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Journal of Virology logoLink to Journal of Virology
. 2016 Jan 15;90(3):1444–1454. doi: 10.1128/JVI.02670-15

A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner

Alexander Koehler a, Larissa Kolesnikova a, Ulla Welzel a, Gordian Schudt a, Astrid Herwig a, Stephan Becker a,b,✉
Editor: D S Lyles
PMCID: PMC4719610  PMID: 26581998

ABSTRACT

Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling.

IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.

INTRODUCTION

Filoviruses, including Ebolaviruses (EBOV) and Marburg virus (MARV), are enveloped, nonsegmented, negative-strand RNA viruses (1). These viruses are known to cause severe fevers in humans and nonhuman primates, with case fatality rates of up to 90% (2). Although several antivirals and vaccines currently are being tested in clinical studies, none of them are licensed for human use. Therefore, work with filoviruses is restricted to biosafety level 4 (BSL-4) facilities. The recent EBOV outbreak in Guinea, Sierra Leone, and Liberia demonstrated the potential of filoviruses to cause massive and prolonged outbreaks with high lethality rates (3).

Remarkably, filovirus infection in rodents leads only to transient nonlethal illness. The sequential passaging of filoviruses in rodents results in the selection of viruses able to induce lethal disease (4). The duration of filovirus passaging in rodents and the number of detected mutations in the lethal variants are different for mice and guinea pigs. For example, 23 to 28 passages of MARVRavn or MARVAngola were necessary to select for highly pathogenic viruses in mice. In guinea pigs, only 8 passages of MARVMusoke resulted in a variant that induced lethal disease (5–8). Whereas 11 (MARVAngola) and 14 to 19 (MARVRavn) amino acid mutations in five or four viral genes were detected in the lethal mouse variants, four amino acid mutations in two viral genes were found in the lethal guinea pig MARV (5–8).

Among all of the detected mutations in rodent-adapted MARV, only the mutation in the viral matrix protein VP40 (D184N) occurred in both mice and guinea pigs. Moreover, sequential sequencing of the passages of lethal mouse MARVRavn revealed that the D184N mutation in VP40 occurred first and then was followed by mutations at nine other residues in VP40 (5). The early appearance of the D184N mutation in VP40 and its presence in both lethal mouse and lethal guinea pig MARVs suggested that this amino acid change was important for viral replication in a new host. The impact of the D184N mutation on MARV replication in guinea pig cells is of special interest, because it was the only mutation in this viral gene that was detected in lethal guinea pig MARV (6).

In the MARV genome, which encodes seven viral structural proteins (NP, VP35, VP40, GP, VP30, VP24, and viral polymerase L), the VP40 gene is located at the third position. Within the filamentous MARV particle, the viral matrix protein VP40 is located at the inner side of the viral envelope in which the viral surface glycoprotein GP is inserted (9). The viral envelope covers the filamentous nucleocapsid, consisting of the viral RNA encapsidated by the nucleocapsid proteins NP, VP35, VP30, VP24, and L (10). MARV VP40 is a peripheral membrane protein that is synthesized as a soluble protein and then recruited to membranes (11). The accumulation of VP40 was observed upon its ectopic expression in filamentous plasma membrane protrusions; the fission of these protrusions results in the release of filamentous virus-like particles (VLPs) into the supernatant (12, 13). The coexpression of VP40 with NP or with the viral surface protein GP induces the redistribution of NP and GP to the VP40-enriched plasma membrane protrusions and their subsequent incorporation into released VLPs (14–17). Based on these observations, it is currently thought that VP40 functions as the main driver of MARV particle assembly and release. Interestingly, filovirus VP40 proteins have a poorly understood regulatory function on replication and transcription in the context of a minigenome assay (17, 18). In addition, MARV VP40 has an inhibitory function on interferon (IFN) signaling (19).

The goal of our study was to analyze whether the single mutation D184N in VP40 influenced the capacity to produce progeny virions in guinea pig cells or, in other words, whether this mutation improved viral fitness (20). In addition, we wanted to elucidate whether the known functions of VP40 were affected by this single mutation in a species-specific manner.

We have shown that recombinant MARV encoding VP40D184N [rMARVVP40(D184N)] shows increased infectivity and accelerated growth compared to that of the recombinant MARV encoding wild-type VP40 (rMARVWT). This effect was more pronounced in guinea pig cells than in human cells. Comparative analyses of the functions of VP40D184N and VP40 showed that some VP40 functions remained unaltered, such as the inhibitory function of VP40 on interferon signaling in human and guinea pig cells. In addition, we detected specific functions of VP40 that were enhanced with VP40D184N exclusively in guinea pig cells. These consisted of the production and budding of VP40-induced VLPs, recruitment of NP into infectious VLPs (iVLPs), and infectivity of iVLPs. Finally, the suppression of viral transcription and replication in the minireplicon system was much less pronounced with VP40D184N than with wild-type VP40.

MATERIALS AND METHODS

Cells.

Human embryonic kidney cells (HEK293), human hepatoma cells (Huh-7), African green monkey kidney cells (VeroE6), and guinea pig colorectal adenocarcinoma cells (GPC-16) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, l-glutamine, and a penicillin-streptomycin solution (Gibco, Karlsruhe, Germany). The guinea pig fetal carcass-derived fibroblast-like line (104C1) was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, l-glutamine, and a penicillin-streptomycin solution (Gibco, Karlsruhe, Germany). HEK293 cells were used for flotation, reporter gene, VLP, and iVLP assays because of their high transfection efficiency. Huh-7 and VeroE6 cells, which are more susceptible to infection with MARV than HEK293 cells (21, 22), were used for virus titration and analysis of viral growth kinetics and for immunofluorescence analysis.

Cloning and rescue of recombinant viruses.

The cloning of recombinant MARVs was achieved by ligating three fragments that together contained the full sequence of MARV Musoke (GenBank accession number NC_001608) and a minimal pBlueScript vector which is under the control of the T7 polymerase (23). The plasmids containing the MARV fragment in three individual pBlueScript plasmids were digested on unique restriction sites, where fragment 1 (FR1) contained the sequences of T7 leader-NP-VP35-VP40-GP, fragment 2 (FR2) contained GP-VP30-VP24-L, and fragment 3 (FR3) contained L-Trailer-Ribozyme. To distinguish recombinant virus from wild-type virus, silent mutations at positions 6498 (C>T) and 7524 (A>G) were introduced, resulting in the replacement of a KpnI restriction site with a SacII restriction site. The mutation in VP40 in FR1 was inserted via multisite-directed mutagenesis to introduce the mutation GAC to AAT at positions 8104 to 8106, resulting in the amino acid alteration of aspartic acid to asparagine.

For the rescue of recombinant viruses, VeroE6 and Huh-7 cells were mixed in a one-to-one ratio in 6-well plates and grown to approximately 50% confluence. The transfection with the necessary support as well as the full-length plasmids, containing the full genomic sequence of MARV, was performed as previously described (21, 24). The sequence integrity of rescued viruses was confirmed by sequencing of the viral RNA from rMARVWT and rMARVVP40(D184N), as well as the inserted mutation GAC to AAT (Asp to Asn) in the VP40 gene of rMARVVP40(D184N). Recombinant viruses were propagated and titrated by PFU assay and 50% tissue culture infectious doses (TCID50) on VeroE6 cells. Infections with recombinant MARVs were performed under biosafety level 4 (BSL-4) conditions at the Institute of Virology, Philipps University Marburg.

Antibodies.

Monoclonal mouse antibodies against MARV NP, GP, and VP40, goat serum anti-GP, and guinea pig serum anti-NP were used for the detection of viral proteins in Western blot and immunofluorescence analyses.

Flotation assay.

Flotation assay was performed as described previously (22, 25). Briefly, HEK293 or 104C1 cells were transfected with plasmid encoding VP40 or VP40D184N. At 24 h posttransfection (p.t.), cells were washed with phosphate-buffered saline (PBS) at 4°C, scraped off the dish, and transferred in lysis buffer (10 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 200 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride). Lysis was achieved by 10 strokes through a 26-gauge needle. Nycodenz was added to the lysates to a final concentration of 40% (wt/wt). The sample was placed at the bottom of an SW60 centrifuge tube and overlaid with 3 ml 30% Nycodenz in TNE (Tris-HCl, NaCl, and EDTA) buffer and 500 ml TNE buffer. The step gradient was centrifuged at 52,000 rpm for 4 h. Five fractions were collected from the top to the bottom. Equal amounts of each fraction were analyzed by SDS-PAGE and Western blotting (22).

Indirect immunofluorescence analysis.

Cells were grown on glass coverslips and fixed with 4% PFA at the time indicated in the figure legends after transfection or infection. After fixation, cells were processed as described previously (22). Antibodies used are indicated in the figure legends. To monitor the intracellular location of cholesterol by fluorescence microscopy, a freshly prepared 50 μg/ml filipin solution was applied together with fluorescently labeled secondary antibodies as described previously (26, 27). Microscopic analysis was performed with a Zeiss fluorescence microscope and Leica SP5 confocal laser scanning microscope. Cross-sectional areas of viral inclusions were measured using Leica Application Suite X software.

Reporter gene assay.

HEK293 cells or 104C1 cells were cotransfected with pGL3-Mx1P-FF-Luc reporter plasmid expressing firefly luciferase under the control of the Mx1 promoter (28, 29) cloned into pGL3 (Promega), pGL 4.73 encoding Renilla luciferase reporter construct, and either VP40 or VP40D184N. At 24 h p.t., cells were washed with PBS and incubated further for 24 h alone or with a recombinant IFN hybrid (interferon-α/β B/D hybrid; 500 U/ml), which has been shown to have broad-host-range activity in an antiviral assay (30). After IFN treatment, cells were harvested using Dual-Luciferase passive lysis buffer (Promega, Madison, WI), and firefly luciferase activities were measured.

iVLP assay.

The MARV iVLP assay was performed as described previously (17), with small modifications. Briefly, HEK293 cells and 104C1 cells were transfected with plasmids encoding all MARV structural proteins and either VP40 or VP40D184N, a MARV-specific artificial minigenome encoding the Renilla luciferase reporter gene, and the T7 DNA-dependent RNA polymerase. Culture supernatants were collected for purification of iVLPs at 72 h p.t., and iVLPs were purified via centrifugation through a 20% sucrose cushion. Simultaneously, producer cells (p0) were lysed in Dual-Luciferase passive lysis buffer (Promega, Madison, WI). To evaluate the infectivity of iVLPs, iVLPs were used for the infection of target cells. HEK293 or 104C1 cells were either pretransfected with plasmids encoding MARV NP, VP30, VP35, and L (p1 tr) (17) or not pretransfected (p1), and 24 h p.t. they were infected with iVLPs. The amount of iVLPs was controlled by Western blotting, and comparable amounts of iVLPs (by normalizing the amount of incorporated VP40) were used for the infection of target cells. At 48 h postinfection (p.i.), infected target cells were harvested and lysed in 100 μl Dual-Luciferase lysis buffer. The measurements of Renilla reporter activity of producer and target infected cells (p0, p1tr, and p1) was performed using the Promega Dual-Luciferase reporter assay and a Berthold LB 960 Centro luminometer (Bad Wildbad, Germany).

Determination of viral genome copies.

Quantitative reverse transcription-PCR (qRT-PCR) was used to determine the number of viral genomes or minigenomes present in the supernatants of virus-infected cells or iVLP-producing cells. Minigenome RNA extraction and qRT-PCR were performed as described previously (22). Virus-containing supernatants were inactivated with AVL buffer (Qiagen) and 100% ethanol. Viral RNA was isolated by following the manufacturer's instructions (QIAmp viral RNA kit; Qiagen), and qRT-PCR was performed using the OneStep RT-PCR kit (Qiagen). The primers and probe were specifically designed to detect a region of the MARV polymerase L gene.

Minigenome assay.

A minigenome assay was performed as previously described (31). Briefly, HEK293 cells and 104C1 cells were transfected with plasmids encoding MARV L, NP, VP30, and VP35, a MARV-specific minigenome carrying the Renilla luciferase reporter gene, and the T7 DNA-dependent RNA polymerase. To test the inhibitory effect of VP40 on the transcription and replication of the minigenome, cells were cotransfected with the plasmid encoding either VP40 or VP40D184N. Cells were lysed at 24 h p.t., and the reporter activity of cell lysates was determined as described above.

SDS-PAGE and Western blotting.

SDS-PAGE and Western blot analysis were performed as described previously (32). The intensity of bands was quantified using the ImageLab software package (Bio-Rad).

Electron and immunoelectron microscopic analyses.

Huh-7 and 104C1 cells infected with either rMARVWT or rMARVVP40(D184N) were fixed by adding an equal volume to the culture media of double-concentrated fixative solution [120 mM piperazine-N,N′-bis(2-ethanesulfonic acid), 50 mM HEPES, 4 mM MgCl2, 20 mM EGTA, 8% paraformaldehyde, 0.2% glutaraldehyde, pH 6.9] in the BSL-4 laboratory. After fixation for 30 min at room temperature, cells were scraped, pelleted, and overlaid with 4% paraformaldehyde. After virus inactivation by fixation for at least 48 h, the cells were removed from the BSL-4 laboratory and processed for embedding in Epon and Araldite as described previously (32). Ultrathin sections (60 to 90 nm) of cells were made using an ultramicrotome (Leica EM UC6) and stained with uranyl acetate and lead citrate. For immunoelectron analysis of rMARVWT and rMARVVP40(D184N), virus suspensions fixed with 4% paraformaldehyde were used. Indirect immunostaining of viral surface glycoprotein was performed as described previously (15), using a goat serum anti-GP and a donkey anti-goat IgG coupled with colloidal gold (12-nm-diameter gold beads). Samples were negatively stained with 2% phosphotungstic acid solution. Ultrathin sections and negatively stained virus samples were analyzed by using a JEM 1400 transmission electron microscope at 120 kV. Electron micrographs were recorded using a 4,000- by 4,000-pixel TemCam-F416 (TVIPS, Germany) camera.

Statistical analysis.

The presented data represent the mean values and standard deviations from at least three independent experiments. The statistical significance was determined using Student's t test. Statistically significant differences are indicated with asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

RESULTS

The D184N mutation in VP40 provides a replicative advantage for rMARVVP40(D184N) in a species-specific manner.

Mutations in viral genes appear regularly when MARV is serially passaged in rodents, and these mutations are accompanied by a lethal phenotype (5–8). Upon passaging in guinea pigs, MARV particles were selected to carry three mutations in L and a mutation of D184 to N in VP40 (VP40D184N) (6). To evaluate whether the D184N mutation provides a replicative advantage over the VP40 wild type in guinea pig cells, a recombinant MARV, rMARVVP40(D184N), was constructed and rescued that contained the D184N mutation in VP40. For the control, recombinant MARV containing the wild-type genome of MARV Musoke, rMARVWT, was constructed. Both viruses were rescued and viral stocks were prepared in VeroE6 cells. Viruses were purified from the supernatant of infected cells by centrifugation through a sucrose cushion and analyzed by electron microscopy and silver staining of virus lysates. Electron microscopy analysis after immunogold staining with a goat polyclonal anti-GP antibody revealed that the relative portion of filamentous, hook-shaped, 6-shaped, and ring-like particles in rMARVWT and rMARVVP40(D184N) preparations were similar, as was the density of immunogold-labeled GP (Fig. 1A). The evaluation of protein composition in the silver-stained SDS-PAGE gel of rMARVWT and rMARVVP40(D184N) particles showed no substantial changes in the protein composition of either virus (Fig. 1B). To investigate the propagation of rMARVWT and rMARVVP40(D184N), the two viral stocks were used for the infection of human Huh-7 and guinea pig 104C1 cells at an MOI of 0.01; the titers of the respective viruses were determined in the supernatant (Fig. 1C). At day 1 p.i., titers of released rMARVWT and rMARVVP40(D184N) were higher in Huh-7 cells than in 104C1 cells, which indicated that the cell lines differ in their intrinsic susceptibility and extent of viral replication. Interestingly, both human and guinea pig cell lines infected with rMARVVP40(D184N) released more viral particles than did cells infected with rMARVWT at days 2 and 3 p.i. The difference in titers was higher in guinea pig cells. After day 4 p.i., the difference between the viruses was detected only in 104C1 cells and was lost in Huh-7 cells (Fig. 1C). We compared the ratio of NP to VP40 in the virions and observed that the relative amount of NP was higher in rMARVVP40(D184N) particles released from human and guinea pig cells (Fig. 1D), suggesting more efficient production of infectious virions. To investigate further the replication capacities of rMARVVP40(D184N) and rMARVWT in different cell types, human and guinea pig cells were infected with either of the two viruses at an MOI of 3, and viral genome copy numbers in the viral particles released into the cellular supernatants were measured by qRT-PCR at days 1 and 3 p.i. (Fig. 1E). The copy number of the viral genomes in the supernatants of human cells infected with rMARVVP40(D184N) or rMARVWT did not notably differ, while the number of viral genomes in the supernatant of guinea pig cells infected with rMARVVP40(D184N) was 4.7-fold higher at day 1 p.i. and 7.8-fold higher at day 3 p.i. than levels for rMARVWT-infected cells (Fig. 1E).

FIG 1.

FIG 1

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D184N mutation in VP40 provided a replicative advantage for rMARVVP40(D184N) in a species-specific manner. (A) Comparison of rMARVWT and rMARVVP40(D184N) morphology. Virus particles were fixed, immunogold labeled with GP-specific antibody (12-nm gold particles), and negatively stained with 2% phosphotungstic acid. Upper panels show representative images of rMARVWT and rMARVVP40(D184N) particles. Gray boxes indicate single particles which are shown at higher magnification in the lower panels, and some of the gold particles are indicated by arrows. Bars in the upper panels, 500 nm; bars in the lower panels, 100 nm. (B) SDS-PAGE and silver staining of rMARVWT and rMARVVP40(D184N). (C) Growth kinetics of rMARVWT (black circles) and rMARVVP40(D184N) (gray circles) in Huh-7 and 104C1 cells infected with viruses at an MOI of 0.01. (D) Western blot analysis of viral protein levels in culture supernatants of Huh-7 or 104C1 cells infected at an MOI of 0.01 with rMARVWT or rMARVVP40(D184N). Supernatants were collected at the indicated time points and analyzed by SDS-PAGE and Western blotting using NP- and VP40-specific antibodies. (E) Viral genome copy number per microliter present in the supernatants of Huh-7 or 104C1 cells infected at an MOI of 3 with rMARVWT (black columns) or rMARVVP40(D184N) (gray columns) at days 1 and 3 p.i. Shown are the means and standard deviations from two independent experiments performed in duplicate. *, P < 0.05; ***, P < 0.001.

To compare the infectivity of rMARVWT and rMARVVP40(D184N) via a single-step growth curve, Huh-7, 104C1, and VeroE6 cells were infected at an MOI of 1, fixed at 19 h p.i., and analyzed by immunofluorescence for the presence of NP (Fig. 2A). The numbers of infected cells were counted. A comparison of the ratios of infected to noninfected cells showed that 34.8% of human cells were infected with rMARVWT, and 76.8% were infected with rMARVVP40(D184N) (Fig. 2A, upper, and B). VeroE6 cells showed almost equal susceptibility to infection with rMARVWT and rMARVVP40(D184N) (35.7% and 40.6% of infected cells, respectively) (Fig. 2A, lower, and B). Guinea pig cells were less susceptible to MARV infection (Fig. 2A, middle, and B), and we detected 2.4% of rMARVWT- versus 13.1% of rMARVVP40(D184N)-infected cells (5.5-fold difference).

FIG 2.

FIG 2

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Comparison of rMARVWT and rMARVVP40(D184N) infectivity in VeroE6, Huh-7, and 104C1 cells. (A) Viruses were titrated in VeroE6 cells and used for the infection of cells grown on glass coverslips at an MOI of 1. Cells were fixed at 19 h p.i. and stained with an NP-specific antibody (red) for the detection of infected cells by immunofluorescence analysis. Nuclei were stained with DAPI (blue). Bars, 20 μm. (B) Graphics show the ratio of infected to uninfected cells; the relative number of cells infected with rMARVWT was normalized to 1 in Huh-7, 104C1, and VeroE6 cells. Shown are the means and standard deviations of the results from three independent experiments. The cells were counted in 10 random fields per sample. ***, P < 0.001.

In addition, infection with rMARVVP40(D184N) resulted in the formation of larger viral inclusions specifically in guinea pig cells (Fig. 3A and B). The size measurement of viral inclusions at 19 h p.i. showed that the total cross-sectional area of viral inclusions per cell, as well as the cross-sectional area of single viral inclusions (Fig. 3B), were 1.7- and 1.5-fold larger in guinea pig cells infected with rMARVVP40(D184N) than in cells infected with rMARVWT. In human cells infected with either of the viruses, the sizes of the inclusions were similar. Supporting this observation, electron microscopic analysis at 24 h p.i. showed that the overall morphology of perinuclear viral inclusions was similar for rMARVWT- and rMARVVP40(D184N)-infected human cells. Viral inclusions in rMARVVP40(D184N)-infected guinea pig cells displayed a larger amount of mature nucleocapsids than those in rMARVWT-infected cells (Fig. 3C). These results indicated that rMARVVP40(D184N) replication was more efficient in guinea pig cells.

FIG 3.

FIG 3

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Analyses of viral inclusions formed in human and guinea pig cells upon infection with rMARVWT or rMARVVP40(D184N). (A) Viruses were titrated in VeroE6 cells and used for the infection of Huh-7 and 104C1 cells grown on glass coverslips at an MOI of 1. Cells were fixed at 19 h p.i. and stained with an NP-specific antibody (red) and VP40-specific antibody (green), and nuclei were stained with DAPI (blue). Maximum-intensity projections are shown. Yellow arrows indicate viral inclusions. Bars, 20 μm. (B) Graphics show sizes of viral inclusions. Images were obtained as described for panel A and analyzed with Leica Application Suite X software. The cross-sectional area of single viral inclusions and the total cross-sectional area of inclusions per cell were measured. Shown are the means and standard deviations of the measurements in 30 cells. **, P < 0.01. (C) Electron microscopy analysis of viral inclusions formed by rMARVWT or rMARVVP40(D184N) in human or guinea pig cells. Virus-infected cells were fixed at 24 h p.i. and embedded in epoxy resin. Ultrathin sections of infected cells were contrasted with uranyl acetate and lead citrate. Arrows indicate some of the mature nucleocapsids with an electron-dense wall. Bars, 500 nm.

The D184N mutation in VP40 does not affect the IFN-antagonistic function of VP40 in human and guinea pig cells.

We next were interested in which functions of VP40 were affected by the D184N mutation. To clarify whether the VP40 IFN-antagonistic function was influenced by the D184N mutation, we transfected human or guinea pig cells with a reporter plasmid carrying the firefly luciferase gene under the control of the Mx1 promoter (28, 29) and a plasmid encoding VP40, VP40D184N, or an empty vector. At 24 h after transfection, cells were incubated with an IFN hybrid, which activates the IFN receptors of multiple species (30). Treatment with the IFN hybrid increased reporter gene activity in human cells by 30-fold and in guinea pig cells by 6-fold (Fig. 4). The expression of VP40 or VP40D184N inhibited the IFN-induced activation of the Mx1 reporter completely in human cells. Likewise, VP40 and VP40D184N reduced the IFN-induced reporter signal in guinea pig cells, indicating that the D184N mutation did not impair the IFN-antagonistic function of VP40 in either human or guinea pig cells.

FIG 4.

FIG 4

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D184N mutation in VP40 did not affect the IFN-antagonistic function of VP40 in human and guinea pig cells. Human (HEK293) and guinea pig (GPC-16) cells were cotransfected with the pGL3-Mx1P-FF-Luc reporter plasmid expressing firefly luciferase under the control of the Mx1 promoter, pGL 4.73 encoding Renilla luciferase, and plasmids encoding VP40D184N, VP40, or an empty vector. At 24 h p.t., cells were washed and incubated with IFN hybrid (500 U/ml) for 24 h. After IFN hybrid treatment, cells were lysed and analyzed for firefly and Renilla luciferase activity. The reporter activity was determined by normalizing the firefly signal to the Renilla signal. Shown are the means and standard deviations from three independent experiments. **, P < 0.01.

The D184N mutation slightly enhanced membrane binding of VP40.

MARV VP40 is a peripheral membrane protein, polymers of which form large patches beneath the plasma membrane and cover the inner surface of the virions (11, 22). The patches of VP40 at the plasma membrane were positive for lipid raft markers (33) and represented sites where long filamentous VLPs are released into the cell supernatant (12, 13). Flotation assays were performed to analyze whether the D184N mutation influenced the membrane binding capabilities of VP40 (Fig. 5A). Compared to VP40, slightly larger amounts of VP40D184N were associated with the cellular membranes both in human and guinea pig cells. These results indicated that the D184N mutation in VP40 did not affect the membrane binding function of VP40 in the analyzed cell types.

FIG 5.

FIG 5

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D184N mutation in VP40 slightly enhanced membrane binding and VLP productive function. (A) Membrane binding of VP40 and VP40D184N as determined by flotation analysis in human (Huh-7) and guinea pig (104C1) cells. Cells expressing VP40 or VP40D184N were subjected to flotation assay at 24 h p.t. (Upper) Fractions (1 to 5, top to bottom) were analyzed by Western blotting using a VP40-specific antibody. (Lower) Graphics show the percentage of membrane-bound VP40 (black columns) or VP40D184N (gray columns) in human and guinea pig cells. Shown are the means and standard deviations of the results from three independent experiments. (B) Accumulation of VP40 and VP40D184N in the plasma membrane subdomains enriched in cholesterol. Human (Huh-7) and guinea pig (104C1) cells were transfected with plasmids encoding VP40 or VP40D184N and were fixed 24 h p.t.; VP40 was detected using a VP40-specific antibody (red). Intracellular cholesterol was visualized using filipin III staining. The filipin signals were pseudocolored in green in the merged images. Bars, 20 μm. (C) Production of VLPs induced by VP40 or VP40D184N in human and guinea pig cells. VLPs were purified from the supernatants of cells expressing VP40 or VP40D184N at 72 h p.t. by centrifugation through a sucrose cushion. (Left) Equal amounts of cell lysates and VLPs were subjected to SDS-PAGE and Western blot analysis. (Right) Graphics represent the means and standard deviations of relative VLP production (amount of VP40 in VLPs compared to the amount of VP40 in cell lysates) from three independent experiments. *, P < 0.05.

To examine whether the D184N mutation influenced the ability of VP40 patches to recruit cholesterol, human and guinea pig cells expressing VP40 or VP40D184N were stained with filipin and a specific antibody for VP40 (Fig. 5B). Both cell types displayed clusters of VP40 or VP40D184N beneath the plasma membrane, forming large bubble-like protuberances or bundles of long, thin protrusions (Fig. 5B, left column). Remarkably, VP40- and VP40D184N-positive plasma membrane protrusions were highly enriched in cholesterol in both human and guinea pig cells, which indicated that VP40D184N is able to accumulate in plasma membrane subdomains and recruit cholesterol in a manner similar to that of VP40 (Fig. 5B, middle and right columns).

The D184N mutation improved VP40-induced VLP production and the iVLP assembly function of VP40 in guinea pig cells.

To monitor the influence of the D184N mutation on the VP40-intrinsic budding function, we analyzed the amount of VLPs in the supernatants of human or guinea pig cells expressing either VP40 or VP40D184N. The relative VLP release (ratio between VP40 signal in VLPs and VP40 signal in cell lysates) was slightly reduced in human cells but was enhanced in guinea pig cells expressing VP40D184N (Fig. 5C), demonstrating that the budding function of VP40 is enhanced in a species-specific manner by the D184N mutation.

We then tested whether D184N influenced the assembly function of VP40 using the iVLP assay (17). For this purpose, human or guinea pig cells were transfected with plasmids encoding VP40 or VP40D184N and all other structural proteins of MARV, an artificial MARV-specific minigenome encoding the Renilla luciferase reporter gene, and the T7 DNA-dependent RNA polymerase. At 72 h p.t., human and guinea pig cells were lysed and subjected to the Renilla luciferase reporter assay (Fig. 6A). In addition, iVLPs were purified from the cell supernatants, and normalized amounts of iVLPs then were used to infect human or guinea pig cells. As shown in Fig. 6A, human cells transfected with VP40D184N showed slightly lower reporter activity than cells with VP40. In contrast, guinea pig cells expressing VP40D184N showed a 1.7-fold increase in reporter activity compared to that of cells with VP40. It then was analyzed whether the protein composition of released iVLPs changed as a consequence of the D184N mutation in VP40. Western blot analyses of the protein composition of iVLPs showed that the ratio of GP to VP40 was comparable in iVLPs formed by VP40 and VP40D184N in human and guinea pig cells. However, the ratio of NP to VP40 was 1.3-fold higher in iVLPs released from guinea pig cells that expressed VP40D184N than from VP40-expressing cells (Fig. 6B). To analyze whether the increased amount of nucleocapsid-like structures in iVLPs correlated with the amount of incorporated minigenomes, quantitative RT-PCR of purified iVLPs was performed. We detected a slight but not statistically significant increase in the copy number of minigenomes in iVLPs induced by VP40D184N in guinea pig cells (Fig. 6C). Together, these results indicated that D184N mutation in VP40 enabled higher levels of transcription and replication of the MARV-specific minigenome only in guinea pig iVLP-producing cells and improved the incorporation of nucleocapsid-like structures into VP40D184N-induced iVLPs.

FIG 6.

FIG 6

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D184N mutation in VP40 improved iVLP assembly function of VP40 in guinea pig cells. (A) Human (HEK293) or guinea pig (104C1) cells were transfected with all plasmids for the iVLP assay, as well as with plasmids encoding VP40 or VP40D184N, as indicated. At 72 h p.t., p0 cell lysates were harvested and reporter activity was measured. (B) Western blot analysis of iVLPs. (Left) Purified iVLPs were subjected to Western blot analysis using NP-, VP40-, and GP-specific antibodies. (Middle and right) The relative amount of GP to that of VP40 and of NP to that of VP40 in iVLPs was quantified using the intensity of the band. (C) The amount incorporated into iVLP minigenomes was measured by qRT-PCR. (D) Equal amounts of iVLPs were used for the infection of naive (p1) human and guinea pig cells (normalized to the amount of incorporated VP40). Cells were either pretransfected with plasmids encoding MARV NP, VP35, VP30, and L before infection with iVLPs (p1 tr) or infected without pretransfection (p1 naive). Infected cells were lysed at 24 h p.i., and Renilla reporter activity was determined to monitor iVLP infectivity. Data represent the means and standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01.

Because the VP40D184N-induced iVLPs from guinea pig cells contained more nucleocapsids, we presumed that they also induced more reporter gene activity in target cells than iVLPs induced by VP40. Therefore, iVLPs induced by either VP40 or VP40D184N were used to infect target cells that were either pretransfected with plasmids encoding NP, VP35, VP30, and L (p1 tr) or were left untreated (p1 naive). At 48 h p.i., cells were subjected to a Renilla luciferase reporter assay. VP40D184N-induced iVLPs showed a lower infectivity in human cells independently of whether the cells were pretransfected or left untreated (Fig. 6D). In contrast, the infectivity of VP40D184N-induced iVLPs was enhanced in guinea pig cells, which supports the idea that the enhanced transcription and replication of minigenomes in the presence of VP40D184N leads to the formation of more mininucleocapsids, which can be incorporated into iVLPs (Fig. 6D).

D184N mutation in VP40 affected its suppressive function on minigenome-based transcription and replication in guinea pig cells.

The suppressive effect of the ectopically expressed VP40 on minigenome-based transcription and replication has been described previously (18). In addition, it was shown that filovirus VP24 inhibited viral replication and transcription (34). To investigate whether the effect that was observed for the VP40D184N mutant in guinea pig cells could be attributed to VP40 directly or was mediated by VP24, we used the minigenome system for MARV (expressing NP, L, VP30, and VP35) and additionally expressed increasing amounts of either VP40 or VP40D184N (Fig. 7). A dose-dependent decrease in Renilla activity mediated either by VP40 or VP40D184N was observed in both human and guinea pig cells. While in human cells the reporter gene activity of the MARV minigenome system was reduced by VP40 and VP40D184N to similar levels, significant differences were detected in guinea pig cells. At 100 ng transfected plasmid encoding VP40, minigenome activity was diminished by 59%, and no reduction was observed by transfection of the plasmid encoding VP40D184N. At higher doses, plasmid encoding VP40D184N also showed suppressive effects on reporter gene activity in guinea pig cells; however, at all tested concentrations, the suppression by equal amounts of wild-type VP40 encoding plasmid was twice as high (Fig. 7A). The expression control showed that VP40 and VP40D184N were synthesized (Fig. 7B). These results indicated that the D184N mutation in VP40 influenced the suppressive function of VP40 on transcription and replication directly and was not mediated by VP24 or GP. This loss-of-function effect of VP40D184N was specific for guinea pig cells.

FIG 7.

FIG 7

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D184N mutation in VP40 affected its suppressive function on minigenome-based transcription and replication in guinea pig cells. (A) Human (HEK293) and guinea pig (104C1) cells were transfected with all minigenome-based plasmids with or without plasmids encoding L and different amounts of plasmids (pC-VP40s) encoding either VP40 (black column) or VP40D184N (gray columns). At 24 h p.t., cells were lysed and reporter gene activity (reflecting minigenome transcription and replication) was measured. Reporter gene activity without expression of VP40 was set to 100%. Shown are the means and standard deviations of the results from three independent experiments. *, P < 0.05. (B) Western blot analysis of protein levels in the HEK293 or 104C1 cells transfected as described for panel A. Cell lysates were analyzed by SDS-PAGE and Western blotting using alpha-tubulin (Tub)- and VP40-specific antibodies. VP40, lanes 1, 3, and 5; VP40D184N, lanes 2, 4, and 6.

DISCUSSION

We constructed recombinant MARV encoding VP40D184N and found this virus to be significantly improved in terms of replication in guinea pig cells, which suggests that the single mutation of D184 to N in VP40 was sufficient to change viral fitness. Further analyses using MARV-specific iVLP and minigenome systems revealed that VP40D184N differed from VP40 specifically in its budding function and in its capacity to attract nucleocapsids into infectious particles, which were enhanced in guinea pig cells, as well as the ability to suppress MARV replication and transcription, which was reduced in guinea pig cells. All other functions of VP40, including its ability to inhibit IFN activity, were either unchanged or changed in human and guinea pig cells.

Mutations in viral matrix proteins in viruses other than filoviruses have been described to be associated with host cell adaptation. For example, the adaptation of the measles virus to cotton rats was accompanied by a single mutation in the matrix protein, and the adaptation of chimpanzee immune deficiency virus to human lymphoid tissue also was dependent on a point mutation in the gag matrix protein (35, 36). The mechanisms of how the mutated matrix proteins mediated the effect on replicative fitness in a new host remain largely unknown.

Point mutations in VP40 also were observed when MARV was adapted to mice. Some of these mutations have been shown to modulate the IFN-antagonistic function of VP40 in mouse cells (37, 38). Therefore, we first analyzed whether the D184N mutation improved the ability of VP40 to overcome the IFN-induced antiviral response in guinea pig cells. However, our experiments showed that the D184N mutation did not impact the IFN-antagonistic function of VP40 (Fig. 4). This was in line with a previously published study revealing that the efficient control of the IFN response in mouse cells required mutations detected in lethal mouse MARVRAVN at residues 57 and 165 of VP40, while a mutation at residue D184N was not important (37).

Mutations in the viral matrix protein of swine influenza A virus have been shown to induce changes in virus shape (39) and to increase virulence in mice (40). Thus, we suggested that the D184N mutation influences the shape of MARV particles and/or the incorporation of the surface glycoprotein GP and explains the increased infectivity of rMARVVP40(D184N) in guinea pig cells compared to that of rMARVWT-infected cells. However, we did not find differences in the shapes or amounts of incorporated GP between rMARVVP40 and rMARVVP40(D184N).

Interestingly, viral inclusions in guinea pig cells infected with rMARVVP40(D184N) were increased in size compared to those of cells infected with rMARVWT. This suggested that the D184N mutation influenced the transcription and replication efficiency in guinea pig cells. Indeed, our results indicated that the D184N mutation reduced the ability of VP40 to suppress viral transcription and replication (Fig. 6A and 7). To our knowledge, this is the first time that adaptive mutations have influenced this particular function of matrix proteins. The suppressive effect of matrix proteins on viral replication and transcription is a well-known phenomenon (41). Cellular proteins that likely are involved in the mediation of this suppressive effect are unknown. Our data suggest that the D184N mutation influenced the interaction between host cell factors and VP40, thereby regulating viral RNA synthesis.

Our results further suggested that the better propagation of rMARVVP40(D184N) in guinea pig cells is based in part on the improved budding and assembly functions of VP40D184N in guinea pig cells. The molecular mechanism of the increased production of VP40D184N-induced VLPs in guinea pig cells is enigmatic because the late domain motif, which supports ESCRT-dependent virus budding, is located in the N terminus of MARV VP40 and therefore likely is not impaired by the D184N mutation (42). However, it has been shown that MARV budding is not completely dependent on ESCRT-based mechanisms (43). The fact that the budding activity of VP40D184N was increased specifically in guinea pig cells suggests that other currently unknown mechanisms supporting viral release benefit from the D184N mutation in a species-specific manner. Using iVLP assays, we found that the particles induced by VP40D184N in guinea pig cells contained a higher NP-to-VP40 ratio, indicating that more mininucleocapsids were incorporated which were responsible for the higher infectivity of iVLPs released from VP40D184N-expressing cells.

Taken together, our study showed that the D184N mutation in VP40 provided MARV with a clear advantage for replication in guinea pig cells. We were able to show that the D184N mutation altered several VP40 functions in a species-specific manner. (i) A higher viral RNA synthesis rate was facilitated by reducing the suppressive effect of VP40 on replication/transcription. (ii) The budding function of VP40 was improved, allowing for a more efficient release of viral particles. (iii) The assembly function of VP40 was improved, allowing for more efficient incorporation of nucleocapsid-like structures into infectious particles.

ACKNOWLEDGMENTS

All work with MARV was performed in the BSL-4 facility of the Philipps University of Marburg. We thank Friedemann Weber for providing the interferon-α/β B/D hybrid, Hubert Schäfer (Robert Koch Institute) for providing the guinea pig cell line 104C1, Markus Eickmann for facilitating experiments conducted in the BSL-4 laboratory, and Katharina Kowalski as well as Sonja Heck for technical assistance.

Funding Statement

Alexander Köhler was additionally funded through a stipend of the Jürgen Manchot Stiftung, and the European Union, Sixth Framework Programme “EVIDENT,” funded Gordian Schudt and Stephan Becker.

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