Structure and assembly of a paramyxovirus matrix protein.
ABSTRACT Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.
- SourceAvailable from: Monika Bajorek[Show abstract] [Hide abstract]
ABSTRACT: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide; however there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study we focus on M protein and identify a key phosphorylation site (Thr(205)) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable Alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic Aspartate (Asp) resulted in a non-viable virus which could only be recovered with an additional mutation in M (Serine to Asparagine at position 220), strongly implying that Thr(205) is critical for viral infectivity. Experiments in vitro showed that mutation of Thr(205) does not affect M stability or the ability to form dimers, but implicate an effect on higher order oligomer assembly. In transfected and infected cells, Asp substitution of Thr(205) appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using EM. Our data thus imply for the first time that M oligomerization, regulated by negative charge at Thr(205), may be critical to production of infectious RSV. We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine (Thr(205)), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or virus-like filament formation per se, but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus, rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T(205)-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge, and hence of considerable interest for vaccine approaches in the future.Journal of Virology 03/2014; · 5.08 Impact Factor
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ABSTRACT: In this issue of Structure, Leyrat and colleagues provide the first structural analysis of the human metapneumovirus (HMPV) matrix protein, a key regulator of viral assembly. Though structurally similar to other matrix proteins, two calcium binding sites suggest intriguing differences in regulation.Structure 01/2014; 22(1):5-7. · 5.99 Impact Factor
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ABSTRACT: A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious.Current opinion in virology. 01/2014; 5C:16-23.
Structure and assembly of a paramyxovirus
Anthony J. Battistia,1, Geng Menga,1, Dennis C. Winklerb, Lori W. McGinnesc, Pavel Plevkaa, Alasdair C. Stevenb,
Trudy G. Morrisonc, and Michael G. Rossmanna,2
aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907-2032;
Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-8025; and
Physiological Systems/Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655-0122
bLaboratory of Structural Biology Research, National Institute of
cDepartment of Microbiology and
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved July 26, 2012 (received for review June 15, 2012)
Many pleomorphic, lipid-enveloped viruses encode matrix proteins
that direct their assembly and budding, but the mechanism of this
process is unclear. We have combined X-ray crystallography and
cryoelectron tomography to show that the matrix protein of New-
castle disease virus, a paramyxovirus and relative of measles virus,
forms dimers that assemble into pseudotetrameric arrays that
generate the membrane curvature necessary for virus budding. We
show that the glycoproteins are anchored in the gaps between the
matrix proteins and that the helical nucleocapsids are associated in
register with the matrix arrays. About 90% of virions lack matrix
arrays, suggesting that, in agreement with previous biological
observations, the matrix protein needs to dissociate from the viral
membrane during maturation, as is required for fusion and release
of the nucleocapsid into the host’s cytoplasm. Structure and
sequence conservation imply that other paramyxovirus matrix pro-
teins function similarly.
crystallography and cryoelectron microscopy, but less is known
about the atomic structure of viral proteins in the context of
pleomorphic viruses. Paramyxoviridae, Rhabdoviridae, Filoviridae,
and Bornaviridae are pleomorphic viruses belonging to the order
Mononegavirales. These enveloped viruses all have a linear, non-
segmented, negative-sense, 10- to 20-kb–long RNA genome that
is encapsidated by a nucleocapsid protein into a helical assembly
(1). Paramyxoviruses include human pathogens that infect the re-
spiratory system. Newcastle disease virus (NDV) is an avian para-
myxovirus that is the prototypical species of the genus Avulavirus,
belonging to the Paramyxovirinae subfamily. Poultry are especially
susceptible to NDV infections, and some virulent strains have
been classified by the US Department of Agriculture as select
agents (2, 3).
Paramyxoviruses (Fig. 1) are pleomorphic and encode at
least six proteins (1). They all have two transmembrane glyco-
proteins—the attachment protein termed HN (approximately
75 kDa), G, or H (depending upon the virus) and the F protein
(approximately 60 kDa)—that form spikes protruding from the
lipid bilayer. HN is a dual-function hemagglutinin/neuramini-
dase, capable of binding to cell surface sialic acids (4). F is re-
quired for fusion with the host cell plasma membrane (5–7). The
nucleocapsid protein, NP (approximately 50 kDa), together with
the genomic RNA forms a helical structure that encapsidates and
protects the viral RNA genome. The helical parameters of the
nucleocapsid structure exhibit variability among paramyxoviruses
(8, 9). The matrix (M) protein of many pleomorphic, membrane-
enveloped viruses directs assembly and budding (10–14). The
structures of three matrix proteins of viruses belonging to the
order Mononegavirales [respiratory syncytial virus (RSV) (14),
Ebola virus (12), and Borna virus (13)] have been determined.
These structures are built of one or two domains that have similar
β-sandwich folds, suggesting gene duplication during evolution.
The dimers of paramyxovirus M protein (approximately 40 kDa)
can form a grid-like array on the inner surface of the viral mem-
he structure of icosahedral viruses has been extensively stu-
died by X-ray crystallography and by a combination of X-ray
brane (15–18), and probably interact with both the cytoplasmic
tails of the HN and F glycoproteins (19, 20) as well as the nucleo-
capsid (21–24) to initiate virus assembly and budding (20). The
NDV M protein has greater than 20% sequence identity with
other paramyxovirus M proteins including measles, mumps, and
the parainfluenza viruses. However, the NDV M protein has no
sequence homology to any proteins with known structure.
Using X-ray crystallography in combination with electron tomo-
graphy we determined the pseudoatomic structure of assembled
M protein arrays in NDV and showed how the matrix protein
arrays organizethe glycoproteins and nucleocapsid and suggest the
of the few structural investigations of whole pleomorphic virions
and a unique description of a matrix array in atomic detail.
Results and Discussion
Morphology of Newcastle Disease Virions. Consistent with previous
observations on paramyxoviruses (1), the morphology and size of
NDV, as seen here in tomographic reconstructions (Fig. 1A–C,
Left), varied from approximately spherical to ellipsoidal. The
diameters of the spherical virions ranged from about 100 to
250 nm, whereas the ellipsoidal virions were sometimes as long
as 350 nm and as narrow as 125 nm. On most virions, a layer of
glycoprotein spikes extended about 12 to 18 nm beyond the sur-
face of the 4–5 nm thick membrane. The electron tomographic
results reported here show more detail of the paramyxovirus
matrix proteins and nucleocapsid proteins in the virus than re-
ported previously (17).
Within the virions, the nucleocapsids have both linear and
bend segments. When viewed end-on, they have a ring-like cross-
section with an outer diameter of about 20 nm and an inner
diameter of 4–5 nm (Fig. 1A and C). For straight segments, the
nucleocapsid has an approximately 7 nm repeating structure
along its long axis, representing a helical arrangement of the
nucleocapsid (Fig. 1B and Fig. S1).
Some virions have an additional layer of density on the inner
surface of the viral membrane (Fig. 1A and B, Left). This layer is
about 4 to 5 nm thick and represents the matrix protein. Although
this layer was visible in only about 10% of the virions, SDS-PAGE
Author contributions: A.J.B., G.M., and M.G.R. designed research; A.J.B., G.M., D.C.W., and
L.W.M. performed research; L.W.M. and T.G.M. contributed new reagents/analytic tools;
A.J.B., G.M., D.C.W., L.W.M, P.P., A.C.S., T.G.M., and M.G.R. analyzed data; and A.J.B., G.M.,
and M.G.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Theatomic coordinatesof theNewcastle diseasevirus M crystalstructures
have been depositedwith the Protein Data Bank, www.pdb.org(PDB ID codes4G1G, 4G1L,
and 4G1O); the averaged cryoelectron tomography map of the matrix layer has been
deposited with the Electron Microscopy Data Bank, www.emdatabank.org (EMDB ID code
1A.J.B. and G.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
13996–14000 ∣ PNAS ∣ August 28, 2012 ∣ vol. 109 ∣ no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1210275109
showed that the matrix protein was in approximately the same
abundance as the other structural proteins (Fig. S2). In those
NDV particles where there is at least a partial matrix layer, the
surface glycoproteins and internal nucleocapsids associate with
the matrix array (Fig. 1A and B).
The Averaged Matrix Protein Array and Intercalating Glycoproteins.
Tomographic sections tangential to the viral membrane show that
the matrix layer forms a grid-like array (Fig. 1D, Left). An im-
proved map representing the matrix protein and membrane was
generated by averaging over repeating matrix subunits (SI
Materials and Methods). This showed that the repeating unit of
matrix protein has a nearly square shape, 5 nm on edge, which
forms an array with a 7 nm repeat in orthogonal directions along
the diagonals of the square (Fig. 1D, Right). Although visual
inspection of the tomographic sections tangential to the virion
surface did not show any obvious organization for the HN and F
glycoprotein ectodomains, averaging the glycoprotein layer using
the periodicity of the matrix protein showed the existence of an
extracellular array with high densities in register with the gaps
between repeating units in the matrix array on the inner side of
the membrane (Fig. 2). The volume of the gaps between the
matrix protein units is about equal to the volume of the repeating
matrix protein unit itself, equivalent to about 70 kDa (SI Materials
tomographic section through the center of a nearly spherical virion, which has
an approximately 4–5 nm thick matrix layer lining the inner surface of the viral
membrane (Left). The white asterisks indicate the absence of glycoproteins and
nucleocapsids where the virus lacks a matrix layer. The red arrowhead indicates a
portion of the nucleocapsid as viewed end-on. The virion is also depicted sche-
matically (Right). The HN and F glycoproteins are colored in red and magenta,
respectively. The nucleocapsid is depicted in yellow and the matrix protein and
membrane are colored dark blue and light blue, respectively. (B) A tomographic
section through the center of an elongated virion (Left) and the corresponding
schematic representation (Right). The white asterisks indicate the absence of gly-
coproteins and nucleocapsids where the virus lacks a matrix layer. The green
arrowhead indicates a portion of the nucleocapsid that lies in plane. (C) A tomo-
graphic section through the center of a spherical virion that lacks an organized
matrix protein layer (Left). The red arrowhead indicates the nucleocapsid as
viewed end-on. Presumably, the matrix layer has dissociated from the viral mem-
brane (Right). (D) The array of matrix proteins is evident in tomographic sections
tangential to the viral membrane for some virions (Left). Subtomographic aver-
aging generated an improved structure for a portion of the matrix protein layer
(Right). The averaged map, as viewed from the inside of the virion, is colored
according to the radius of a cylinder such that the density attributed to the mem-
brane is light blue and the matrix protein subunits are dark blue. The on-edge
and diagonal dimensions of a matrix protein subunit are indicated. For all the
tomographic sections, strong density is black and the scale bar represents 75 nm.
The tomographic images (A–C, Left) represent the average of three layers of
voxels over a thickness of approximately 4.5 nm. The tangential section (D, Left)
from the same virion represented in B.
Tomographic and diagrammatic representations of NDV virions. (A) A
protein array, which lines the inside of the viral membrane in the averaged
tomographic map. (B) A section through the averaged map indicating an
array on the extracellular side of the membrane. The extracellular densities
were averaged assuming the same periodicities that were determined for the
matrix protein array. The high densities in B oppose the low-density regions
in A, indicating that the HN and F glycoproteins are anchored in the spaces
between adjacent proteins in the matrix layer. (C) A cross-section of the aver-
aged map, orthogonal to the ordered arrays. The white and black arrows
indicate the planes from which A and B are derived, respectively. (D) A sche-
matic diagram showing the relative locations of the matrix protein dimers
(blue squares) and intercalating extracellular densities (red circles). For A–C,
black represents high density.
Periodic glycoprotein densities. (A) A section through the matrix
Battisti et al. PNAS
August 28, 2012
and Methods). The volume of this gap would be ample to accom-
modate the cytoplasmic tails of the tetrameric HN or trimeric F
glycoproteins. Modeling of the NDV HN (25) or parainfluenza
virus F (6) structures into the glycoprotein array shows that the
glycoprotein ectodomains have the correct size to fit into neigh-
boring sites consistent with the matrix protein periodicity.
Although the head of the HN protein can be accommodated in
arrays along one direction, the spaces in the orthogonal direction
would only be able to accommodate the F protein (Fig. 3).
The Tomographic Nucleocapsid Density. The tomograms of NDV
showed long regions of nucleocapsids that have a repeating pat-
tern (Fig. S1). This pattern was shown to correspond to a helix
with a pitch (the axial translation for one turn of the helix) of
7 nm, consistent with that of other paramyxovirus nucleocapsids
(8, 9, 26). The twist (or number of NP repeats per turn of the
helix) could not be determined because of the limited resolution
of the tomographic maps (SI Materials and Methods and Fig. S1).
The nucleocapsids observed in the tomograms were often in reg-
ister with the matrix protein dimers, such that the 7 nm pitch
along the long axis of the nucleocapsid was placed along the same
axis as the 7 nm repeat along the diagonal of the matrix array
(Fig. S3). However, a helically organized matrix protein envelop-
ing the nucleocapsid structure as described for measles virus (24)
was not observed.
Structure of the Dimeric Matrix Protein Determined by X-Ray Crystal-
lography. The full-length NDV M protein, containing 364 amino
acids, was recombinantly expressed, purified, and crystallized.
Elution peaks from size-exclusion chromatography indicated a
dimeric matrix protein (Fig. S4). The structure was determined
in three different crystal forms to at least 2.2 Å resolution
(Table S1). Each crystal form had two monomers in the asym-
metric unit related by the same dimer axis. The M protein mono-
mer has two similarly folded domains (Fig. 4 A and B) joined by
a 16 amino acid linker and related by a roughly fourfold axis
coincident with the dimer twofold axis (Fig. 4C and SI Materials
and Methods). The N-terminal domain extends from residues 1
to 181 and the C-terminal domain stretches from residues 198 to
364. Each domain consists of a β-sandwich in which the β-strands
in the opposing β-sheets are approximately orthogonal to each
structures of NDV HN (red) (25), prefusion parainfluenza virus F (magenta)
(6), and NDV M (navy blue) (present results) are shown (Top) and represented
schematically (Bottom). The structures are shown such that the viral mem-
brane would be in the plane of the page. (B) Three possible packing arrange-
ments (Top, Middle, and Bottom) of the HN protein (Left) and F protein
(Right) relative to the spaces between the matrix proteins. The size of the HN
globular head would restrict the placement of other HN proteins in some
neighboring spaces. Because the globular head of F is smaller than that of HN,
the placement of the F glycoprotein within the array would not be restricted.
(Lower Right) The HN and F glycoproteins as they might be situated relative
to the matrix layer. Black stars indicate spaces between matrix proteins that
could not be occupied by a glycoprotein because of steric hindrance.
The relationship between matrix and glycoproteins. (A) The crystal
bon drawing of themonomericstructureshown as a stereo diagram.The NDV
matrix protein monomer consists of two similarly folded β-sandwich domains
(magenta) that are flanked by a number of α-helices (cyan). (B) A schematic
diagram representing the secondary structure elements of the matrix protein
monomer. (C) The surface of the matrix protein dimer that faces the viral
membrane is positively charged and has a nearly square envelope. The two
monomers (blue and gold) are shown as a ribbon diagram (Left). The surface
charge is represented on a space-filling model (Right), with the positively
charged regions shown in blue and the negatively charged regions in red.
The matrix protein structure from X-ray crystallography. (A) The rib-
www.pnas.org/cgi/doi/10.1073/pnas.1210275109Battisti et al.
β-sandwich domains (Fig. 4A). The domains can be superimposed
with a rmsd of 3.8 Å between 74 equivalent Cα atoms of 167 re-
The dimeric M protein crystal structure (Fig. 4C) was fitted
into the subunit density in the matrix protein layer of the tomo-
grams (Fig. 5 and Table S2) using the program EMfit (27). The
volume of the tomographic density was consistent with the
volume of the crystallographic dimer structure (SI Materials and
Methods and Table S2). The good quality of the fit verified that
the crystallographic dimer is the physiologically relevant unit.
Furthermore, the side of the matrix protein facing the membrane
is highly positively charged as is required to associate with the
negatively charged membrane surface (Fig. 4C, Right).
Structural Comparison with Other Mononegavirales Matrix Proteins.
A Dali search (28) gave a significant match between the NDV
and the RSV matrix proteins as indicated by a Z score of 18 and
a rmsd of 3.0 Å for 242 out of 254 residues. However, the crystal
structure of RSV is a monomer (14), not a dimer as in NDV. This
may be because the crystallized RSV matrix protein is missing the
25 carboxyl-terminal residues, including what would be helix α13
in NDV that is involved in the monomer-to-monomer contact
within the dimer. The greater conservation of residues in the
dimer contact surface as compared to elsewhere in the protein
(Fig. S5) provides further evidence that the biologically relevant
assembly unit is dimeric for paramyxoviruses. The Dali search
also showed that the matrix protein of Borna virus consists of
a single domain similar in fold to each β-sandwich domain of
the NDV matrix protein. These domains are related by an exact
fourfold axis in Borna virus (13) corresponding to the approxi-
mate fourfold axis in the NDV matrix protein. Ebola virus matrix
protein, VP40, also consists of two β-sandwich domains (12), but
their rotational relationship to each other is different than that
in the NDV matrix protein. Although the sequence divergence
between these different Mononegavirales matrix proteins is great,
their function and tertiary structure are well-conserved (Fig. S6
and Table S3), as has frequently been observed in other proteins
The Matrix Protein Environment. The result of fitting the crystal
structure into the tomographic density shows that there are two
separate contact areas (Fig. 5) related by quasi-fourfold symme-
try between neighboring matrix protein dimers in the virus. The
first contact region consists of helix α9 interacting with the
antiparallel helix α9 of a neighboring dimer. The other contact
region consists of helix α2 interacting with the antiparallel helix
α2 of a different neighboring dimer. The first contact region also
occurs between dimers related by a crystallographic twofold axis
in the monoclinic crystal structure. This contact involves hydro-
gen bonding and electrostatic interactions between Asp 255 and
Arg 263 as well as Arg 262 and Glu 258. The angle between the
noncrystallographic symmetry (NCS) dimer axes of the abutting
dimers in the crystal is 20° (Fig. 5C), giving a similar curvature as
is produced by the 6° angle between neighboring dimers in virions
(Fig. 5B). Previously it was reported that the assembly of the
matrix protein array is essential for virus budding (21). As is
shown here, the contact between dimers controls the approximate
curvature of the assembled matrix array and thus the virion’s
shape while budding.
Implications for the Virus Life Cycle. The contacts between the
dimeric matrix proteins will determine the curvature of the matrix
arrays as shown above and, hence, that of the budding virus mem-
brane. The matrix protein is also required for recruiting the
nucleocapsid and glycoproteins into the virus (21). The relatively
few particles that were observed to have at least a partial matrix
protein layer presumably represent the virus immediately after
budding, because this event requires the matrix protein arrays
for the generation of membrane curvature (33). A similar obser-
vation has been hinted for the distribution of viral proteins in the
organization of Sendai virus (17). Therefore, other particles that
did not show distinct matrix protein arrays probably represent the
virus structure sometime after budding. These particles still con-
tain the matrix protein (Fig. S2) but the arrays have disassembled,
either as a result of the normal virus life cycle or the purification
procedure. The signal for the dissociation of the matrix protein
arrays in vivo might be, for instance, recognition of a receptor
molecule by the glycoproteins. This disassembly is necessary for
infection when the glycoproteins must be freed from the matrix
array, allowing the F glycoprotein to undergo the conformational
change required to transform from a pre- to postfusion state (34).
In addition, the matrix protein has to be released from the mem-
brane for the formation of fusion pores and the release of the
nucleocapsid into the host’s cytoplasm. Thus, the matrix protein
orders the maturation and infection process in time and space.
Materials and Methods
NDV was purified and vitrified for cryotomography (SI Materials and
Methods). Tilt-series images were acquired and used to reconstruct 3D
tomographic maps. Using the periodicity of the matrix protein seen in the
reconstructed virions, an improved structure of the matrix protein array was
generated by averaging subtomographic densities. The pitch of the helical
nucleocapsid was determined by measuring the repeat distance along the
matrix protein crystal structure (cyan and gold ribbon diagram) into the
matrix protein density (gray mesh), which was generated by subtomographic
averaging. The matrix protein array lies in the plane of the page. The green
arrow indicates the contacts between helix α9 of adjacent dimers. The red
arrow indicates the contacts between helix α2 of adjacent dimers. The scale
bar represents 5 nm. (B and D) Cross-sections of the viral membrane and
matrix layer. B shows the contacts between adjacent dimers generated by
helix α9. The dashed line demarcates the membrane and matrix layer inter-
face and illustrates the membrane curvature generated by the matrix protein
array. D shows the contacts between adjacent monomers generated by helix
α2. The scale bars represent 5 nm. (C) Contacts between matrix dimers similar
to those shown in B are made between neighboring dimers in the monoclinic
crystal structure. The NCS dimer axes related by a crystallographic twofold
subtend an angle of 20° between them. Similar interdimer contacts are made
between neighboring dimers in the virus (B), which subtend an angle of
about 6° between them.
The matrix protein environment in the virus. (A) Fit of the dimeric
Battisti et al.PNAS
August 28, 2012
length of the helix in the tomographic data. In addition, the full-length NDV
matrix protein was recombinantly expressed, purified, and crystallized (SI
Materials and Methods). The structure was determined by X-ray crystallogra-
phy using single-wavelength anomalous dispersion data. The dimeric crystal-
lographic structure was then fitted into the tomographic density and
compared to the previously determined structures of homologous matrix
ACKNOWLEDGMENTS. We thank Anastasia Aksyuk and Giovanni Cardone for
helpful comments and suggestions regarding image processing; Ken Hibler
and Zhiheng Yu for assistance with the FEI Titan Krios Microscope; Paul Chip-
man, Siyang Sun, and Ye Xiang for helpful comments and suggestions
throughout the course of this project; Xinzheng Zhang for assistance with
electron microscopy; and Sheryl Kelly for help in the preparation of this
manuscript. Use of the Advanced Photon Source was supported by the US
Department of Energy, Office of Science, Office of Basic Energy Sciences
under Contract DE-AC02-06CH11357. We thank National Institutes of Health
for both an individual research award (AI11219 to M.G.R.) and support via
the intramural research program of National Institute of Arthritis and Mus-
culoskeletal and Skin Diseases (to A.C.S.). Support for A.J.B. was provided by a
Biophysics Training Grant (T32 GM008296-20) from the National Institutes of
Health/General Medicine (C.V. Stauffacher, principal investigator) and a Bils-
land Dissertation Fellowship from Purdue University Graduate School.
1. Lamb RA, Parks GD (2007) Paramyxoviridae: The viruses and their replication. Fields
Virology, eds DM Knipe and PM Howley (Lippincott Williams and Wilkins, Philadel-
phia), 5th Ed,, pp 1449–1496.
2. McGinnes LW, Pantua H, Reitter J, Morrison TG (2006) Newcastle disease virus: Propa-
gation, quantification, and storage. Curr Protoc Microbiol Chapter 15:Unit 15F.2.
3. National Select Agent Registry, http://www.selectagents.gov. Accessed August 2012.
4. Scheid A, Caliguiri LA, Compans RW, Choppin PW (1972) Isolation of paramyxovirus
glycoproteins. Association of both hemagglutinating and neuraminidase activities
with the larger SV5 glycoprotein. Virology 50:640–652.
5. Yin HS, Paterson RG, Wen X, Lamb RA, Jardetzky TS (2005) Structure of the uncleaved
ectodomain of the paramyxovirus (hPIV3) fusion protein. Proc Natl Acad Sci USA
6. Yin HS, Wen X, Paterson RG, Lamb RA, Jardetzky TS (2006) Structure of the parain-
fluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439:38–44.
7. Nagai Y, Klenk H-D, Rott R (1976) Proteolytic cleavage of the viral glycoproteins and its
significance for the virulence of Newcastle disease virus. Virology 72:494–508.
8. Bhella D, Ralph A, Yeo RP (2004) Conformational flexibility in recombinant measles
virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-
space helical reconstruction. J Mol Biol 340:319–331.
9. Egelman EH, Wu SS, Amrein M, Portner A, Murti G (1989) The Sendai virus nucleocap-
sid exists in at least four different helical states. J Virol 63:2233–2243.
10. Harris A, Forouhar F, Qiu S, Sha B, Luo M (2001) The crystal structure of the influenza
matrix protein M1 at neutral pH: M1–M1 protein interfaces can rotate in the oligo-
meric structures of M1. Virology 289:34–44.
11. Rao Z, et al. (1995) Crystal structure of SIV matrix antigen and implications for virus
assembly. Nature 378:743–747.
12. Dessen A, Volchkov V, Dolnik O, Klenk H-D, Weissenhorn W (2000) Crystal structure of
the matrix protein VP40 from Ebola virus. EMBO J 19:4228–4236.
13. Neumann P, et al. (2009) Crystal structure of the Borna disease virus matrix protein
(BDV-M) reveals ssRNA binding properties. Proc Natl Acad Sci USA 106:3710–3715.
14. Money VA, McPhee HK, Mosely JA, Sanderson JM, Yeo RP (2009) Surface features of a
Mononegavirales matrix protein indicate sites of membrane interaction. Proc Natl
Acad Sci USA 106:4441–4446.
15. Russell PH, Almeida JD (1984) A regular subunit pattern seen on non-infectious New-
castle disease virus particles. J Gen Virol 65:1023–1031.
16. Heggeness MH, Smith PR, Choppin PW (1982) In vitro assembly of the nonglycosylated
membrane protein (M) of Sendai virus. Proc Natl Acad Sci USA 79:6232–6236.
17. Bachi T (1980) Intramembrane structural differentiation in Sendai virus maturation.
18. Hewitt JA (1977) Studies on the subunit composition of the M-protein of Sendai virus.
FEBS Lett 81:395–397.
19. Waning DL, Schmitt AP, Leser GP, Lamb RA (2002) Roles for the cytoplasmic tails of the
fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus
simian virus 5. J Virol 76:9284–9297.
20. Pantua HD, McGinnes LW, Peeples ME, Morrison TG (2006) Requirements for the
assembly and release of Newcastle disease virus-like particles. J Virol 80:11062–11073.
21. Harrison MS, Sakaguchi T, Schmitt AP (2010) Paramyxovirus assembly and budding:
Building particles that transmit infections. Int J Biochem Cell Biol 42:1416–1429.
22. Ghildyal R, Mills J, Murray M, Vardaxis N, Meanger J (2002) Respiratory syncytial virus
matrix protein associates with nucleocapsids in infection cells. J Gen Virol 83:753–757.
23. Schmitt PT, Ray G, Schmitt AP (2010) The C-terminal end of parainfluence virus 5 NP
protein is important for virus-like particle production and M–NP protein interaction.
J Virol 84:12810–12823.
24. Liljeroos L, Huiskonen JT, Ora A, Susi P, Butcher SJ (2011) Electron cryotomography of
measles virus reveals how matrix protein coats the ribonucleocapsid within intact
virions. Proc Natl Acad Sci USA 108:18085–18090.
25. Yuan P, et al. (2011) Structure of the Newcastle disease virus hemagglutinin–neurami-
nidase (HN) ectodomain reveals a four-helix bundle stalk. Proc Natl Acad Sci USA
26. Tawar RG, et al. (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNA
complex of respiratory syncytial virus. Science 326:1279–1283.
27. Rossmann MG, Bernal R, Pletnev SV (2001) Combining electron microscopic with x-ray
crystallographic structures. J Struct Biol 136:190–200.
28. Holm L, Rosenström P (2010) Dali server: Conservation mapping in 3D. Nucleic Acids
29. Perutz MF, et al. (1960) Structure of haemoglobin. A three-dimensional Fourier synth-
esis at 5.5-Å resolution, obtained by X-ray analysis. Nature 185:416–422.
30. Rossmann MG,Moras D, Olsen KW (1974) Chemicaland biologicalevolution of nucleo-
tide-binding protein. Nature 250:194–199.
31. Rossmann MG, et al. (1985) Structure of a human common cold virus and functional
relationship to other picornaviruses. Nature 317:145–153.
32. Nandhagopal N, et al. (2002) The structure and evolution of the major capsid protein
of a large, lipid-containing DNA virus. Proc Natl Acad Sci USA 99:14758–14763.
33. Shnyrova AV, et al. (2007) Vesicle formation by self-assembly of membrane-bound
matrix proteins into a fluidlike budding domain. J Cell Biol 179:627–633.
34. Lamb RA, Jardetzky TS (2007) Structural basis of viral invasion: Lessons from paramyx-
ovirus F. Curr Opin Struct Biol 17:427–436.
www.pnas.org/cgi/doi/10.1073/pnas.1210275109 Battisti et al.