Structural dissection of Ebola virus and its assembly
determinants using cryo-electron tomography
Tanmay A. M. Bharata, Takeshi Nodab, James D. Richesa, Verena Kraehlingc, Larissa Kolesnikovac, Stephan Beckerc,
Yoshihiro Kawaokab,d,e,f, and John A. G. Briggsa,1
aStructural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany;bInternational Research Center
for Infectious Diseases anddDivision of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo,
Tokyo 108-8639, Japan;cInstitut für Virologie, Philipps-Universität Marburg, 35043 Marburg, Germany;eExploratory Research for Advanced Technology
Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama 332-0012, Japan; andfDepartment of Pathobiological Sciences,
School of Veterinary Medicine, University of Wisconsin, Madison, WI 53711
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved February 6, 2012 (received for review December 12, 2011)
Ebola virus is a highly pathogenic filovirus causing severe hemor-
rhagic fever with high mortality rates. It assembles heterogenous,
filamentous, enveloped virus particles containing a negative-sense,
single-stranded RNA genome packaged within a helical nucleocap-
sid (NC). We have used cryo-electron microscopy and tomography
to visualize Ebola virus particles, as well as Ebola virus-like particles,
in three dimensions in a near-native state. The NC within the virion
forms a left-handed helix with an inner nucleoprotein layer deco-
rated with protruding arms composed of VP24 and VP35. A com-
parison with the closely related Marburg virus shows that the
N-terminal region of nucleoprotein defines the inner diameter of
the Ebola virus NC, whereas the RNA genome defines its length.
Binding of the nucleoprotein to RNA can assemble a loosely coiled
NC-like structure; the loose coil can be condensed by binding of
the viral matrix protein VP40 to the C terminus of the nucleopro-
tein, and rigidified by binding of VP24 and VP35 to alternate copies
of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are
necessary and sufficient to mediate assembly of an NC with
structure, symmetry, variability, and flexibility indistinguishable
from that in Ebola virus particles released from infected cells.
Together these data provide a structural and architectural de-
scription of Ebola virus and define the roles of viral proteins in its
structure and assembly.
Mononegavirales|single-stranded RNA virus|virus structure|
Filoviruses are highly pathogenic, causing severe hemorrhagic
fever in monkeys and humans, with high mortality rates (1).
Because of the lack of approved vaccines and antiviral drugs,
both EBOV and MARV are categorized as biosafety level-4
The order Mononegavirales also contains several other patho-
gens of clinical importance, such as rabies virus (RABV), mumps
virus, measles virus (MeV), and respiratory syncytial virus (RSV)
(2). All members of the order possess a nonsegmented, negative-
sense RNA genome, which is encapsidated by the viral nucleo-
protein (NP). The NP–RNA complex acts as the template for
genome replication and assembles into a helical nucleocapsid
(NC) along with accessory proteins (3). This characteristic links
genome replication mechanisms of mononegaviruses to their
NC structure. The NC is recruited to the plasma membrane by
the viral matrix protein, where it buds through the membrane
to form an enveloped virion. All mononegaviruses share these
EBOV virions contain an RNA genome and seven viral pro-
teins: NP, VP35, VP40, GP (glycoprotein), VP30, VP24, and an
RNA-dependent RNA polymerase (L). NP, VP30, VP35, and L
are known to associate with the transcription and replication-
competent NC (4–6). VP24 is additionally required for NC as-
sembly (7, 8). VP40, the viral matrix protein, binds directly to the
viral envelope. Expression of VP40 alone in mammalian cells can
bola virus (EBOV) and Marburg virus (MARV) constitute
the family Filoviridae within the order Mononegavirales.
lead to formation and release of enveloped, filamentous virus-
like particles (VLPs) (9–12). Expression of NP alone leads to the
formation of narrow, tubular structures in the cytoplasm of the
cell (13). These narrow structures can be recruited into VLPs by
coexpression of VP40 (14). If NP is expressed together with
VP24 and VP35, cytoplasmic clusters of NC-like structures are
formed that are similar to those seen in infected cells (13). These
structures are also recruited into VLPs when VP40 is coex-
pressed (7, 13–15). Together these studies suggest that a direct
interaction between VP40 and NP can recruit NP into released
VLPs and that formation of an NC with diameter similar to that
in native virions requires co-expression of NP, VP24, and VP35.
Recent cryo-electron microscopy (cryoEM) investigations of
MARV described the 3D structure of the MARV NC (16). The
MARV NC is a left-handed helix, with the viral NP forming the
innermost layer of the structure. Each NP binds to six bases of
RNA. Arm-like structures protrude from alternate interfaces
between NPs, and immuno-electron microscopy analysis locates
VP24 and VP35 to these protrusions. The NC is incorporated
into virions by envelopment at the plasma membrane initiated at
one end of the NC (16, 17).
In the present study, EBOV virions were imaged using cryoEM
and cryo-electron tomography (cryoET) to describe their struc-
ture in a near-native state. Image-processing techniques were
applied to define the 3D structure of the NC within the virion.
The EBOV NP shares ≈40% sequence homology with MARV
NP (18, 19). Comparison of the morphological parameters and
NC structures of EBOV with MARV allowed us to dissect the
roles played by the RNA genome and filovirus NPs in deter-
mining NC structure.
In addition, Ebola VLPs were produced with different combi-
nations of viral proteins and studied using biochemical, cryoEM,
and cryoET techniques. These studies define roles for viral pro-
teins in determining the structure of EBOV virions and their NCs,
which range from mediating initial coiling of the NC helix, to
helical condensation, to rigid helix formation, to NC envelopment
CryoEM and CryoET of EBOV. Zaire EBOV virions were harvested
from infected Vero cells 1 d after infection in a BSL-4 laboratory.
The inactivated virus pellet was released from the BSL-4 labo-
ratory and then imaged using cryoEM. Long, filamentous mem-
J.D.R., V.K., and L.K. performed research; T.A.M.B., T.N., J.D.R., L.K., S.B., Y.K., and J.A.G.B.
analyzed data; and T.A.M.B. and J.A.G.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: CryoEM data reported in this paper have been deposited with the
Electron Microscopy Data Bank (accession no. EMD-2043).
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 13, 2012
| vol. 109
| no. 11
brane-bound particles could be observed along with spherical
particles and other irregularly shaped vesicles (Fig. 1A). Several
virions possessed previously described “moth-eaten” membranes
(1). Some filamentous particles lacked NC structures, resulting in
a smaller diameter (Fig. 1 A and B, black arrow). Many virions
displayed an intact membrane and a clearly visible NC (Fig. 1B,
white arrow). This variable morphology of EBOV is consistent
with previous negative staining EM analysis (1).
CryoEM allows excellent preservation of the specimen in
a near-native environment: this allowed us to accurately measure
morphological parameters of virions. The distribution of virus
lengths (Fig. 1C, Fig. S1A, and SI Materials and Methods) showed
a major population of virus particles with a length of 1,028 ± 69
nm (n = 37), consistent with previously reported values of 970–
1,200 nm for the average EBOV virion (20, 21). We also found
a secondpopulation with a meanlength of1,978± 112 nm(n=8)
(Fig. 1C), as well as some longer particles (Fig. S1A). The di-
ameter of filamentous EBOV particles that had a continuous
membrane and an internalized NC was 90 ± 3 nm (n = 50)
(Fig. 1C, Right), which is slightly smaller than that of MARV
particles (92 ± 4 nm) (Fig. 1D, Right).
To understand the 3D arrangement of the virion, we per-
formed cryoET. A slice through a representative tomogram is
shown in Fig. 2A. The viral NC appears as a cylinder-like density
within the particle center (white arrow in Fig. 2A, Movie S1),
similar in appearance to the MARV NC (16). Regular repeats at
a pitch of ≈7 nm could be observed along the length of the NC.
To resolve the structure of the EBOV NC in more detail, we
applied subtomogram averaging methods on the tomography data
(SI Materials and Methods), as described previously (16, 22). All of
the reconstructed helices were left-handed with an inner layer dec-
orated with arm-like protrusions in the outer layer. Of NCs whose
symmetry could be unambiguously assigned, all were found to
the NC helices with the same symmetry into one single subtomo-
gram averaging reconstruction enabled refinement up to 4.1 nm
resolution. We also performed iterative real-space helical recon-
struction using helical segments extracted from 2D cryoEM images
of EBOV (Fig. S2, Left) and again obtained a reconstruction with
a resolution of 4.1 nm. In contrast, subtomogram averaging and it-
erative real-space helical reconstruction resolved the MARV NC to
better resolutions of 3.4 nm and 2.5 nm, respectively (16). This
suggests that the EBOV NC has a higher amount of conformational
variability or flexibility than the MARV NC.
We therefore identified the subset of NC helices that aligned
successfully (SI Materials and Methods) and had a symmetry of
11.8 subunits per turn and combined them into one final re-
construction (Fig. 2 B and C, Left) with an improved resolution
of 3.6 nm. The final subtomogram averaging reconstruction
shows the EBOV NC helix to be left-handed with a pitch of
≈7.4 nm (Fig. 2 B and C, Left) close to that of MARV (7.5 nm)
(16). An inner layer is observed with a diameter of ≈28 nm.
Boomerang-shaped densities protrude outward from this inner
layer, and the diameter of the entire structure is ≈40 nm. The
protrusions have two lobes. A left-handed helix with a pitch of
EBOV. Protein density is black. Filamentous particles of varying lengths,
spherical particles, and other irregularly shaped particles are observed. (B)
CryoEM image of a filamentous EBOV virion. White arrow, EBOV virion with
an NC. Black arrow, a thin particle without an internalized NC. (C) Histo-
grams of virion length (Left) and diameter (Right) for filamentous EBOV
virions containing an NC. (D) Corresponding histograms for MARV. More
details in Fig. S1 and SI Materials and Methods.
CryoEM of EBOV. (A) Low-magnification cryoEM images of purified Fig. 2.
averaging. (A) A slice through a reconstructed, filtered tomogram of EBOV.
Protein density is black. White arrowhead indicates the rod-like NC within
the virion. (B) Reconstruction of the EBOV NC from cryoET and subtomogram
averaging (Left) compared with the MARV NC reconstruction (Right) (16).
Isosurfaces have been contoured at 1.5 σ away from the mean, and the
helical axis is vertical in the plane of the paper. (C) The same reconstructions
as B, viewed along the helical axis.
CryoET and 3D reconstruction of the EBOV NC from subtomogram
| www.pnas.org/cgi/doi/10.1073/pnas.1120453109Bharat et al.
≈7.5nm, with an inner layer from which boomerang-shaped
densities protrude outward, has been observed previously for the
MARV NC (Fig. 2 B and C, Right) (16), indicating close struc-
tural similarity between the two filovirus NCs. By analogy with
MARV, the inner layer likely represents NP, and the protrusions
likely contain VP24 and VP35 (16).
Relationship Between Genome Length, NC Symmetry, and NC Length.
A comparison of MARV and EBOV genome lengths, NC sym-
metries, and NC lengths is informative. In MARV, there are
13.8, 14.8, or 15.8 boomerang-shaped protrusions per turn of the
NC helix (16), but in EBOV there are only 11.8 or 12.8 pro-
trusions per turn. Because there are two NP monomers for each
boomerang-shaped protrusion (16), on an average this translates
into 29.6 MARV NPs per turn but only 24.6 EBOV NPs per
turn. Both filovirus NPs have a similar molecular mass (83.2 vs.
77.8 kDa). The smaller number of EBOV NPs per turn is
reflected in the smaller diameter of the EBOV NC helix (com-
pare Fig. 2C, Left vs. Right).
The genome lengths of Zaire EBOV and Lake Victoria
MARV are very similar (18,961 vs. 19,111 bases). Because there
are fewer NP molecules per turn of the EBOV NC than in the
MARV NC, the EBOV NC would have to be longer to package
the entire genome at the same density. The mean lengths of
MARV (876 nm) and EBOV (1,028 nm) virions confirm this
expectation (Fig. 1 C and D, Fig. S1, and SI Materials and
Methods). On the basis of the average length of EBOV, and on
the number of subunits per turn of the NC helix, we calculate that
a virion of 1,028 nm in length contains ≈3,200 EBOV NP mol-
ecules per virion (SI Materials and Methods). This means that for
each EBOV NP molecule, there are 5.9 ± 0.4 RNA bases. Like
MARV (16), EBOV therefore likely packages six RNA bases per
copy of NP. The longer virions, with a length of 1,978 nm, would
contain ≈6,450 copies of the NP and therefore probably package
two copies of the genome (SI Materials and Methods).
Formation of the Inner NC Helix. After describing the structure of
the EBOV NC, we wanted to understand the roles of different
viral proteins in assembling the NC. To determine the minimum
assembly component of EBOV NC, we purified full-length
EBOV NP from mammalian cells. This sample has been pre-
viously shown to assemble together with cellular RNA, and
appears by negative staining EM as coil-like structures (18).
Using cryoEM, we confirmed that the sample formed loose coil-
like structures (Fig. 3A). The diameter of the coils was roughly
30–40 nm but varied slightly between individual coils.
The C-terminal parts of NPs from other members of Mono-
negavirales like MARV and MeV are known to contain large
disordered regions (16, 23, 24). Deletion of the C-terminal dis-
ordered region of the MARV NP allowed it to assemble con-
densed helical rods with a diameter of ≈33 nm (16). To test
whether this was also the case in EBOV, we expressed and pu-
rified a C-terminal deletion mutant of the EBOV NP containing
only the first 451 amino acid residues [NP(1-451)]. This construct
is known to be sufficient to bind RNA and assemble an NC coil
(18). In contrast to the full-length NP, we found that NP(1-451)
mostly formed condensed helical rods with a defined diameter
and pitch (Fig. 3B). We extracted short helical segments from
cryoEM images of the NP(1-451) mutant and carried out 2D
alignment and averaging. The average image (Fig. 3B, Inset)
shows that the diameter of the helix is ≈28 nm and that the pitch
of the helix is ≈7.4 nm. A reconstruction of the NP(1-451) helix
using real-space helical reconstruction techniques was obtained
(Fig. S2, Center) and compared with the EBOV NC re-
construction. The N-terminal 451 residues of NP assemble into
a helical structure that is similar to the innermost layer of the
complete EBOV NC, suggesting that these residues form the
core of the helical NC.
These data show that NP–NP oligomerization on cellular RNA
forms a loose coil. In contrast, the first 451 residues of EBOV NP
can oligomerize on RNA to form condensed helical rods in which
both the diameter and helical pitch are the same as the inner layer
of EBOV NC in virions. The N-terminal region of NP is thus
sufficient to form the interactions around and along the helix,
which define the pitch and inner diameter of the EBOV NC.
Because VP40 has been shown to bind to the C terminus of NP
(25), we wanted to test whether co-expression of NP with VP40
could also lead to the formation of condensed helices. We
therefore expressed both full-length NP and VP40 in mammalian
cells (Materials and Methods), which leads to the formation and
release of VLPs containing NC-like structures (13, 15, 25). VLPs
were collected, their membranes were disrupted, and the NCs
were then isolated by ultracentrifugation for imaging by cryoEM.
Whereas full-length NP purified from cells in the absence of
VP40 formed only loose coils, we observed that the NC helix
purified from NP+VP40 VLPs formed short stretches of con-
densed helices punctuated by short coil-like regions (Fig. 3C).
2D averaging of the condensed helical segments showed a helix
with a diameter of ≈28 nm and pitch of ∼7.5 nm (Fig. 3C, Inset).
The NC helix purified from NP+VP40 VLPs is therefore very
similar to the NC helix purified from cells expressing NP(1-451)
in the absence of VP40. This similarity is further highlighted by
quantification of the number of condensed helices and coils
found in the three samples (Fig. 3D, Table S1, and SI Materials
and Methods). These data support a model whereby the C-ter-
minal part of NP disrupts helix condensation, and interaction of
VP40 with the C-terminal part of NP relieves this disruptive
effect to allow NP–NP contacts to form between turns of the
helix, leading to NC condensation.
of purified full-length EBOV NP. Protein density is black. (B) Image of puri-
fied NP(1-451). Inset: 2D average of extracted helical segments. Width of box
720 nm, protein density white. (C) Corresponding images of the NC helix
purified from NP+VP40 VLPs. (D) Comparison of proportion of condensed
helices (green) and loose coils (yellow) observed in the three samples. Data
values are in Table S1.
Minimum assembly component of the EBOV NC. (A) CryoEM image
Bharat et al.PNAS
| March 13, 2012
| vol. 109
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Order of Protein Assembly and Formation of a Rigid NC Helix. We
next prepared a series of VLPs by expression of different com-
binations of viral proteins along with VP40 in mammalian cells:
NP+VP40, NP+VP24+VP40, NP+VP35+VP40, and NP+
VP24+VP35+VP40. Previous thin-section EM analyses have
indicated the presence of an NC-like structure in these VLPs (13–
15). We analyzed recruitment of viral proteins into released VLPs
using Western blot analysis with anti-NP, anti-VP24, anti-VP35,
coexpression with VP40 alone (Fig. 4A). When NP, VP35, and
VP40 were coexpressed, all three proteins could be detected in
VLPs. These observations are consistent with previous observa-
tions that VP40 can recruit VP35 and NP independently into
VLPs (13, 26). We found that only a low amount of VP24 was
recruited into VLPs when it was coexpressed with NP and VP40.
However, VP24 was detected in large amounts when VP35 was
additionally expressed (Fig. 4A). These results indicate that NP
can be directly recruited into VLPs by VP40, that VP24 and VP35
can be recruited by NP and/or VP40, and that VP35 significantly
enhances the recruitment of VP24 into VLPs.
All filamentous VLPs were subjected to cryoEM and cryoET.
CryoEM was used to quantify the number of VLPs with and
without an internalized NC. CryoET was used to divide VLPs that
contained NCs into two structural classes by a visual inspection of
the filtered tomograms. The first class had an NC with short
stretches of condensed helix broken at multiple points (Fig. 4C).
The second class contained a rigid, largely continuous NC struc-
ture with outer protrusions (Fig. 4D). We compared the frequen-
cies of the different classes of NCs found in the VLP samples from
tomograms (Table S2) with those in EBOV virions. Together the
cryoEM and cryoET data showed that in NP+VP40 VLPs, 64%
of the VLPs were empty (Fig. 4E), and 36% contained broken,
discontinuous NCs. A rigid, continuous NC could not be observed
in any of the NP+VP40particles.A verysimilar pattern was found
in NP+VP24+VP40 VLPs (73% empty, 27% broken) and in
NP+VP35+VP40 VLPs (66% empty, 34% broken) (Fig. 4E).
Although the percentage of empty particles (68%) in NP+
VP24+VP35+VP40 VLPs was similar to the other analyzed
VLP samples, the NC, when present, was predominantly rigid:
30% of VLPs contained a rigid NC structure, whereas only 2%
contained a broken or discontinuous NC. These numbers are
comparable to our observations of authentic virions, in which we
found that 63% of the particles were empty and 34% contained
continuous rigid NCs (Fig. 4E). Unlike authentic virions, the
length of the NP+VP24+VP35+VP40 VLPs was not well de-
fined (Fig. S1C). To summarize, in the absence of NP, VP24, or
VP35, a rigid NC-like structure was never observed. When NP,
VP24, VP35, and VP40 were coexpressed, VLPs were obtained
with rigid NCs that were morphologically similar to the full
EBOV NC. These data indicate that NP, VP24, and VP35 are all
required to form a rigid, continuous NC structure.
Structural Characterization of the VLPs. To detect differences in the
NCs between various VLP samples, we next performed 2D
classification and averaging of helical segments extracted from
cryoEM images of the VLPs (SI Materials and Methods). The NP+
VP40 VLPs contained an NC helix with a diameter of ≈28 nm,
lacking the arm-like protrusions observed in authentic virions
(Fig. 5A). Because of discontinuities in the NC helix, the NP
layer in the average image appears blurred. Average NC images
from NP+VP24+VP40 VLPs and NP+VP35+VP40 VLPs had
the same appearance (Fig. S3).
(Fig. 5B, arrows), appearing similar to NC from authentic virions
(Fig. 5C). Weperformed 3D reconstructionofNCsfrom theNP+
VP24+VP35+VP40 sample using subtomogram averaging and
real-space helical reconstruction techniques, exactly as described
above for the NC within EBOV virions. The NC helix in the VLPs
adopted the same symmetries (11.8 and 12.8 protrusions per turn)
and structure as the NC helix in virions (Fig. 5 D and E), with an
inner layer decorated with boomerang-shaped outer protrusions.
The resolution of the real-space helical reconstruction was 4.1 nm
(Fig. S2, Right), and a selected subset of the NCs combined with
subtomogram averaging reached a resolution of 3.9 nm (Fig. 5 D
and E). In both cases the reconstructions are the same as the NC
reconstruction from virions with the same resolution (compare
Figs. 2 B and C with 5D and E and Fig. S2A, Left vs. Right). Thus,
viral proteins in respective VLPs. Purified VLPs were collected, and Western
blot analysis using rabbit anti-NP, -40, -35, and -24 antibodies was per-
formed. (B) A tomographic slice through an empty VLP. Protein density is
black. (C) Slice through a VLP with a broken NC. Points of breakages in the
NC helix have been highlighted with white arrows. (D) A VLP with a rigid NC.
(E) Proportion of particles observed with a rigid NC (dark green), with an
overall broken NC (orange), and without an NC (gray) in different samples.
Data values are in Table S2.
Protein recruitment and formation of a rigid NC. (A) Detection of
NC from NP+VP40 VLPs. (B) 2D class averages of the NC from NP+VP24+VP35+
VP40 VLPs. (C) 2D class averages of the NC from EBOV virions. Black arrows
indicate protrusions. (D) Subtomogram averaging reconstruction of the NC
helix from NP+VP24+VP35+VP40 VLPs. Isosurfaces have been contoured at
1.5 σ away from the mean, and the helical axis is vertical in the plane of the
paper. (E) The same reconstruction viewed along the helical axis.
Location of viral proteins in the EBOV NC. (A) 2D class averages of the
| www.pnas.org/cgi/doi/10.1073/pnas.1120453109Bharat et al.
the NC helices from NP+VP24+VP35+VP40 VLPs and from
EBOV virions are indistinguishable in structure, symmetry, and
Architecture of EBOV Virions and the EBOV NC. We found that
EBOV particles were largely filamentous, but other morpholo-
gies, including spherical particles and particles without an in-
ternalized NC, were also observed. Such variable morphology is
consistent with earlier observations by negative staining EM (1).
Within cryoEM images we could see that straight sections of
virions contain a cylindrical NC along the center of the virus
particle. Most filamentous EBOV virions had a length of ≈1,028
nm, although longer viruses were also observed with lengths that
were approximate multiples of this length, suggesting they con-
tain multiple NCs.
A comparison of the EBOV with the recently presented cryoET
structure of MARV NC (16) sheds light on factors affecting virus
assembly. Many features are shared between the two NCs. The
pitch of the EBOV NC helix (7.4 nm) is almost identical to that
described by cryoEM for the MARV NC (7.5 nm). The 3D struc-
ture of the EBOV NC reveals a left-handed helical structure, just
up of the viral NP, which is decorated by boomerang-shaped pro-
trusions. By analogy with MARV, one protrusion emanates from
every two NPs in the inner layer. Binding of one copy of the viral
phosphoprotein to two copies of the NP has also been observed by
x-ray crystallography of a purified rhabdoviral complex (27).
There are also differences between the EBOV and MARV
NCs. In all our analyses the EBOV NC was consistently more
flexible than the MARV NC. This suggests higher intrinsic
conformational flexibility in the repeating asymmetric unit of the
EBOV NC. The symmetry of the two filovirus NCs differs: the
EBOV NC has fewer NP subunits per turn of the helix but has
more turns of the helix per virion, so that EBOV virions are
longer than MARV virions. This means that the total number of
NPs is approximately the same in EBOV and MARV, and the
number of RNA bases per copy of NP is also the same, with each
NP binding six RNA bases.
Genome replication in Mononegavirales is tightly linked to NC
with previous observations that only multiples of six bases can be
added or removed from the replication promoter region while
maintaining function (29). Binding to a multiple of six RNA bases
per NP monomer is also observed in paramyxoviruses like Sendai
virus and MeV (30, 31), and like EBOV (29) these viruses also
have bipartite replication promoters. These facts together suggest
that genome replication mechanisms of filoviruses are likely
rhabdoviruses like VSV and RABV, which package different
numbers of RNA bases per NP (32–34).
Structural Roles of EBOV Components in Determining NC Structure.
The expression of VP40 along with NP leads to recruitment of
NP into VLPs. This is likely due to binding of VP40 to the C
terminus of NP (25). Expression of NP and VP40 together allows
recruitment of VP35 into VLPs. For efficient VP24 recruitment
into VLPs, NP, VP35, and VP40 must be expressed. This is
consistent with previous morphological studies that asserted that
NP, VP24, and VP35 are all necessary for NC assembly (7, 13).
The EBOV NP alone, upon binding to RNA, forms a loosely
coiled helix. Removal of the C-terminal 288 residues of NP,
which are predicted to contain large disordered regions, leads
to formation of condensed helices instead of loose coils. The
C-terminal region of NP therefore prevents condensation of the
N-terminal region of NP into helices. CryoEM observations on
MARV and MeV NPs have also shown that purified NP samples
could form loose helices, and that C-terminally deleted NPs
could assemble condensed helices (16, 23), suggesting that this is
a general property shared with other mononegaviruses. The
disordered C-terminal domain of EBOV NP contains binding
sites for VP40. NCs purified from VLPs produced by coex-
pression of NP and VP40 are condensed helices indistinguish-
able from those formed by C-terminally deleted NP. We
therefore propose that binding of VP40 to the C-terminal region
of NP during virus assembly relieves its inhibitory effect, allowing
the N terminus of NP to assemble a condensed helix. The con-
densed EBOV NP(1-451) helices have a diameter of ≈28 nm,
which is the same as the 28-nm diameter of the inner NC helix in
the authentic EBOV particle. MARV NP(1-390) assembles into
a condensed helix with a diameter of 33 nm, which is the same as
the 33-nm diameter of the inner NC helix in the authentic
MARV particle (16). This comparison suggests that the N-ter-
minal domain of NP in filoviruses is alone sufficient to define the
diameter of the NC helix.
The condensed NC helix retains some flexibility and is punc-
tuated by breaks when packaged into VLPs, or by regions of
loose coil when purified. This contrasts with the viral NC, which
we found to form rigid helices. Coexpression of both VP24 and
VP35 with NP and VP40 was required to release VLPs con-
taining rigid NCs, suggesting that binding of VP24 and VP35
leads to rigidification of the helix. These two proteins form
boomerang-shaped protrusions emanating from the inner NP
layer. NP, VP24, VP35, and VP40 together are sufficient to as-
semble an NC that has the same symmetry, structure, variability,
and flexibility as the NC within the virion.
The NCs in NP+VP24+VP35+VP40 VLPs do not have
a defined length, contrasting with the NC in authentic EBOV
and MARV virions that consistently has exactly the length re-
quired to package one viral genome at a density of six RNA
bases per NP. In some cases more than one NC can be in-
corporated into a single virion, giving a virus particle with double
the samples described in this study and their assembly properties. Assembly
of a virus particle is indicated by the thick arrow. Initial condensation of
the NP-RNA complex can be achieved in vitro by removal of the disordered
C-terminal, or in cells by coexpression with VP40 (thin arrows). The con-
densed helix can be converted into a rigid NC-like helix inside VLPs only if all
NP, VP24, VP35, and VP40 are expressed. The resulting NC helix is in-
distinguishable from that in EBOV virions.
Steps involved in EBOV NC assembly. A schematic illustration of
Bharat et al.PNAS
| March 13, 2012
| vol. 109
| no. 11
or triple the expected length. These observations imply that NC
length is determined by genome length.
By analysis of purified proteins, VLPs, and virions, we can
propose distinct structural roles for these components in EBOV
assembly. We suggest that formation of a virus particle requires
packaging of the RNA genome by N-termini of NP to form
a loose coil with a length defined by total genome length. We
suggest that it requires condensation of the loose coils into
a helix with diameter defined by the N-terminal region of NP and
that this can be mediated by binding of VP40 to the C terminus
of NP. We suggest that it requires rigidification of the condensed
coils into a tight helix with arm-like protrusions by binding of
VP24 and VP35 to alternate NPs and that these components are
sufficient to define the mature EBOV NC structure (Fig. 6).
Materials and Methods
Purification of Recombinant EBOV NP. NP or its (1-451) truncation mutant were
purified from transfected HEK 293 cells using CsCl gradient centrifugation
(SI Materials and Methods). All samples were prepared in duplicate to con-
trol for differences between sample preparations.
Preparation of VLPs and Virus. EBOV proteins were coexpressed with VP40 in
HEK 293 cells. Two days after transfection, VLPs were fixed with 1% para-
formaldehyde (PFA) and pelleted by ultracentrifugation through a 20% (wt/
wt) sucrose cushion. The pellet was resuspended in PBS and stored at 4 8C
until further investigation.
All work with infectious EBOV was performed under highest safety pre-
cautions in the BSL-4 facility at the Institut für Virologie, Philipps-Universität
Marburg. Particles of EBOV that were released from infected Vero cells were
collected1 d after infection,purified by centrifugationthrougha 20%sucrose
cushion, andfixedwith4% PFAto inactivate thevirus completely (SI Materials
and Methods). All samples were prepared in duplicate and initially analyzed
separately to control for differences between preparations.
Western Blot Analysis. Purified VLPs were lysed in SDS sample buffer and
separated on a PAGE Tris/glycine gel. Blots were incubated with rabbit anti-
NP, anti-VP24, anti-VP35, or anti-VP40 serum as primary antibodies, and with
HRP-conjugated anti-rabbit IgG antibody as a secondary antibody. Bands
were detected with ECL Plus Western Blotting Detection Reagents (GE
Healthcare) and visualized using VersaDoc Imaging System (Bio-Rad).
CryoEM and Image Analysis. ForcryoEMstudies,vitrifiedsampleswereimaged
under standard low-dose conditions in a FEI CM120 Biotwin microscope
used. Tomographic tilt ranges were typically from +608 to −608, with a total
dose of 6,000–10,000 e−/nm2. For each VLP sample, 8–20 tomograms were
collected, and for virions more than 20 tomograms were collected.
2D data were analyzed using Bsoft (35) and Spider (36). Helical re-
construction was carried out using the real space reconstruction technique
(37) implemented in the Spider package (SI Materials and Methods). Tomo-
were extracted along the length of NCs and iteratively aligned in six dimen-
sions, taking into account the missing wedge as described previously (16, 39).
Visualization of image data was carried out in Amira (Visage Imaging) and
ACKNOWLEDGMENTS. This work was funded by Deutsche Forschungsge-
meinschaft Grants SPP 1175 (to J.A.G.B. and S.B.) and SFB 593 (to S.B.). This
work was supported by National Institute of Allergy and Infectious Diseases
Public Health Service research grants (to Y.K.). This study was technically
supported by the use of the European Molecular Biology Laboratory IT Ser-
vice unit. T.N. was supported by a Grant-in-Aid for Young Scientists from the
Japan Society for the Promotion of Science.
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| www.pnas.org/cgi/doi/10.1073/pnas.1120453109 Bharat et al.