recombinant marburg virus expressing egfp allows rapid

S U P P L E M E N T A R T I C L E

Recombinant Marburg Virus Expressing EGFPAllows Rapid Screening of Virus Growth andReal-time Visualization of Virus Spread

Kristina Maria Schmidt,1,2,3,a Michael Schumann,3,a,b Judith Olejnik,1,2,3 Verena Krahling,3 and Elke Muhlberger1,2,3

1Department of Microbiology, Boston University School of Medicine, and 2National Emerging Infectious Diseases Laboratories Institute, Boston,Massachusetts; and 3Department of Virology, Philipps University of Marburg, Germany

The generation of recombinant enhanced green fluorescent protein (EGFP)–expressing viruses has

significantly improved the study of their life cycle and opened up the possibility for the rapid screening of

antiviral drugs. Here we report rescue of a recombinant Marburg virus (MARV) expressing EGFP from an

additional transcription unit (ATU). The ATU was inserted between the second and third genes, encoding

VP35 and VP40, respectively. Live-cell imaging was used to follow virus spread in real time. EGFP expression

was detected at 32 hours postinfection (hpi), and infection of neighboring cells was monitored at 55 hpi.

Compared to the parental virus, production of progeny rMARV-EGFP was reduced 4-fold and lower protein

levels of VP40, but not nucleoprotein, were observed, indicating a decrease in downstream protein expression

due to the insertion of an ATU. Interestingly, EGFP concentrated in viral inclusions in infected cells. This was

reproduced by transient expression of both EGFP and other fluorescent proteins along with filovirus

nucleocapsid proteins, and may suggest that a general increase in protein synthesis occurs at viral inclusion

sites. In conclusion, the EGFP-expressing MARV will be a useful tool not only to monitor virus spread and

screen for antiviral compounds, but also to investigate the biology of inclusion body formation.

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 in-

clusions 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

Potential conflicts of interest: none reported.Presented in part: XIV International Conference on Negative Strand Viruses,

Brugge, Belgium, 20–25 June 2010; and New England Regional Center ofExcellence (NERCE) Sixth Annual Retreat, Newport, RI, 14–15 November 2010.

aK. M. S. and M. S. contributed equally to this work.bPresent address: CSL Behring GmbH, Emil-von-Behring-Straße 76, 35041

Marburg, Germany.Correspondence: Elke Muhlberger, PhD, Department of Microbiology, Boston

University School of Medicine, 72 East Concord St, Boston, MA 02118([email protected]).

The Journal of Infectious Diseases 2011;204:S861–S870� The Author 2011. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected] (print)/1537-6613 (online)/2011/204S3-0016$14.00DOI: 10.1093/infdis/jir308

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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.

MATERIALS AND METHODS

Cell Lines and VirusesVero E6 (African green monkey kidney), HT-1080 (human fi-

brosarcoma), and U2OS (human osteosarcoma) were main-

tained in Dulbecco’s modified Eagle’s medium supplemented

with penicillin (50 units/mL), streptomycin (50 mg/mL), and

10% fetal calf serum (FCS). MARV strain Musoke and re-

combinant Marburg viruses were propagated in Vero E6 cells as

described previously [9]. All work with infectious MARV was

performed under biosafety level 4 conditions at the Institute of

Virology, Philipps University of Marburg, Marburg, Germany.

Generation of an Infectious MARV Clone Expressing EGFPThe MARV strain Musoke cDNA clone pMARV(1) described

in [9] was used as a template to insert an ATU encoding EGFP

between the second and third genes. The intergenic region be-

tween VP35 and VP40 genes spanning 5 nucleotides (CTATG)

was mutated by in vitro mutagenesis, generating an AvrII re-

striction site (CCTAGG; inserted or substituted nucleotides

underlined). The AvrII restriction site was then used to insert the

ATU consisting of the EGFP open reading frame (ORF) flanked

by authentic MARV transcription start and stop signals [22].

Virus rescue was performed as previously described [10].

Stable integration of the ATU in the viral genome was verified

by reverse transcription–polymerase chain reaction (RT-PCR).

Vero E6 cells were infected with rMARV-EGFP and total RNA

was isolated from cells and supernatants at 6 days postinfection

(dpi) using TRIZOL reagent (Invitrogen). The isolated RNA was

subjected to RT-PCR (OneStep RT-PCR, Qiagen) using primers

flanking a 362–base pair (bp) PCR fragment of the EGFP gene.

TransfectionsHT-1080 cells, grown on glass coverslips, were transfected using

FuGeneHD (Roche), and U2OS cells were transfected using

TransIT-LT1 (Mirus) according to the suppliers’ protocols.

Unless otherwise stated, cells were transfected with 50 ng ex-

pression plasmid for EGFP, or red fluorescent proteins TagRFP,

DsRed, or mCherry in the absence of or along with plasmids

encoding NP (500 ng) and VP35 (500 ng).

Immunofluorescence Analysis of Infected Cells105 Vero E6 cells per well of a 6-well plate were infected with

rMARV-EGFP at a multiplicity of infection (MOI) of 0.05. At 2

and 5 dpi, cells were fixed in 4% (w/v) paraformaldehyde for at

least 24 hours and permeabilized with a mixture of acetone and

methanol (1:1, v/v) for 5 minutes at 220�C. As primary anti-

bodies, a rabbit antiserum directed against the nucleocapsid

complex of MARV (anti-NC antiserum) or a goat anti-MARV

antiserum were used. Antibody binding was visualized by using

Alexa Fluor 568-conjugated and Alexa Fluor 594-labeled sec-

ondary antibodies (Invitrogen). In addition, the cells were

stained with 100 ng/mL 4#-6-diamidino-2-phenylindole (DAPI)

for 10 minutes.

Virus titration was performed by counting foci of infected cells.

Vero E6 cells were infected with recombinantMARV at anMOI of

0.05. Supernatants were collected at 2 and 6 dpi, purified by low-

speed centrifugation, and 500 lL of the supernatants was used for

infection of 105 Vero E6 cells per well of a 6-well plate. Cells were

fixed and permeabilized at 2 dpi as described above. Staining of

infected cells was performed using the anti-NC antiserum. Foci of

infected cells were counted by UV fluorescence microscopy.

Immunofluorescence Analysis of Transfected CellsHT-1080 or U2OS cells were transfected as described above and

subjected to immunofluorescence analysis at 1 day post trans-

fection (dpt). Cells were fixed with 4% (w/v) paraformaldehyde

and permeabilized with 0.1% (v/v) Triton X100. A MARV anti-

NC rabbit antiserum was used to detect MARV proteins. For the

detection of ZEBOV and Reston ebolavirus (REBOV) proteins,

a goat anti-ZEBOV serum that cross-reacts with REBOVNP was

used. Alexa Fluor 594–conjugated antibodies were used for vi-

sualization. The cell nuclei were stained with DAPI.

Western Blot AnalysisVero E6 cells seeded in 6-well plates were infected with recombi-

nant MARV at an MOI of 0.05. At 2 and 5 dpi, cells were scraped

into 200 lL radioimmunoprecipitation assay (RIPA) buffer (20

mM Tris–HCl, pH 7.5; 150 mMNaCl; 10 mM EDTA; 0.1% (w/v)

SDS; 1% (v/v) Triton X100; 1% (v/v) deoxycholate; 10 mM

iodacetamide) and subjected toWestern blot analysis usingmouse

monoclonal antibodies directed against EGFP (B-2; Santa Cruz

Biotechnology), MARV NP, MARV VP40, or b-actin (Abcam).

As a secondary antibody, an IRDye800-conjugated antibody was

used (Rockland). Protein bands were quantified using an Odyssey

imaging system (LI-COR) and standardized to b-actin.

Live-Cell ImagingVero E6 cells were infected with rMARV-EGFP at an MOI of

0.05 in a l-Dish35mm (Ibidi). At 1 hpi, the inoculum was re-

placed by GIBCO Leibovitz’s L-15 Medium (Invitrogen) con-

taining 20% (v/v) FCS. The cell monolayer was analyzed with

a DM16000B Leica inverted fluorescence microscope. EGFP

fluorescence and phase contrast images were captured every

hour for a period of 9 days. Images were taken with a 203

objective. A Zeiss Axiovert 200 M inverted microscope was used

for live-cell imaging of the transfected cells. Fluorescence and

phase contrast images were taken with a 403 objective.

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RESULTS

Rescue of Recombinant Marburg Virus Expressing EGFPAn additional ATU encoding EGFP was inserted into a full-

length antigenomic cDNA plasmid pMARV(1) of MARV strain

Musoke [9, 23] between the second and third genes, encoding

VP35 and VP40, respectively (Figure 1A).

Successful rescue of the MARV containing the EGFP gene

(rMARV-EGFP) was confirmed by RT-PCR, detection of EGFP

expression in infected cells, Western blot analysis, and immu-

nofluorescence analysis. Stable integration of the EGFP gene in

the viral genome was verified by amplification of a 362-bp PCR

fragment of the EGFP gene using template RNA isolated from

rMARV-EGFP-infected cells (Figure 1B) and by sequencing.

ExpressionofEGFP in livingVeroE6cells infectedwith rMARV-

EGFPwas analyzedbyphase contrast andfluorescencemicroscopy.

Foci formation of green fluorescent cells was initiated at 1–2 dpi

without inducing visible cytopathic effects (CPE). The initial signs

of CPE were observed at 5 dpi, when EGFP was detected in

clusters of infected cells (Figure 1C). These data show that

rMARV-EGFP productively infects susceptible cells and can be

used as a sensitive marker to visualize virus spread over time.

To assess the replication efficiency of rMARV-EGFP com-

pared with recombinant wild-type virus (recMARV; described

in [9]), supernatant fluids of Vero E6 cells infected with either

virus were collected at 6 dpi and used for infection of Vero E6

cells. At 2 dpi, cells were subjected to immunofluorescence

analysis using an antiserum directed against MARV nucleo-

capsid proteins (anti-NC antiserum). Foci of infected cells were

counted by fluorescence microscopy (Figure 1D). Progeny virus

production of rMARV-EGFP was reduced approximately 4-fold

compared with wild-type virus.

To further address this, we compared the protein expression of

both viruses. Vero E6 cells were infected as described above, har-

vested at 2 and 5 dpi, and lysates were analyzed by quantitative

Western blot analysis using antibodies directed against EGFP,

MARVNP,MARV VP40, and actin. While viral proteins could be

readily detected at 2 dpi, EGFP accumulated to detectable levels

only at 5 dpi, which might be due to differences in the sensitivity

of the used antibodies (Figure 1E). NP levels were similar at all

time points, whereas VP40 levels were reduced in rMARV-EGFP-

infected cells at 2 dpi and to a lesser extent at 5 dpi. Since the EGFP

gene is located downstream of NP and upstream of VP40 gene

(Figure 1A), the reduced VP40 expression in rMARV-EGFP

indicates that the presence of the ATU causes a decrease in

downstream protein expression, thereby explaining the slightly

growth-restricted phenotype of rMARV-EGFP.

Cell-to-Cell Spread of MARV-EGFP Observed by Live-CellImagingNext we examined the spread of rMARV-EGFP in cell

culture by live-cell imaging. Vero E6 cells were infected with

rMARV-EGFP and the cell monolayer was analyzed by collect-

ing EGFP fluorescence and phase contrast photomicrographs.

Photomicrographs of 25 different positions were captured every

hour from 1 hpi for a period of 9 days (Supplementary Video;

online only). Single infected cells expressing EGFP were ob-

served at 26 hpi. In most of the infectious centers, EGFP ex-

pression in neighboring cells was detected 20–30 hours later with

a mean value of 24.6 hours, which correlates with the MARV

Musoke replication cycle of approximately 21 hours [24].

However, in some infectious centers, EGFP expression in sur-

rounding cells was observed as late as 48 hpi. Intriguingly, even

late in infection, EGFP fluorescence was not homogenously

distributed throughout the monolayer but restricted to in-

dividual foci, suggesting that virus spread occurred by direct

cell-to-cell contact rather than by release of viral particles.

Typically, individual infected cells were observed early in in-

fection, and later on, the infection spread to cells in close

proximity to the primarily infected cell (Figure 1C and

Supplementary Video). In addition, virus spread was promoted

by viral replication in actively dividing cells (Figure 2A and

Supplementary Video).

After the first signs of CPE appeared at 5 dpi, the cell

monolayer began to disintegrate at 6–7 dpi, followed by cell

rounding and blebbing of EGFP-expressing cells, which corre-

lates with impending cell death (Figure 2B, arrows). Some, but

not all, fluorescent cells formed large intracytoplasmic vacuoles

resembling vacuolated degenerating cells as described for non-

apoptotic forms of cell death [25] (Figure 2C).

Higher magnification of infected cells revealed that

EGFP was homogenously distributed in the nucleus and in

the cytoplasm, but unexpectedly was also observed in

intracytoplasmic aggregates (Figure 1C, bottom panel). Since

MARV infection leads to the formation of inclusions in in-

fected cells, we examined whether the EGFP aggregates were

localized with nucleocapsid-derived inclusions. Therefore, at

2 and 5 dpi, rMARV-EGFP-infected cells were examined by

indirect immunofluorescence using anti-NC antiserum rec-

ognizing the nucleocapsid proteins. EGFP autofluorescence

was assessed in parallel. Cytoplasmic EGFP aggregates colo-

calized with MARV-induced inclusions (Figure 3A). In-

terestingly, immunofluorescence analysis revealed infected

cells that were stained with the virus-specific antiserum but

lacked detectable EGFP expression at 5 dpi, indicating that

immunodetection using virus-specific antibodies is more

sensitive than EGFP detection. To exclude the possibility of

‘‘cross-talk’’ or nonspecific binding of antibodies, rMARV-

EGFP-infected cells were stained with a goat anti-MARV an-

tiserum that predominantly recognizes the MARV surface

protein GP. Surface staining of infected cells was observed

with the GP-specific antibody (Figure 3B, middle panels, red

staining). However, green fluorescent inclusions were also

visible (Figure 3B, left panels).


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http://jinfdis.oxfordjournals.org/cgi/content/full/jir308/DC1

http://jinfdis.oxfordjournals.org/cgi/content/full/jir308/DC1

EGFP Accumulates in Nucleocapsid Protein-Derived InclusionBodies

To further investigate the nature of the EGFP-positive viral in-

clusions, EGFP was expressed in the absence or presence of

MARV nucleocapsid proteins NP and VP35, and cells were

monitored by live-cell imaging. We used U2OS cells for the

transfection experiments because these cells are large and flat,

resulting in high-quality images. U2OS cells were transfected

Figure 1. Characterization of recombinant MARV containing an ATU encoding EGFP. A, Scheme of rMARV-EGFP genome. The EGFP coding sequence isflanked by conserved MARV transcription start and stop signals. The EGFP ORF was inserted between the VP35 and VP40 gene via a newly createdAvr II restriction site within the intergenic region (IR). The intergenic region spanning 5 nucleotides (CTATG) was altered to CCTAGG. B, Detection ofrecombinant genomes in supernatants and cell lysates of rMARV-EGFP-infected Vero E6 cells by RT-PCR. RT-PCR was conducted using primers binding inthe EGFP ORF. Cellular RNA from Vero E6 cells transiently expressing EGFP was used as a positive control. C, Fluorescence microscopy of rMARV-EGFP-infected cells. Living cells were analyzed by phase contrast and fluorescence microscopy. Images were collected at 2 and 5 dpi. Bottom panel showsrMARV-EGFP-infected cell at higher magnification. Inclusions are indicated by an arrow. D, Comparison of progeny production of recombinant wild-typeMARV (recMARV) and rMARV-EGFP. Vero E6 were seeded on glass coverslips and infected with recMARV or rMARV-EGFP. At 2 dpi, cells were subjectedto immunofluorescence analysis using a MARV-specific antibody, and foci of infected cells were counted. The experiment was performed in triplicate andthe bars represent mean values, including standard deviations. E, Quantitative Western blot analysis of virus protein and EGFP levels in Vero E6 cellsinfected with recombinant wild-type recMARV or rMARV-EGFP. Assays were performed in triplicate and standard deviations are shown.


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with 500 ng or 50 ng EGFP expression plasmids along with

plasmids encoding MARV NP and VP35 genes. When 50 ng of

EGFP plasmid was used for transfection, EGFP accumulated in

the nucleus but was also observed in the cytoplasm of transfected

cells, where it was concentrated in inclusion-like aggregates

surrounded by homogenously distributed protein (Figure 4A).

We also observed cells in which EGFP was not concentrated in

intracytoplasmic inclusions. However, since it was not possible

to verify the expression of the nucleocapsid proteins in the live-

cell imaging studies, it is not clear whether these cells expressed

NP and VP35. When 500 ng of EGFP plasmid was used for

transfection, it was difficult to distinguish between concentrated

EGFP and nonspecifically distributed EGFP due to the high

intensity of overexpressed EGFP. Inclusions were only observed

in few cells with lower EGFP expression (Figure 4A). EGFP

inclusions were not detected in cells expressing EGFP in the

absence of NP and VP35 (Figure 4A, bottom panels).

Next, the distribution of EGFP in transfected cells was

analyzed by indirect immunofluorescence. Since concentrated

EGFP could not be differentiated from the homogenous

nonspecific distribution when 500 ng EGFP plasmid was used

for transfection, U2OS cells were transfected with 50 ng EGFP

plasmid along with plasmids for NP and VP35 stained

with anti-NC antiserum. As shown in the upper panels of

Figure 4B, EGFP was distributed in a punctate pattern and

colocalized with intracytoplasmic nucleocapsid inclusions.

Interestingly, the amount of intracytoplasmic homogenously

distributed EGFP was reduced compared with the live-cell

imaging data, which might be due to fixation and/or per-

meabilization effects or due to the fact that EGFP is constantly

Figure 2. Time-lapse fluorescent microscopy of rMARV-EGFP spread. Vero E6 cells were infected with rMARV-EGFP at an MOI of 0.05, and EGFPfluorescence and phase contrast images were captured every hour for a period of 9 days. A, Cell division of infected cells. B and C, Cytopathic effects atlate stages of infection. Blebbing cells are indicated by arrows. Time points postinfection when images were taken are indicated.


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being formed in the live cells, leading to limited bleaching. To

exclude the possibility of antibody cross-reactivity, antibody

staining was omitted. Fluorescence analysis revealed that

EGFP was distributed in large cytoplasmic aggregates (Figure 4B,

middle panels). In contrast, EGFP was homogenously distri-

buted when expressed in the absence of NP and VP35

(Figure 4B, bottom panels). The transfection experiments were

repeated using HT-1080 cells with a similar outcome (data not

shown).

To analyze whether the association with filovirus inclusions is

restricted to EGFP or can also be observed with other fluorescent

proteins, we expressed MARVNP and VP35 proteins along with

various fluorescent proteins from a range of different taxa and

exhibiting different physicochemical features. We selected

TagRFP and DsRed as genealogically different proteins, which

share about 20% amino acid sequence identity with EGFP

[26, 27]. In addition, the monomeric mCherry derivative of the

tetrameric DsRed was included [28]. Intriguingly, each of the

fluorescent proteins colocalized with nucleocapsid-derived

inclusions, when coexpressed with NP and VP35 (Figure 5A).

Similar to EGFP, fluorescent inclusions were also observed when

antibody staining was omitted (Figure 5A, right panels). As

a control, the examined fluorescent proteins were expressed in

the absence of NP and VP35, and were found to be homoge-

neously distributed in the cells (data not shown).

Next, we addressed the question of the observed relocalization

of EGFP in virus-derived inclusions was specific for MARV or

could also be observed with other filovirus species. Therefore,

EGFP was coexpressed with either REBOV or ZEBOV NP and

VP35 proteins. Cells were subjected to immunofluorescence

analysis at 1 dpi using an anti-ZEBOV antiserum that cross-

reacts with REBOV NP. Figure 5B shows that EGFP colocalized

with both ZEBOV and REBOV inclusions, demonstrating that

EGFP accumulation in inclusion bodies is not restricted to

MARV. These data demonstrate that the accumulation of

coexpressed proteins in inclusion bodies is neither restricted to

MARV nor EGFP, but occurs irrespective of filoviral species or

fluorescent proteins used in the assay.

Figure 3. Fluorescence microscopy analysis of EGFP and immunohistochemically labeled viral proteins in rMARV-EGFP-infected cells. Vero E6 cellswere infected with rMARV-EGFP and subjected to immunofluorescence analysis at 2 and 5 dpi. Antibodies were directed against (A) intracellular viralproteins or (B) viral surface proteins. Antibody staining is indicated by red color; EGFP autofluorescence, green; and DAPI staining of the nuclei, blue.


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DISCUSSION

Here we report the generation of a recombinant MARV ex-

pressing EGFP from an ATU inserted between the VP35 and

VP40 genes. We chose this position for the insertion of the ATU

to avoid altering the balance between NP (first gene product)

and VP35 (second gene product), since previous results with

a MARV minigenome system suggested that the ratio of NP to

VP35 is critical for efficient replication and transcription [5].

EGFP has been expressed from the closely related ZEBOV from

different positions in the genome, and insertion of the ATU

between the NP and VP35 genes did not lead to significant

growth defects in cell culture. However, the virus was attenuated

in a STAT-1 knockout mouse model [14, 15]. Similar effects

were observed with a ZEBOV variant containing the ATU be-

tween the VP30 and VP24 genes (fifth and sixth genes).

This virus showed no or mild growth defects in cell culture

depending on the cell line used for propagation, was moderately

attenuated in the mouse model, and was severely attenuated in

a nonhuman primate model [15]. A recombinant ZEBOV in

which the ATU was added between the VP35 and VP40 genes

(second and third genes) could be rescued and propagated in

cell culture [16]. Taken together, these data show that ZEBOV

tolerates the addition of a foreign gene at different positions,

although the insertion of extra nonviral genetic material may

lead to reduced virulence in animal models.

Although replication of the recombinant MARV expressing

EGFP was reduced 4-fold in cell culture, it was successfully used

to monitor viral spread in living cells. Our data suggest that virus

spread in the infectious centers occurred predominantly

through cell-to-cell-contact. Release of viral particles in MARV-

infected cells takes place at filamentous protrusions, the filo-

podia [29]. Since filopodia act as sensory cellular organelles to

explore the extracellular environment, including neighboring

Figure 4. Accumulation of EGFP in MARV inclusions formed by NP and VP35. U2OS cells were transfected with an EGFP expression construct alone oralong with plasmids encoding MARV NP and MARV VP35, as indicated. A, Live-cell imaging of transfected cells. EGFP autofluorescence is shown ingreen. Intracytoplasmic EGFP aggregates are indicated by arrows. B, Cells were stained using an antiserum directed against MARV nucleocapsid proteins(anti-NC; red) and DAPI (blue).


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cells [30], it has been suggested that MARV particles may bud

into adjacent cells via filopodia-mediated cell-to-cell contact

[29]. Besides cell-to-cell-contact, virus replication in actively

dividing cells seems to be an important mechanism of MARV

spread in cell culture. Cell division was not inhibited by MARV

infection, indicating that MARV does not interfere with cell

cycle progression. The collected data clearly illustrate the

strength of live-cell imaging.

Toward the end of the observation period of 9 days, cell

rounding, blebbing, then detachment of infected cells was ob-

served. Some of the infected cells formed large intracytoplasmic

vacuoles. While blebbing is associated with both apoptotic and

necrotic cell death [31], vacuolization of dying cells has been

described for nonapoptotic cell death, such as necrosis or au-

tophagy [25], suggesting that MARV-infected cells might not

undergo apoptosis late in infection. Although the induction of

apoptosis in the context of filovirus infection has been observed

in bystander cells, there are conflicted data on the induction of

apoptosis in infected cells [32–36].

Surprisingly, we found that EGFP accumulates in filoviral

inclusions. A similar observation was reported for some mem-

bers of the nucleorhabdoviruses, where green fluorescent pro-

tein colocalized with viral nucleocapsid protein in loci within

and around the nuclei [37]. In contrast, EGFP was found to be

homogenously distributed in the nuclei and cytoplasm of cells

infected with EGFP-expressing measles virus, which also pro-

duces intracytoplasmic inclusions [38, 39]. The intracellular

distribution of EGFP was examined by live-cell imaging and

immunofluorescence analysis using infected and transfected

cells. In both infected and transfected cells, the fluorescence

intensity of homogenously distributed EGFP surrounding the

intracytoplasmic EGFP aggregates was higher in living cells,

making it difficult to distinguish between EGFP aggregates and

nonspecific-distributed EGFP. Moreover, when large amounts

Figure 5. Accumulation of coexpressed fluorescent proteins (FPs) in nucleocapsid protein-induced inclusions. A, Fluorescence microscopy analysis ofHT-1080 cells expressing TagRFP (red), DsRed (orange), or mCherry (pink), along with MARV NP and VP35. Cells were either stained with anti-NCantiserum (green) and DAPI (blue) or DAPI alone (right panels). B, Fluorescence microscopy analysis of HT-1080 cells coexpressing EGFP along with ZEBOV(upper panel) or REBOV (lower panel) NP and VP35 proteins. Cells were stained with DAPI (blue) and an antibody detecting the nucleocapsid proteins ofboth virus species (red).


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of EGFP plasmid were used for transfection (500 ng), the

punctate pattern of EGFP was not observed in cells coexpressing

MARV NP and VP35, suggesting that the EGFP aggregates were

masked when overall EGFP expression was high, illustrating the

importance of making observations in cells that do not express

too much of a protein. In fixed and permeabilized cells, punctate

EGFP was clearly visible in both infected cells and transfected

cells coexpressing NP and VP35, indicating that the intensity of

EGFP autofluorescence was reduced by the treatment of the

cells.

Intriguingly, EGFP did not only colocalize with MARV in-

clusions but also with ZEBOV and REBOV inclusions formed by

NP and VP35. In addition, fluorescent proteins other than EGFP

also accumulated in MARV inclusions. These data indicate that

the accumulation of ectopic proteins in filoviral inclusions is

most likely not mediated by direct protein–protein interaction,

and future studies are planned to elucidate the underlying

mechanisms. For now, the observed colocalization of ectopic

fluorescent proteins with filovirus inclusions may be useful to

investigate nucleocapsid maturation and transport in infected

cells without tagging viral proteins.

In conclusion, this study describes the generation and

characterization of an EGFP-containing MARV, which will be

a useful tool for imaging-based antiviral drug screening as-

says. High-throughput screening assays based on the expres-

sion of EGFP are currently the preferred approaches for

testing potential therapeutic compounds against EBOV in-

fection [19], and the availability of an EGFP-expressing

MARV facilitates the development of similar assays for

MARV. Furthermore, we demonstrated that rMARV-EGFP

can be used to study important steps of the viral replication

cycle in living cells, including virus spread and infection-

related morphological changes. Since EGFP and other fluores-

cent proteins colocalize with the viral inclusions, the putative

sites of viral replication, rMARV-EGFP could also be used to

study the temporal and spatial regulatory steps involved in the

formation of these virus-derived intracytoplasmic structures.

Future studies are planned to use rodent-adapted recombinant

Marburg viruses expressing EGFP or luciferase as valuable tools

for the development of whole-animal imaging assays and

pathogenesis studies.

Supplementary Data

Supplementary video available at The Journal of Infectious Diseases

online.

Funding

This work was supported by the Manchot Foundation (to K. M. S. and J.

O.); by funds from the German Research Foundation (SFB 535); by National

Institutes of Health (NIH; grants AI082954 and AI057159; New England

Regional Center of Excellence–Kasper, subaward 149047-0743); and by start-

up funds from Boston University.

Acknowledgments

The authors are grateful to O. Dolnik, Philipps University of Marburg,

Germany, for rescue of rMARV-EGFP, J. Connor, Boston University,

Boston, MA, for his help with carrying out live-cell imaging, and W. P.

Duprex, Boston University, for critical review of the manuscript. We also

thank N. Kedersha and P. Anderson, Brigham And Women’s Hospital,

Harvard Medical School, Boston, MA. for providing the U2OS cells, V.

von Messling, University of Quebec, Quebec, Canada, for the mCherry

plasmid, and S. Becker, Philipps University of Marburg, for kindly pro-

viding anti-MARV antiserum, anti-EBOV antiserum, and anti-MARV

VP40 monoclonal antibody.

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