprisingly, immunouorescence and scanning electron microscopy revealed that the transient transfection of HeLa and various other cell lines (Supplementary Fig. 1) with FLAG-GP2 constructs resulted in the drastic formation of laments at the plasma membrane (Fig. 1b,c and Fig. 2). The transfection of FLAG-GP1,2 led to the formation of dot-like structures (Fig. 2a,b, left panel) that likely resemble the previously described GP1,2-induced pleiomorphic structures36. The FLAG-GP2-induced laments were, on average, 14.70.44 mm in length, and B50 nm in diameter (Supplementary Fig. 3a). The observed lament length is consistent with the dimensions observed for the virus-like particles produced through VP40 and GP1,2 (ref. 36).

The observed reduction in diameter (80 nm for virions) might reect the lack of other viral proteins. The laments exhibited a high extent of peripheral branching, with ne and widespread extensions (Fig. 1b,c). Strikingly, a strong reduction in the volume of the cells was observed concomitant with the production of these laments and frequently resulted in rod-like cell remnants (Fig. 1b; Supplementary Movies 13).

Cytotoxicity of GP is encoded in its GP2 subunit. Next, we analysed the potential induction of HeLa cell detachment through FLAG-GP2. Although HEK cells are frequently used to study

GP-induced detachment6,32, we performed quantitative measurements in HeLa cells, which adhere more strongly and are less prone to detachment than HEK cells. HeLa cells were transfected with either FLAG-GP2 or FLAG-GP1,2 constructs for various times, and the ratio of detached to adherent cells was determined through ow cytometry (Fig. 1d). As a control, we employed plasma membrane-resident (Supplementary Fig. 3f) asialoglycoprotein receptor 1 (ASGR1), which forms trimers similar to Ebola GP. At 36 h post transfection, B60% of the

FLAG-GP2-transfected cells were detached, whereas ASGR1-transfected cells did not exceed 10% detachment, although the protein levels were higher (Fig. 1d). Importantly, FLAG-GP1,2-

transfected cells achieved a similar degree of detachment (B70%,

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Figure 1 | The GP2 subunit of Ebola GP leads to lament formation at the plasma membrane of HeLa cells and their detachment. (a) Constructs. All constructs are based on ZEBOV GP. For GP1,2 constructs, a linker-anked FLAG tag was positioned between the signal sequence and the GP1 subunit. For GP2, the signal sequence of GP was fused to a linker-anked FLAG tag, followed directly by GP2. GP variants are abbreviated as GP2-LXXXL ( FLAG-

GP2-G211LA215L), and GP1,2-GXXXL ( FLAG-GP1,2-A215L). The respective domains are drawn to scale. SP, signal peptide; FLAG (epitope); MLR,

mucin-like region; FP, fusion peptide. Gly and Ala residues seaming the TMD are highlighted (yellow boxes). (b) Immunouorescence analysis of a HeLa cell transiently transfected with FLAG-GP2. Green, FLAG-GP2; red, actin; blue, nucleus. Scale bar, 10 mm. (c) Scanning electron microscopy of HeLa cells transiently transfected with FLAG-GP2 at different magnications. Dashed lines (in b,c): areas of inset. Scale bars indicate 10 mm (top), 5 mm (middle)

and 1 mm (bottom). (d) Detachment of HeLa cells transiently transfected with either FLAG-GP1,2, -GP2, -ASGR1 (as a control) or mock-transfected cells within 36 h. Data points represent the means.d. of three independent experiments (N 3). The ratio of adherent to detached cells was

determined by ow cytometry. Black diamonds, GP1,2-transfected cells; light black triangles, GP2-transfected cells; dark grey squares, ASGR1-transfected cells; light grey circles, mock-transfected cells.

after 36 h), which indicated that the propensity to induce detachment is attributed to the GP2 subunit and not the mucin-like domain within GP1 as previously reported6.

A GXXXA motif in the TMD of GP impacts cytotoxicity. Single G/A/S-XXX-G/A/S motifs are able to mediate TMDTMD interactions39. We therefore speculated that the presence of multiple repeats of these motifs, aligned on one side of the TMD of GP (Supplementary Fig. 5b), would enhance GP oligomerization and potentially inuence the extent of lament formation and detachment. To this end, we changed the central GXXXA motif in FLAG-GP2 to LXXXL (as highlighted in Fig. 1a and Supplementary Fig. 5b). This mutant was analysed through immunouorescence and scanning electron microscopy. Notably, lament formation was strongly inhibited, with the average lengths reduced to 4.70.33 mm (Supplementary Fig. 3a). When the propensity of the mutant to confer detachment was analysed, a signicant B50% reduction was observed compared with

wild-type FLAG-GP2 (Fig. 3b), which suggested that the GXXXA motif contributes to the extent of lament formation and GP2-mediated cell detachment. Strikingly, a single point mutation in full-length FLAG-GP1,2, yielding GP1,2-GXXXL, exhibited a drastic reduction of 450% in detachment compared with the wild-type construct (Fig. 3a).

On the basis of the immunouorescence analysis of cells expressing FLAG-GP2 and co-stained for actin, we observed microlaments at the basis of the FLAG-GP2-induced laments (Fig. 1b and Fig. 2a,b). We therefore incubated the cells with cytochalasin B (CB), an inhibitor of actin lament polymerization, and observed that GP2-transfected cells no longer exhibited GP-induced laments, although FLAG-GP2 was signicantly localized to the plasma membrane (Supplementary Fig. 3d), which suggested a role for actin in the observed phenotype, that is, potentially acting as a scaffold for GP-induced laments.

Cholesterol impacts GP-induced cytotoxicity. Notably, the endomembrane system in FLAG-GP1,2- and FLAG-GP2-

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FLAG-GP1,2

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Figure 2 | The TMD of GP2 affects the morphology of GP2-induced laments. (a) HeLa cells were transiently transfected with FLAG-GP1,2, -GP1,2-GXXXL, -GP2 or -GP2-LXXXL for 24 h. The cells were xed, and FLAG-tagged proteins were visualized by immunouorescence. Green, a-FLAG; red, actin; blue, nucleus. Dashed lines, area of inset. Scale bar, 10 mm. (b) Magnications of the areas indicated in a. Scale bar, 5 mm. (c) Scanning electron microscopy images of cells transfected with the indicated constructs. Scale bar, 5 mm. (d) MBCD abolishes GP2-induced laments at the plasma membrane.

Immunouorescence analysis of HeLa cells transiently transfected with FLAG-GP2 for 24 h, and incubated with a nal concentration of 0.5 mM MBCD 6 h post transfection. Scale bar, 20 mm. (e) GP variants exhibit differences in their ability to bind cholesterol. Photoafnity labelling of full-length FLAG-GP1,2,

-GP1,2-GXXXL, -GP2 or -GP2-LXXXL using tritiated, photolabile cholesterol. HeLa cells were transiently transfected with the indicated constructs for 24 h. After 16 h, tritiated photocholesterol was added. The cells were then ultraviolet irradiated, lysed, subjected to immunoprecipitation against the FLAG epitope and analysed by western blot and digital autoradiography (Supplementary Fig. 8). AR, autoradiography; WB, western blot.

transfected cells appears unaffected (Supplementary Fig. 2a,b). The partitioning of GP into (cholesterol-dependent) detergent-resistant membranes40 suggested that in addition to the GXXXA motif, the oligomerization of GP might be affected through the increased level of cholesterol at the plasma membrane. A cholesterol gradient exists across the secretory pathway41 (low in the endoplasmic reticulum (ER) and high in the plasma membrane), and would hence allow the formation of laments at the plasma membrane, but not in the endomembrane system.

To analyse the specic interactions of GP with cholesterol, we utilized a well-established photoafnity-labelling assay4245. Radioactively labelled (tritiated) photoactivatable cholesterol was fed to HeLa cells expressing either FLAG-GP1,2, FLAG-GP2 or their respective variants. At 24 h post transfection, the cells were irradiated with ultraviolet, thereby crosslinking the photolabile cholesterol analogue to the proteins and lipids in the immediate proximity (r3 ). As a control, the equally trimeric and plasma membrane-resident protein ASGR1 was employed. Although ASGR1 exhibited weak labelling despite high expression levels (Supplementary Fig. 3e), both FLAG-GP1,2 and

FLAG-GP2 were highly and efciently labelled (Fig. 2e). SDS-resistant oligomers of FLAG-GP2 corresponding to dimers and trimers were observed. Strikingly, a strong reduction in photocholesterol labelling was observed for both FLAG-GP1,2-

GXXXL and FLAG-GP2-LXXXL (Fig. 2e). The reduction in cholesterol labelling of the GXXXA motif point mutants was correlated with a strong decrease in cell detachment of B50%, respectively (Fig. 3a,b). Therefore, we next analysed the effect of lowering cholesterol in the plasma membrane of FLAG-GP2 and

FLAG-GP1,2-transfected cells with regard to lament production and cell detachment. Methyl-b-cyclodextrin (MBCD) is a well-established tool for lowering the cholesterol content in the plasma membranes of mammalian cells46. We exposed cells transfected with full-length FLAG-GP1,2, FLAG-GP2 or variants mutated in the central GXXXA motif to 0.5 mM MBCD at 6 h post transfection, which signicantly lowered the levels of cholesterol at the time of analysis 24 h post transfection (425%;

Supplementary Fig. 4a). Subsequently, we measured the extent of detachment induced through the respective proteins in the presence and absence of MBCD using ow cytometry and

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a d

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Figure 3 | MBCD suppresses the detachment of cells induced by Ebola GP. (a,b) HeLa cells were transfected with either FLAG-GP1,2, its mutant FLAG-

GP1,2-GXXXL, FLAG-GP2 or FLAG-GP2-LXXXL for 24 h. Six hours post transfection, 0.5 mM MBCD was added. The ratio of adherent to detached cells was determined by ow cytometry. (c) The inhibitory effect of MBCD on GP-induced detachment is overcome by the addition of exogenous cholesterol. Cells transfected with FLAG-GP1,2 were treated with 5 mM MBCD for 30 min before transfection. Cholesterol was replenished by incubation with 100 mM

MBCD-complexed cholesterol for 30 min. (d) Detachment is reduced in statin-treated cells. Cells were cultured in 2 mM simvastatin for 4 days. Cells transfected with FLAG-GP1,2 are compared with cells without statin treatment. (e) Exogenous cholesterol increases GP-induced detachment. Cells transfected with FLAG-GP1,2 were treated 6 h post transfection with 100 mM MBCD-complexed cholesterol for 30 min. Data points represent the means.d. from three independent experiments (N 3). Detachment of ASGR1-transfected cells was subtracted as unspecic detachment. The results of

unpaired, two-tailed t-tests are given. *Po0.05; **Po0.01; ***Po0.001. (f) Titration of MBCD to GP-expressing cells. Cells were transfected with GP1,2 or

GP2 and exposed to different concentrations of MBCD as indicated. The data points represent the mean of three independent experiments (N 3). Error

bars represent the s.e.. Best-t values for a sigmoidal dose response of GP1,2 (black solid line) and GP2 (dashed grey line) are given. The IC50 of MBCD-mediated inhibition of detachment by GP1,2 was 197.5 mM25.44 s.e., and 295.1 mM98.52 s.e. for GP2. (g) Cholesterol enriches in GP2-induced plasma membrane protrusions. HeLa cells were transfected with FLAG-GP2 or FLAG-GP2-LXXXL, and treated with 0.5 mM MBCD 6 h post transfection. Cells were xed and stained with lipin (cholesterol) and anti-FLAG antibodies. Green, FLAG-GP2/FLAG-GP2-LXXXL; red, cholesterol. Scale bar, 20 mm.

immunouorescence. Strikingly, MBCD signicantly reduced (450%) the level of detachment induced through FLAG-GP1,2

and FLAG-GP2 constructs (Fig. 3a,b), whereas for FLAG-GP1,2-GXXXL and FLAG-GP2-LXXXL, detachment was not affected after treatment with MBCD (Supplementary Fig. 3b). Treatment with MBCD alone did not signicantly induce the detachment of cells, changes in morphology or interfered with expression of GP (Supplementary Figs 3c, 4c and 7). Accordingly, when we incubated the cells with the statin simvastatin before transfection, a signicant reduction in the cholesterol levels was

observed (430%; Supplementary Fig. 4b), and GP-induced detachment was signicantly reduced B40% (Fig. 3d). Interestingly, the addition of cholesterol to cells using cholesterol-loaded MBCD strongly increased GP-induced cell detachment (Fig. 3e). Furthermore, the inhibitory effect of MBCD on GP-induced detachment was restored when the cells were incubated with MBCD-complexed cholesterol (Fig. 3c).

Next we expanded the analysis of loviral GP2 subunits to Marburg virus (MARV) and Lloviu cuevavirus (LLOV), the only two remaining genera of Filoviridae47,48. Comparisons of

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the TMDs of the respective GP2 subunits of these viruses (Supplementary Fig. 5a) revealed that EBOV and LLOV share a high extent of sequence identity, including the G/A/S-XXX-G/A/S motif, but that MARV lacks a comparable sequence (Supplementary Fig. 5b). Analogous to EBOV GP2, we examined

FLAG-LLOV- and FLAG-MARV- GP2 for the induction of cell detachment and production of plasma membrane protrusions. MARV FLAG-GP2 induced neither virus-like-lament formation at the plasma membrane, nor signicant cell detachment, while the GXXXA motif containing LLOV FLAG-GP2 triggered cell protrusions and cell detachment comparable to EBOV FLAG-GP2, and this effect was susceptible to treatment with

MBCD (Supplementary Fig. 5c,d). These data highlight the correlation between the presence of G/A/S-XXX-G/A/S motifs in the TMD of loviral GP, cell detachment and cholesterol.

Next we employed the cholesterol-specic probe lipin to examine the enrichment of cholesterol in GP2-induced cell protrusions49. Although GP2-expressing cells are efciently labelled at the plasma membrane and its lamentous protrusions, staining of the plasma membrane is strongly reduced in MBCD-treated cells (concomitant with the absence of protrusions), and FLAG-GP2-LXXXL-expressing cells exhibit low lipin co-staining (Fig. 3g). We speculate that the co-aggregation of cholesterol into Ebola virions and the concomitant depletion of plasma membrane cholesterol might also contribute to pathogenicity, as the sequestration of cholesterol would also likely affect the function of cholesterol-interacting receptors and signalling proteins in the plasma membrane, such as the T-cell receptor or MHC class II molecules50,51.

Altogether, these data suggest that the GXXXA motif mediates interactions among GP trimers, generating higher, cholesterol-competent, GP oligomers. Thus, the mutation of this motif correlates with a loss of MBCD sensitivity and a marked reduction in detachment. The titration of MBCD revealed a sigmoidal dose response for both GP1,2 and GP2 constructs, with half-maximal inhibition of detachment at similar concentrations (FLAG-GP1,2:

197.5 mM25.4 s.e., and FLAG-GP2: 295.1 mM98.5 s.e.; Fig. 3f). Thus, we concluded that the cytotoxicity conferred through GP depends on cholesterol and is additionally inuenced by the presence of a GXXXA motif in the TMD. Although these data strongly implicated cholesterol as the major lipid involved in cytotoxicity, we cannot exclude that other lipids were indirectly affected in these experiments. A reduction in the cholesterol levels at the plasma membrane might reect differences in the composition of specic lipid microdomains targeted through GP, for example, polyunsaturated phospholipids resulted in deformations of the plasma membrane52, and phosphatidylserine levels in the cytoplasmic leaet might also be affected.

Nonetheless, these data suggest that cholesterol-reducing treatments are potential candidates for therapeutics to counteract the pathogenicity induced through Ebola GP; these treatments could also be employed after infection with Ebola virus as an addition to the established post exposure regimens5360. Cyclodextrins have been patented as antiviral agents for the treatment of infections resulting from various enveloped viruses61. Treatment with MBCD is even more promising because the IC50 reported here is B20-fold lower than those determined for the herpes, vaccinia, EpsteinBarr and hepatitis C viruses61.

FLAG-GP2-induced laments form by retraction. We next monitored the production of the FLAG-GP2-induced laments through live-cell imaging using a TIRF-DIC (total internal reection uorescence-differential interference contrast) microscope set-

up. As depicted in Fig. 4c and in Supplementary Movie 1, after 370 min and within the course of B70 min, the cells contract with lamentous structures left behind while the cell body retracts, which suggests that the laments are formed through retraction rather than outgrowth. Moreover, bubbles were observed underneath the cell, and the cells rounded up before eventual detachment, which suggests a correlation between the production of plasma membrane protrusions and cell detachment.

A subset of host proteins exhibits a GP-like TMD arrangement. Subsequently, we analysed the amino-acid sequences of host single-spanning plasma membrane-resident proteins to characterize the seam-like arrangement of Gly and Ala residues present in the TMD of GP. Strikingly, only 26 proteins exhibited more than ve Gly, Ala or Ser residues within a seam-like arrangement in the TMD. A total of 14 of these candidates were implicated in cell adhesion or cytoskeleton organization (Fig. 4d, Supplementary Table 1). Among these TMDs are known inter-actors of GP, such as b1-integrin24. Nine proteins involved in the immune response (for example, the a-chain of HLA class II) were also identied. These ndings suggest that GP1,2 might bind to and interfere with these candidates via the GXXXA motif.

DiscussionAs depicted in Fig. 4a, we propose the following model for the formation of GP-induced lamentsat the onset of assembly, GP-TMDs trimerize in a cholesterol-independent manner. During Ebola virus assembly, trimeric GP subunits interact within cholesterol-enriched membrane domains via GXXXA-motifs in the TMDs, generating a lattice-like structure that predominantly contains GP and excludes host proteins (Fig. 4a, centre). When a critical lattice size is formed, this membrane domain collapses around an actin lament that serves as a scaffold for plasma membrane lament formation (Fig. 4b). Matrix protein VP40 participates in this process through oligomerization at the cytoplasmic face of the plasma membrane34,62 (Supplementary Fig. 6), further stabilizing the lamentous structures in the viral context and putatively additionally exerting an outward-directed force on the plasma membrane.

In summary, based on the results obtained in the present study, we propose a novel mechanism for Ebola virus assembly at the plasma membrane, based on cholesterol-dependent interactions among GPs, leading to the collapse of large GP aggregates around actin laments. Rather than through outgrowth, the formation of these laments reects the growth of GP aggregates within the plasma membrane and the subsequent wrapping of these aggregates around actin scaffolds (Fig. 4a,b). This mechanism might be closely associated with cell detachment, and suggests two novel drug targets to counter GP-mediated cell detachment cholesterol, which could be targeted using cholesterol-reducing agents, such as MBCD and statins, and the central GXXXA-motif situated within a seam-like assembly of Gly and Ala residues in the TMD of GP.

Methods

Reagents and antibodies. MBCD, simvastatin, CB, water-soluble cholesterol, phalloidin-conjugated to Atto 565, Filipin III and EZview red anti-FLAG M2 afnity gel were purchased from Sigma-Aldrich. Prolong Gold Antifade reagent containing 40,6-diamidino-2-phenylindole, 5-hexadecanoylaminouorescein and

WGA conjugated with Alexa 647 were purchased from Life Technologies. Cell dissociation buffer was purchased from Gibco. The following antibodies were purchased from Life TechnologiesAlexa Fluor 546 goat anti-mouse, Alexa Fluor 546 goat anti-rabbit, Alexa Fluor 546 donkey anti-sheep, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 488 goat anti-mouse. Anti-FLAG mouse and anti-FLAG rabbit were purchased from Sigma. Anti-GM130 mouse was purchased from BD-

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a

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Figure 4 | A novel role for GP in virus assembly at the plasma membrane. (a) Model depicting the proposed initial steps in the formation of GP2-based domains at the plasma membrane. GP2 trimers begin to associate due to elevated levels of cholesterol at the plasma membrane. Interactions among trimers are stabilized via seam-like arranged Gly and Ala residues present in each TMD (highlighted yellow). Black, membrane lipid; green, cholesterol; red, actin; grey/yellow, trimeric TMD of Ebola GP. The TMD was simulated as a solvent-excluded structure using BallView 1.3 and is depicted in grey. The seam-like assembly of glycine and alanine residues is depicted in yellow. Palmitates covalently attached to cysteines 221 and 223 are depicted in blue. The trimeric ectodomain of GP2 (PDB ID: 1EBO) is depicted as grey ribbons. Dashed arrows indicate the addition of GP2 trimers to a growing cholesterol-dependent aggregate. (b) The cholesterol-dependent GP2-aggregate reaches a critical mass, and collapses around actin laments in vicinity to the plasma membrane. Dashed arrows indicate the continuous addition of GP2 trimers to the aggregate, thereby forcing a contraction around the actin-stabilized nascent lament (indicated by bold grey arrows). Here the trimeric ectodomain of GP2 was omitted for clarity. (c) Live-cell differential interference contrast imaging of HeLa cells transfected with FLAG-GP2. Cells were imaged for 16 h at 10 min intervals, 5 h post transfection. Dashed line indicates the territory of the cell before the emergence of lament-like structures. Scale bar, 10 mm. (d) A subset of host membrane proteins exhibit a GP-like TMD architecture.

Left, helical wheel representation (Wenxiang diagram67) of the TMD of ZEBOV GP. Polar residues are depicted as white circles, hydrophobic residues in black. Gly and Ala residues contributing to the seam-like assembly are depicted as black circles with red outlines. Right, mammalian single-spanning membrane proteins that contain Z5 small side-chained residues (Gly, Ala, Ser) in a 180 angle, as observed within the TMD of GP. The pie chart depicts the distribution of the annotated functions of the candidates. Candidates with a role in cell adhesion and organization of the cytoskeleton are depicted in dark grey (n 14); light grey, candidates involved in immune response (n 9); white, candidates with other functions (n 3).

Transduction, anti-TGN46 sheep was obtained from Serotec and anti-Calnexin rabbit from Abcam.

Plasmids and constructs. FLAG-tagged cDNA for Ebola GP2 and GP were purchased as synthetic genes from GeneArt (Life Technologies) based on the reverse-translated amino-acid sequences of Uniprot Q05320 (VGP_EBOZM) using the most likely codon usage for H. sapiens (Supplementary Table 2). Using EcoRI and NotI sites, the synthetic genes were cloned into pEGFP-N1 vectors, from which EGFP had been removed via EcoRI/NotI digestion. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). FLAG-tagged ASGR1-cDNA was obtained from Origene (clone # RC205686).

Cell culture and transfection. HeLa cells (DSMZ number: ACC 57) were cultured in a-MEM (Applichem) with 10% fetal calf serum (FCS), 2 mM L-glutamine and 100 U ml 1 penicillin, 100 mg ml 1 streptomycin. HEK 293 T (DSMZ number:

ACC 305), COS-7 (DSMZ number: ACC 305) and Vero (ECACC 84113001) cells were cultured in DMEM (Applichem) supplemented with 10% FCS, 4 mM

L-glutamine and 100 U ml 1 penicillin and 100 mg ml 1 streptomycin at 5% CO2 and 37 C. Transfection was performed using FuGENE HD transfection reagent (Promega) according to the manufacturers instructions (3 mg DNA per 9 ml FuGENE HD per well of a six-well plate).

HUVEC cell culture and transfection. Human umbilical vein endothelial cells (HUVECs) from single donors (PromoCell) were cultured in Endopan 3 Kit medium (PAN-Biotech) supplemented with 10% fetal bovine serum, 100 U ml 1

penicillin and 100 mg ml 1 streptomycin (Gibco Life Technologies) in a 5% CO2 humidied incubator at 37 C. HUVECs were used between passage 1 and 5. For microscopy studies, HUVECs were plated onto glass coverslips coated with 0.1% gelatin (from porcine skin; Sigma-Aldrich).

For transfection, the culture medium was removed from a monolayer of 90% conuent cells. The cells were washed with PBS (1 ; PBS, Sigma-Aldrich) and

subsequently trypsinized (0.05% Trypsin-EDTA; Gibco by Life Technologies). A total of 1.5 106 cells were resuspended in a nal volume of 400 ml of

electroporation buffer (RPMI-1640-medium supplemented with 10% fetal bovine serum) and placed in a 0.4 cm electrode gap cuvette (PEQLAB). 15 mg FLAG-GP2 was mixed with the cell suspension and incubated at 4 C for 10 min. Cells were electroporated using an electric pulse generator (BioRad Gene Pulser Xcell) under the following conditionssquare wave electric pulse, 250 V and 40 ms pulse length. Subsequently, the cells were resuspended in the respective growth media and plated for further assays. After 4 h, the culture medium was replaced with fresh complete growth medium. The images were acquired after 24 h through confocal microscopy using a Zeiss LSM 510 unit mounted on an Axiovert 200 inverted microscope with a 1.4 Plan-APOCHROMAT 63 oil objective.

Confocal microscopy. The cells were plated on 35 mm glass-bottomed dishes (MatTek Corp.) and transfected using FuGENE HD. After 24 h, the cells were washed three times with PBS (pH 7.4) and xed with 3.6% paraformaldehyde (Sigma-Aldrich) for 20 min on ice. For endomembrane staining, the cells were permeabilized using 0.5% (w/v) Triton-X-100 for 5 min at RT. After washing three times, for blocking, cells were incubated with 1% bovine serum albumin (BSA; Carl Roth) in PBS for 1 h. Immunouorescence was performed with a dilution of primary

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antibodies at 1:200 and secondary antibodies at 1:1,000 in 0.5% BSA in PBS. The primary antibody dilutions were added to the cells for 1 h, followed by washing three times with PBS for 5 min. Secondary antibody dilutions were subsequently added for 30 min. Alexa647-conjugated WGA was used at nal concentration of 10 mg ml 1 for 20 min, and 10 nmol of Atto565-conjugated phalloidin (Sigma) was added to 1 ml of methanol and incubated with the cells at a dilution of 1:100 in PBS for 30 min. The samples were examined through confocal microscopy using a Zeiss LSM 510 unit mounted on an Axiovert 200 inverted microscope with a 1.3 NA Plan-NEOFLUAR

40 oil objective, equipped with an argon laser. Image processing was performed using Zen 2011 and ImageJ software.

Quantication of cell detachment using ow cytometry. The cells were plated on 6-well dishes (Corning Inc.) and transfected with the indicated contructs using FuGENE HD. At the indicated timepoints, the dishes were placed on ice. The culture medium containing detached cells was transferred to 1.5 ml tubes and centrifuged for 6 min (10,000 r.p.m.). The supernatant was discarded, and the pellet was resuspended in 200 ml serum-free DMEM. The adherent cells were washed once with PBS, and subsequently incubated with 300 ml of EDTA-containing cell dissociation buffer (Gibco) at 37 C for 5 min. Subsequently, 300 ml of serum-free DMEM was added and the resuspended cells were transfered to 1.5 ml tubes. The ratio of detached versus total cells was quantied using a FACSCalibur ow cytometer (Beckton Dickinson).

Fluorescence stain of membrane cholesterol and quantication. A lipin stock solution was prepared in dimethylsulphoxide at a concentration of 50 mg ml 1.

For membrane staining, unpermeabilized paraformaldehyde-xed cells were incubated with a 1:100 dilution of lipin solution in PBS containing 1% BSA for 30 min before incubation with the antibodies (primary antibody: 1:200; secondary antibody: 1:1,000) and then subjected to confocal microscopy. To quantify the membrane cholesterol population, six-well plates with adherent cells were treated accordingly, and lipin uorescence was measured using a MolecularDevices SpectraMax M5 microplate reader at an excitation wavelength of 355 nm and emission wavelength of 460 nm (ref. 49).

Treatment of cells with MBCD and CB. A stock solution of CB was prepared at 5 mg ml 1 in dimethylsulfoxide. To deplete cholesterol and inhibit actin polymerization, MBCD and CB were added to the medium at the concentrations indicated 6 h post transfection and incubated overnight.

Bioinformatics. All proteins annotated as membrane proteins were downloaded from Uniprot63. These proteins were homology reduced to 30% using CD-HIT64. To map the TMDs of these proteins, we used DG-pred65 with the constraint that a TMD shows a D-G score o0. To identify putative TMDTMD interaction interfaces that were enriched in the Gly, Ala and Ser residues, we generated an algorithm that analyses the TMDs from the z axis. On the basis of the assumption of the regular spacing of the residues in these alpha-helical segments, the total number of small side chain-containing residues (Gly, Ala and Ser) and the relative distribution of these residues around the TMD were obtained. Three data sets corresponding to the different subcellular localizations of the membrane proteins (ER, Golgi and plasma membrane) were generated. To evaluate the data, the plasma membrane protein data set was analysed for proteins containing Z5 residues of Gly, Ala or Ser positioned at an angle of 180as observed in the TMD of Ebola GP (Fig. 4d). Falsely annotated entries and those specic to early developmental stages and proteins exclusively present in certain tissues were omitted for clarity.

Depletion and replenishment of cholesterol in the cells. MBCD-complexed cholesterol from Sigma was added to the cells at 6 h after transfection for 30 min at a concentration of 100 mM in normal growth medium. The cells were washed with

PBS and incubated overnight in growth medium. For the rescue experiments, the cells were incubated in 5 mM MBCD for 60 min before transfection and washed three times with PBS. At 6 h after transfection, water-soluble cholesterol was added as indicated.

Treatment of cells with a statin. The cells were cultured in simvastatin at 2 mM for 4 days before experiments in normal growth medium. The cells then were subsequently transfected and treated as described above.

Scanning electron microscopy. The cells were plated onto 35 mm glass-bottomed dishes (MatTek), and transfected for 24 h with different GP variants. The cells were xed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer (Srensen), pH 7.4 for 2 h, rinsed with buffer and dehydrated with an ascending series of ethanol. The samples were critical point dried using carbon dioxide and the slides were coated with gold(5 nm). The samples were analysed using a ZEISS ULTRA 55-eld emission scanning electron microscope, using the EverhartThornley secondary electron detector.

Live-cell imaging. HeLa cells were plated onto 35-mm MatTek glass-bottomed dishes and transfected for 24 h with 3.3 mg FLAG-GP2 and 10 ml FuGENE HD transfection reagent in complete DMEM medium. After 5 h, the cells were washed with PBS and a HEPES-based imaging medium (Invitrogen). Microscopy was performed on a Nikon TIRF-2 system with Eclipse Ti inverted microscope, using a Nikon Apo-TIRF 1.5 NA 60 oil objective in differential interference contrast

mode with a Hamamatsu 1394 ORCA-ERA camera and an incubation chamber adjusted to 37 C and 5% CO2. Imaging was commenced at 5 h post transfection, with an acquisition rate of one image per 10 min, for a total duration of 16 h using NIS-Elements Microscope Imaging Software.

In vivo photoafnity labelling with cholesterol. FLAG-tagged GP2 and GP1,2 constructs and variants thereof and ASGR1, as a negative control, were probed for proteincholesterol interaction in vivo. For this purpose, photoactivatable cholesterol was employed for photolabelling experiments as previously described66. In brief, HeLa cells were grown in six-well dishes and transfected using FuGENE HD (1 mg DNA 3 ml transfection reagent in 100 ml Opti-MEM (Invitrogen) at B60%

conuency. After 16 h, the cells were labelled with 75 mCi of (3H)-photocholesterol in 3 ml of DMEM supplemented with 10% delipidated FCS for 8 h. The cells were ultraviolet irradiated for 10 min on ice and lysed for 1 h in 100 ml lysis buffer(50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton-X-100 (v/v), 0.5% deoxycholate (w/v), and protease inhibitor cocktail (cOmplete mini, Roche)). Post-nuclear supernatants were subjected to immunoprecipitation using M2-(anti-FLAG)-afnity gel (Sigma-Aldrich) according to the manufacturers instructions. The proteins were subsequently eluted in 4 SDSpolyacrylamide

gel electrophoresis sample buffer containing 16% SDS and subjected to SDS polyacrylamide gel electrophoresis (1020% Tris/Tricine gradient gels, Invitrogen), western blotting and the quantitative immunodetection of FLAG-tagged proteins using a LI-COR infrared imager. Radioactively labelled proteins were detected by digital autoradiography (Beta Imager 2000, Biospace Lab), using acquisition times of 16 h.

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Acknowledgements

M.H. was supported by Boehringer Ingelheim Fonds. We thank the Nikon imaging facility Heidelberg for assistance with confocal and live-cell microscopy, and Hilmar Bading for supporting A.H. in performing electron microscopy. pCAGGS-VP40 was a kind gift from Stephan Becker (Marburg).

Author contributions

A.M.E and M.H. designed and performed the experiments. A.H. performed scanning electron microscopy. P.B. performed the bioinformatics analyses. P.H. and C.R.d.A. performed experiments involving primary cell culture. A.M.E, M.H., B.B. and F.W. wrote the manuscript.

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How to cite this article: Hacke, M. et al. Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol. Nat. Commun. 6:7688 doi: 10.1038/ncomms8688 (2015).

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