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Journal of Virology, November 1998, p. 8541-8549, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Structure of Double-Shelled Rice Dwarf
Virus
Guangying
Lu,1
Z.
Hong
Zhou,2
Matthew L.
Baker,3
Joanita
Jakana,4
Deyou
Cai,1
Xincheng
Wei,1
Shengxiang
Chen,5
Xiaocheng
Gu,1 and
Wah
Chiu3,4,*
National Laboratory of Protein Engineering
and Plant Genetic Engineering, College of Life Sciences, Peking
University, Beijing 100871,1 and
Zhejiang Academy of Agricultural Sciences, Hangzhou
310021,5 China, and
Department of
Pathology and Laboratory Medicine, University of Texas
Houston
Medical School,2 and
Program in
Structural and Computational Biology and Molecular
Biophysics3 and
Verna and Marrs McLean
Department of Biochemistry,4 Baylor College
of Medicine, Houston, Texas 77030
Received 10 February 1998/Accepted 14 July 1998
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ABSTRACT |
Rice dwarf virus (RDV), a member of the Reoviridae
family, is a double-stranded RNA virus. Infection of rice plants with
RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-Å three-dimensional structure of the
700-Å-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two
distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1
icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural
comparisons were made between the RDV outer shell trimer and the two
crystal conformations (REF and HEX) of the VP7 trimer of bluetongue
virus, an animal analog of RDV. The low-resolution structural match of
the RDV outer shell trimer to the HEX conformation of VP7 trimer has
led to the proposal that P8 consists of an upper domain of 
-sandwich
motif and a lower domain of 
helices. The less well fit REF
conformation of VP7 to the RDV trimer may be due to the differences
between VP7 and P8 in the sequence of the hinge region that connects
the two domains. The additional mass density and the absence of a known
signaling peptide on the surface of the RDV outer shell trimer may be
responsible for the different interactions between plants and animal
reoviruses.
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INTRODUCTION |
Rice dwarf virus (RDV) is a member
of the genus Phytoreovirus of the family
Reoviridae, which also includes animal reovirus, orbivirus,
and rotavirus. RDV replicates both in insects and in graminaceous plant
cells, but it can be transmitted only by insects such as the
leafhopper (Nephotettix cincticeps or Resilia
dorsalis) (30, 40). Unlike other phytoreoviruses, RDV
does not induce neoplasia. Instead, plants infected with RDV are
stunted, develop characteristic chlorotic flecks, and fail to bear
seeds. This virus is widespread among rice plants in southern China and
other Asian countries, leading to a possible severe decrease in rice production.
Like all other phytoreoviruses, intact RDV is a double-shelled particle
enclosing a double-stranded RNA genome (18, 20, 21, 33, 41,
42). This double-shelled arrangement of the particle in
phytoreoviruses has been shown to differ from structure for other
members of Reoviridae, such as rotavirus, which are triple
shelled (37, 44). The RDV genome is composed of 12 double-stranded RNA segments, designated S1 to S12 in ascending order
of their mobility on a polyacrylamide gel (12). The complete sequences of all of these segments have been determined from a Japanese
isolate (43) and a Chinese isolate (45), which
share more than 90% sequence identity. Seven of the RDV gene products are considered to be structural proteins (29). The P3
(114-kDa) and P8 (46-kDa) proteins account for ~29 and ~52%,
respectively, of the total RDV protein mass (32).
The RDV particle has been crystallized in the cubic space group I23
with a = 789 Å (28), but its
three-dimensional (3D) structure remains unsolved. We have used
electron cryomicroscopy (cryoEM) and computer reconstruction to derive
the low-resolution 3D structure of the RDV particle. By combining the
primary sequence and crystal structures of the major capsid protein
(VP7) of bluetongue virus (BTV), an animal analog of RDV, we were able
to deduce a relatively high resolution structural motif for P8, the
major outer shell protein of RDV.
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MATERIALS AND METHODS |
Virus purification and cryoEM.
The RDV Zhejiang isolate was
maintained and propagated in rice seedlings that were inoculated by the
viruliferous leafhopper (N. cincticeps Uhler). Diseased
leaves were harvested 2 months later, and the virus was purified by
sucrose density gradient centrifugation (33). The purified
RDV specimen was embedded in a thin layer of vitreous ice on a holey
carbon grid with carbon support film, using a standard quick-freezing
procedure (9). The carbon support film was used to prevent
the virus particles from aggregating toward the edge of holey carbon
film. The frozen hydrated intact RDV particles were kept at 
162°C
and imaged at a magnification of ×30,000 in a JEOL 4000 electron
cryomicroscope operated at 400 kV with a dose of 6 to 8 e/Å2 per micrograph. A focal pair was imaged from each
specimen area first at close to focus and then 1-µm farther
underfocused.
Image processing, 3D reconstruction, and visualization.
The
micrographs were digitized on a Perkin-Elmer 1010M microdensitometer.
Figure 1a is an example of a
close-to-focus micrograph. Individual virus particles were boxed out
into particle images of 140 by 140 pixels with a step size of 6 Å/pixel. Quality of the micrographs was evaluated from the
incoherently averaged Fourier transforms of particle images
(47) prior to subsequent data processing. The first zeros of
the contrast transfer functions of three close-to-focus images used in
the final reconstruction were clearly seen at 1/23, 1/18, and 1/17
Å
1, which correspond to defocus values of 3.4, 2.0, and
1.8 µm, respectively.
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FIG. 1.
CryoEM image and shaded surface representation of the
25-Å structure of full RDV. (a) Typical area of one of the electron
cryomicrographs of RDV particles embedded in a thin layer of vitreous
ice, recorded at 400 kV under low-dose conditions. Indicated by dashed
circles are two particles. (b) Shaded surface view of the RDV
reconstruction as viewed along the icosahedral twofold axis. The
numbers 5, 3, and 2 designate the icosahedral five-, three-, and
twofold axes. Highlighted in color are a contiguous group of five
trimers found in each asymmetric unit. (c) Blown-up view of the group
of five trimers computationally extracted from panel b. These trimers
at the distinct quasi-equivalent positions are designated P, Q, S, R,
and T, using a convention set forth for BTV (14).
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The particles from the strongly underfocused micrographs were used to
assist the determination of the center and orientation parameters of
the corresponding particles in the close-to-focus micrographs. The
determination and refinement of center and orientation parameters were
based on the common-lines search method by comparing particle images
and projections from preliminary reconstructions (5, 46,
48). The initial models were reconstructed from particle images
in the underfocused micrographs, and the corresponding particles in the
close-to-focus micrographs were refined against computed projections.
All of the close-to-focus particle images were then combined to
generate a new model followed by an additional cycle of refinement with
the new projections. In the last round of refinement, a global and
simultaneous refinement of the five particle parameters was performed
by minimizing the cross common-line phase residuals across all particle
images (46). The effective resolution of the final map was
assessed by calculating the phase difference and Fourier ring
correlation coefficients of two independent reconstructions
(49). The reconstruction was carried out only to the first
zero of the contrast transfer function, and thus no correction of the
contrast transfer function was made.
To determine the absolute handedness of RDV, we recorded pairs of 0°
and 20° tilt image from the same specimen areas. The
handedness of
the virus capsid was determined by comparing particle
images in 0°
tilt and 20° tilt, using a procedure described previously
(
3,
38). Twenty particle image pairs were then compared to
determine
handedness.
Structural components of interest in the map were computationally
extracted and visualized by using the Explorer software
(NAG Inc.) with
custom-designed modules (
7a). The mass of a
computationally
extracted subunit is estimated by assuming a protein
density of 1.3 g/cm
3. An isosurface value of 1
(standard deviation)
away from the
mean density was used for rendering the surface
representations.
Our data processing was carried out on Iris R4400,
R8000, and
R10000 workstations (Silicon Graphics, Inc.).
Sequence and structural comparison.
The sequence alignment
of P8 of RDV and VP7 of BTV was carried out by using MSA with a PAM 250 weight matrix available from the National Center for Supercomputing
Applications (17). The crystal structure coordinates of HEX
and REF of VP7 (2, 13) were provided by J. Grimes and D. Stuart, and the secondary structure motif of VP7 was obtained from PDB
SUM (23). Following this, the 3D structures of REF and HEX,
rendered in Ribbons (6), were aligned with the
computationally extracted and scaled RDV trimers. Refinement of the
RDV-BTV density fitting was then done to maximize the total density
match based on visual inspection. This fitting was done with the map at
atomic resolution and also the map blurred to 25-Å resolution and
modified by a contrast transfer function corresponding to that in the
RDV data. These densities were then aligned to the RDV trimer.
Interpretation of mismatched density was done by examining the
-carbon trace of the BTV trimers by using RASMOL (R. Sayle, Glaxo
Wellcome Research and Development, Greenford, United Kingdom). Further
analysis of the structural interpretation was done by threading the
primary sequence of the P8 protein through a profile search
(11). Any proteins or protein segments with a Z
score of ~5.0 or greater were considered likely candidate proteins
with similar folds.
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RESULTS |
Outer shell structure (T=13 icosahedral lattice).
The 3D
structure of ice-embedded RDV was determined to 25-Å resolution from
81 particle images (Fig. 1a) recorded in a 400-kV electron
cryomicroscope. The outer shell of RDV, with a 700-Å diameter,
exhibits a T=13 icosahedral lattice, as shown clearly in the
reconstruction map (Fig. 1b), contrary to the previous T=9 lattice
model (21, 42). The absolute handedness was determined to be
left-handed (T=13l) from tilt experiments of the
ice-embedded RDV particles. The mass densities around the fivefold axis
protrude radially ~15 Å further outward than the mass densities
around the threefold axis, resulting in a polyhedral appearance. The most prominent features on the outer shell are the 260 "knobby" trimeric density clusters centered at the local and strict threefold axes (Fig. 1b). These trimeric clusters associate with each other through extensive contacts at a lower radius around the icosahedral fivefold and local sixfold axes. There are also 132 openings, 12 at the
fivefold axes and 120 at the local sixfold axes, which traverse the
entire 69-Å-thick outer shell, becoming narrower at a lower radius
(Fig. 1b).
Group of five trimers and their interactions.
In a T=13
icosahedral particle, there are 13 quasi-equivalent positions per
asymmetric unit (Fig. 2). Each asymmetric
unit contains four and one-third unique trimers (Fig. 1b and c; Fig. 2), which are designated P, Q, S, R, and T. The lettering scheme, based
on the nomenclature used for BTV (14), is indicative of the
relative position of the trimer: one around the fivefold axis (P
[peripentonal]), three around a local sixfold axis (Q, S, and R), and
one at the icosahedral threefold axis (T). To compare the structures of
these trimers in detail, the five RDV trimer types were computationally
extracted from the capsid reconstruction and displayed in equivalent
top and side views (Fig. 3). The top domain of each trimeric knob, which measures 60 Å in diameter, has a
triangular donut shape with a dimple or, in some cases, a hole at its
center (Fig. 3b). The appearance of the dimples is sensitive to the
choice of density threshold, suggesting the densities at these regions
are not as well defined as those in other regions of the trimers. Such
an observation has also been made in BTV, where dimples begin to appear
at a higher density threshold (29a). Each trimer is about 69 Å in height, shorter than the structures of other outer capsid
proteins from double-shelled virus particles in the
Reoviridae (8, 15, 36, 38). Previous biochemical
and immunological studies have indicated that the major outer shell
protein is P8 (26, 32). By drawing an analogy to other
members of the Reoviridae, (8, 15, 36, 38), we
propose that each RDV trimer is made up of three subunits of the major
outer shell protein P8. Thus, there would be 780 copies of the P8
monomeric subunit in the outer shell. This proposition was
substantiated by structural comparison with the BTV capsid protein VP7
(see below).
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FIG. 2.
Schematic diagram of the trimeric subunit organization
within a triangular unit on a T=13l lattice. The five
trimers are labeled P, Q, S, R, and T.
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FIG. 3.
Comparison of trimers at distinct quasi-equivalent
locations. (a) Side views of the five computationally extracted trimers
P, Q, S, R, and T. The numerical labeling on the S trimer identifies a
monomer of P8. The numbering also indicates the putative domain of the
RDV trimer, where 1 is the upper domain and 2 and 3 are both located in
the lower domain. More specifically, 2 marks the leg region and 3 indicates the floor region of the lower domain. (b) Top views of the
five computationally extracted trimers.
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There is no apparent contact among the adjacent trimers in the top
domains regardless of contour display level. As the trimers
of the
outer shell extend toward the inner shell, each knob branches
into
three "legs" toward the adjacent five- or sixfold axes. These
legs
connect with neighboring legs to form an interconnected floor
density
on the outer shell (Fig.
1c). Furthermore, there seems
to be more
variability in the floor region density distribution
than in the knob
region among the trimers. This floor density
network appears to be
essential for maintaining the outer shell
capsid stability since these
are the only regions of interactions
between the neighboring trimers.
Structural motif of P8.
The most structurally defined protein
in an animal reovirus is VP7 of BTV, which consists of an upper domain
with the typical sandwich of viral proteins and a lower -helical
domain, as seen in both the REF and HEX crystal forms (Fig.
4) (2, 13). The major
difference between these two structural isoforms, REF and HEX, is the
relative orientation between these two domains. We have examined both
the primary sequence and the two crystal structures of VP7 of BTV in an
attempt to deduce a higher-resolution structural model of P8 of RDV
from our low-resolution map.
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FIG. 4.
Structures of the BTV VP7 and RDV P8 trimers. (a) Ribbon
diagram of the REF conformation of BTV VP7; (b) side view of the RDV P8
S trimer merged with the REF VP7 ribbon diagram; (c) top view of panel
b; (d) ribbon diagram of the HEX conformation of VP7; (e) side view of
the RDV P8 S trimer superimposed with HEX VP7 ribbon diagram; (f) top
view of panel d.
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High genome sequence homology exists between RDV and other
phytoreoviruses such as rice gall dwarf virus, wound tumor virus,
and
Fijivirus (
4,
41), but no sequence homology between RDV
and
its animal analogs has been reported. The initial primary
sequence
alignment of RDV P8 and BTV VP7 revealed 27.5% homology,
with a fairly
large number of identities, as well as several obvious
gaps. Upon
overlaying the secondary structure of VP7, derived
from the crystal
structure (
2,
13), onto the primary sequence
alignment, we
found that the secondary structure motifs corresponded
well with the
regions of homology (Fig.
5). The two
structural
domains were evident from the alignment, where the middle
region
of the RDV primary sequence corresponded to the
-sandwich
domain
and the two terminal regions corresponded to the lower
-helical
domain. To further quantify the sequence homology between
VP7
of BTV and P8 of RDV, sequence alignments of the helical and the
-sandwich domains were done separately. These segregated alignments
also yielded homologies that were similar to the overall sequence
alignment (Table
1).
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FIG. 5.
RDV P8 and BTV VP7 amino acid sequence alignment.
Homologous residues are shaded; regions of insertion in P8 relative to
VP7 are noted above the RDV sequence. Secondary structure motifs, based
on the VP7 structure, are shown as arrows for sheets, zigzag lines
for helices, and hairpins for turns.
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The crystal structures of the BTV VP7 trimers from both the REF and HEX
isoforms (Fig.
4a to b) were fitted to the RDV trimers
computationally
extracted and scaled from our low-resolution map
to see whether any
structural homology was present. Figures
4b
and c and 4e and f show
examples of the fittings of the two structural
isoforms of BTV trimers
(ribbon representation) with the S trimer
of RDV (shaded
surface representation). Additionally, fitting
of the other four
trimers showed a match similar to that found
in the S trimer. Similar
fits were also found with the trimers
and the VP7 trimers that had been
blurred to 25 Å.
In the top view (Fig.
4c and f), both the REF and HEX conformations of
the BTV trimer appear to fit the triangular donut-shaped
upper domain
of RDV in similar orientations. This good match suggests
that the upper
domain of the P8 subunit in the RDV trimer would
likely have the same
-sandwich motif as that of the VP7 in the
BTV trimer. However, a
small density in the upper domain is seen
in the RDV trimer but not the
BTV trimer. To investigate the cause
of this extra density, we examined
the sequences of both proteins
and found that the amino acid sequence
of P8 of RDV is 71 residues
longer than that of BTV VP7 (Fig.
5). The
extra residues are mostly,
but not entirely, accommodated by gaps
within the alignment both
at the C-terminal end and within the putative
-sandwich domain.
The insertional residues in the putative
-sandwich domain are
located in the gaps between residues 202 and
238 (Fig.
5). Therefore,
the extra density seen in the upper domain of
RDV (represented
by space-filling balls in Fig.
6) might correspond to a total
of 25 insertional residues in the four gaps.
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FIG. 6.
Potential regions of structural variability. (a) Side
view of the VP7 REF monomer of BTV. The space-filling region (residues
200 to 215) marks the region of major insertion in the RDV-BTV sequence
alignment. The solid-line boxes (residues 251 to 253) and dashed-line
boxes (residues 120 to 122) enclose the putative flexible hinge domain.
(b) Top view of the REF VP7 trimer, illustrating the spatial
distribution of the major insertional region.
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While the upper domains of the REF and HEX trimers seem to fit that of
the RDV trimer, the differences in the overall shape
of the BTV trimer
are evident in the lower domain. The REF global
conformation appears to
be a cylinder 80 Å in length with clockwise
twisting of the monomeric
subunits, whereas the HEX conformation
is about 69 Å in length with
L-shaped monomers (Fig.
4a and d).
By examining the side views (Fig.
4b
and e), we found that the
REF conformation is too long and too narrow
to fit the RDV trimer
density. However, the HEX trimer has dimensions
that are nearly
identical to those of the RDV trimer and appears to
accommodate
the helical leg densities extending from the side of the
RDV trimer
(labeled "2" in Fig.
3). This suggests that the lower
domain of
P8 may have the
-helical motif of the HEX conformation.
Nevertheless,
minor mismatches in the bottom regions, including helices
1 and
2 of VP7, are noticeable. A primary sequence alignment of this
region revealed little sequence homology but similar hydrophobicity
plots (
22). It has been suggested that helices 1 and 2 are
highly
hydrophobic and interact with the inner shell proteins in BTV
(
13). Therefore, these regions in P8 may have similar types
of interactions with the inner shell proteins.
To seek further evidence to support our structural motif assignment
based on the primary sequence alignment and 3D fitting
of the RDV and
BTV trimers, we undertook a series of computational
tests with Fischer
and Eisenberg's Fold Recognition server
(
http://fold.doe-mbi.ucla.edu);
this computational method predicts
structural motifs based on
the amino acid sequence and their
biophysical properties (
11).
The algorithm searches for the
best fit between a known protein
or protein segment in the Protein Data
Bank and a queried protein
segment of unknown structure. The results of
this analysis with
the entire P8 sequence as well as various segments
of P8, corresponding
to the putative top, middle, and lower domains,
are summarized
in Table
2. The highest
score for the whole P8 sequence was 4.96
with bacteriophage
X174
capsid protein GP, which contains a
sandwich (
27). When
the putative
-sheet region of P8 (residues
128 to 315 [Fig.
5])
was used, the
Z score was improved to 5.57,
the profile
being similar to that of tumor necrosis factor alpha,
another
-sandwich protein (
10). Upon removal of all insertions
in
the putative
-sheet regions in the aligned RDV sequence (Fig.
5), a
better match was found with the top domain of BTV VP7 (a
Z
score of 12.57) and also the top domain of African horse sickness
virus
VP7, a
-sandwich-containing protein (
1) (a
Z
score of
4.60). These data strongly support the presence of
-sandwich
structure in P8. When the N- and C-terminal domains were
examined
separately, no sufficiently high scoring proteins or protein
segments
were found. As a control, the sequences corresponding to the
lower
-helical domain motif of BTV VP7 were submitted to the Fold
Recognition
server. No other protein with a similar fold was found to
have
a
Z score above 5.0.
Inner shell structure (T=1 icosahedral lattice).
The inner
shell particle of RDV can be chemically purified (32) and
has been studied by 100-kV cryoEM and computer reconstruction (25). The 3D density map of the purified inner shell
particle shows that it is composed of a thin layer, 25 Å thick, with a closely packed mass density arranged on a smooth T=1 icosahedral sphere
approximately 567 Å in diameter. We have used this diameter to
computationally remove the outer shell density in the RDV map (Fig.
7), which confirms that the surface mass
densities of this putative inner shell are relatively smooth and indeed
arranged as a T=1 icosahedral lattice.
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FIG. 7.
Inner shell structure and interaction with trimers on
the outer shell. (a) Inner shell computationally extracted at 590-Å
diameter. It exhibits a T=1 lattice. The dashed triangle designates one
triangular face of the icosahedron. (b) Schematic diagram of
fish-shaped density distribution within a triangle in a T=1 lattice.
(c) Capsid computationally extracted at 604-Å diameter showing the
interface between the outer shell (yellow) and inner shell (blue)
proteins. Color bar shows the color coding as a function of radius. (d)
Schematic diagram illustrating the interaction pattern of the trimers
(yellow triangles) of the outer shell with the fish-shaped densities of
the inner shell.
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The inner shell consists of many small holes connecting the inside to
the outside of the shell. For example, a hole with a
diameter of about
20 Å is located at the fivefold axis, which
is surrounded by five
adjacent holes 20 Å in diameter. These holes,
which line up with those
seen in the outer shell, may serve as
the pathway of transport of RNA
during the assembly and disassembly
process, as is the case in
rotavirus (
24). The most striking
feature of the inner layer
are the fish-shaped mass densities
about 100 Å in length, 30 Å in
width, and 25 Å in thickness (Fig.
7a). In a T=1 icosahedral lattice,
there are two fish-shaped densities
within each asymmetric unit,
yielding a total of 120 copies per
capsid (Fig.
7b).
The second-most-abundant protein in the RDV capsid is the 114-kDa
protein, P3, which is known to be the major protein of the
inner shell
(
19). We propose that each fish-shaped density consists
of a
single P3 molecule. It is interesting that a consensus sequence
of RNA
polymerase activity was found within the P3 sequence, indicating
that
P3 may function as a structural protein in the core, as well
as
directly participating in RNA replication (
40). It is also
noteworthy that a protein of similar size, VP2, which forms the
60 dimers in the inner core shell (
35), exists in rotavirus.
In
aquareovirus, a protein of similar molecular mass, VP3 (126
kDa), has
also been found to form the T=1 icosahedral inner shell
(
38).
There are two minor structural proteins of RDV (P1 and P5) that are
proposed to reside internally. P1 is an RNA-dependent
RNA polymerase
(
39) and is thus likely to be located within
the internal
core of RDV. P5 previously was thought to be an outer
capsid shell
protein but recently was demonstrated to have GTP
binding activity,
suggesting that it may actually be located internally
(
31).
In our present analysis, we were unable to identify the
exact locations
of these two proteins.
Densities between the outer and inner shells.
To examine the
interface densities between the outer and inner shells, the capsid was
computationally extracted at a radius slightly larger (i.e., 597 Å in
diameter) than that of the putative inner shell and displayed with
different colors between the two shells (Fig. 7c). Though the
resolution of the reconstruction is only ~25 Å, at which the
molecular boundaries cannot be unambiguously determined, the smooth
surface density of the biochemically purified inner shell justifies our
choice for the putative boundary between two shells. The trimer legs in
the outer shell floor density comprise the domain of interaction
between the outer and inner shells. The legs of each trimer of the
outer shell join together at the boundary of adjacent fish-shaped
densities in the inner shell (Fig. 7c). Because the 120 fish-shaped
densities are arranged on a T=1 lattice, the number of available
densities varies according to the location (Fig. 7d). For example, on
the icosahedral threefold axis, there are exactly three fish-shaped
densities for the three legs of the trimer to attach. However, there
are only two adjacent fish-shaped densities available for the legs of
the trimers surrounding fivefold axes. Instead of connecting with a
third fish-shaped density, one leg of the peripentonal trimer of the
outer shell is directly attached to a mass density surrounding each
fivefold axis. The pentonal complex of the inner shell protrudes
farther outward about 10 Å; thus, the leg of the peripentonal trimer
extends farther radially, resulting in the polyhedral shape of the
outer shell. Therefore, it is clear from our reconstruction map that the two RDV shells have mismatched lattice symmetries.
To accommodate the mismatched lattice symmetries of the outer and inner
shells, a variable interface region may be required.
One possibility is
that the minor proteins (for example, P2) are
present as linkers at the
interface between the two shells. The
second possibility is that the
structural variations in the floor
domains of P8 facilitate various
types of intersubunit interactions
not only with itself but also with
the inner shell proteins. Though
the chemical identities of the
densities that connect the two
shells remain uncertain, it is
conceivable that the interactions
are made of complementary types of
hydrogen, ionic, and hydrophobic
bonding.
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DISCUSSION |
Most animal virus members of the Reoviridae such as
rotavirus (37), animal reovirus (8), and BTV
(orbivirus) have triple-shelled capsids (15, 16), while RDV
has been found to have a double-shelled capsid. The double-shelled
particles of these viruses are similar in capsid size and identical in
triangulation number and handedness, suggesting possible structural
conservation. The structural match between the HEX form of VP7 trimer
of BTV and the trimer of the outer shell protein of RDV strongly favors
a model of P8 that consists of two-domain motifs, as in VP7. It is
interesting that the P8 trimer of RDV matches better with the HEX
conformation than with the REF conformation, whereas the REF
conformation has been found to match well with the outer shell
structure of the BTV (14). The conformational preference in
RDV could be due to the presence of a particular sequence of residues
in the regions of P8 (residues 251 to 253 and 120 to 122 [Fig. 6]),
which join the upper and lower domains and act as a hinge, favoring the
HEX conformation.
The information on primary sequence alignment and 3D fitting suggests
that the upper domain of the RDV trimer contains a -sandwich domain,
while the lower domain is arranged as a helical network. Combined with
threading, the proposed -sandwich motif in the upper domain of RDV
P8 is further substantiated. However, since the HEX coordinates with
the side chains are not yet in the Protein Data Bank and the structural
motif of the lower domain of VP7 is considered unique, the sequence
threading analysis would not be a reliable method of predicting the
structure of the lower domains of RDV. In addition to the unique nature
of this fold in the database, the sequences for such helical regions
may not be highly conserved, leading to inconclusive results from the threading analysis. Though the Z scores for these putative
helical regions are not above the threshold, this does not discount the structural motif assignments based on the conventional sequence analysis and direct 3D structure fitting.
The quasi-equivalent trimers of RDV appear to be similar but not
identical with respect to mass density distribution (Fig. 1 and 3). For
instance, the helical leg adjacent to the fivefold axis in the P trimer
has an elongated appearance. The central dimples or holes also vary
among the five trimers (Fig. 3). These structural variations seen in
the quasi-equivalent subunits are not observed in the BTV trimers
(14), which indicates that the quasi-equivalence rule is not
strictly applicable to RDV, as in the case of BTV (14). The
breakdown of quasi-equivalence in RDV may be an important mechanism of
maintaining the stability and functions of the capsid.
While the structural motif of outer shell protein appears to be
conserved in reoviruses, virus-host interactions are not. Evident in
the primary sequence alignment between P8 of RDV and VP7 of BTV is the
lack of any known signaling peptide that mediates virus-host cell
interaction. There is no RGD or DGE tripeptide in P8 of both the
Chinese and Japanese isolates, contrary to VP7 of BTV and VP6 of
rotavirus (1, 7). Therefore, different mechanisms of cell
entry for the animal and plant reoviruses are likely. Recent studies
have shown that RDV particles lacking the P2 protein neither enter nor
infect insect vector cells (34). Therefore, it is likely
that the host selectivity stems from the differences in surface
residues (as shown in Fig. 6), alternative signaling peptides, or
additional signaling proteins. Overall, it may be suggested that
regardless of genus, members of the Reoviridae contain a
structurally conserved outer shell that may contain specific regions or
residues on the surface of the capsid that would mediate viral host
specificity.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to this work.
We thank B. V. V. Prasad and Emma Nason for providing
programs for handedness determination and useful discussion; Matt
Dougherty for assisting with graphics display; and David Stuart and
Jonathan Grimes for providing the REF and HEX -carbon coordinates.
This project was supported by grants from the National High Technology
& Development Program of China, the National Institutes of Health
(RR02250, AI38469, and LM07093), and the National Science Foundation
(BIR9413229 and BIR9412521). M.L.B. was supported by the National
Library of Medicine (grant 2T15LM07093) and the W. M. Keck Center
for Computational Biology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Verna and Marrs
McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-6985. Fax: (713) 796-9438. E-mail: wah{at}bcm.tmc.edu.
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Abstract
Introduction
