From the Department of Pathology and Laboratory
Medicine, University of Texas, Houston Medical School, Houston,
Texas 77030, the § Department of Biochemistry, Baylor
College of Medicine, Houston, Texas 77030, and ¶ State Key
Lab for Biocontrol, Institute of Entomology, Zhongshan University,
Guangzhou 510275, China
Received for publication, June 17, 2002, and in revised form, October 1, 2002
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ABSTRACT |
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Viruses in the family Reoviridae are capable of
transcription within the intact capsids. As the only single-shelled and
thus the simplest member of the Reoviridae, cytoplasmic polyhedrosis virus (CPV) provides an attractive system for studying endogenous transcription. We report the structures of the full and empty CPV
determined at 13-Å resolution by electron cryomicroscopy. The
structure of the empty CPV reveals a density attributed to the
transcription enzyme complex, which is attached to the internal surface
of the capsid shell below each of the 12 turrets. The full capsid has
an identical capsid shell but contains additional internal densities
contributed by the genomic double-stranded (ds) RNA. The RNA densities
proximal to the capsid shell are organized into layers with a
dodecahedral appearance, suggesting a genome organization of dsRNA
segments each having a cone shape spooling around a transcription
enzyme complex. Our structures also suggest that the capsid shell
serves as a scaffold for appropriate positioning of the RNA genome,
whereas nascent mRNA release takes place through the constricted
central channel of the turret. Based on these observations, a detailed
moving template transcription mechanism is proposed that may provide
insight into the well coordinated and highly efficient endogenous RNA
transcription of dsRNA viruses.
CPV1 is one of the most
widespread insect pathogens and belongs to the cypovirus genus of the
Reoviridae family (1). Viruses in the Reoviridae are distinctive as
they all have a segmented dsRNA genome that is transcribed within
intact capsids (2). Nascent mRNA is capped before being released
from the intact capsid. The mechanism by which this characteristic
endogenous transcription takes place inside intact dsRNA viruses has
been studied extensively (3-7), and it is generally believed that the
RNA polymerase complex is structurally anchored to the core during
transcription (7-9). The dsRNA template must be flexible enough to
move freely within the densely packed core so that it can slide through
the RNA-dependent RNA polymerase (RDRP) and the capping
enzyme complex to ensure efficient transcription (10). However, the
detailed mechanisms for achieving this efficient endogenous
transcription, the control of dsRNA movement, and its interaction with
the RDRP during this process remain unclear.
Unlike the double-shelled (e.g. rice dwarf virus) or
triple-shelled viruses (e.g. animal reovirus) that are
typical in the Reoviridae family, infectious CPV has only a single
capsid shell and is thus the structurally simplest member of the
Reoviridae (11, 12). This single protein capsid, made up of five
structural proteins with 12 turret-like projections, encompasses a
dsRNA genome of 10 segments. Despite this striking difference in the outer shell organizations, viruses in the Reoviridae share a common core structure within which the RDRP and dsRNA segments are organized into physically and functionally separate units, suggesting a common
dsRNA genome packaging pattern and mechanism of RNA transcription (13).
The genes encoding RDRP that catalyzes the endogenous transcription are
conserved among different dsRNA viruses (14). Therefore, as the
simplest member of the Reoviridae family, CPV provides an attractive
model system for studying the structural and functional organization of
endogenous transcription.
Both x-ray crystallography and electron cryomicroscopy (cryo-EM) have
been employed to study the three-dimensional structures of dsRNA
viruses. In particular, the crystal structure of the animal reovirus
core (15), which is structurally homologous to CPV, and that of the
bluetongue virus sub-core (7, 16) have provided detailed descriptions
of the structural and functional organizations of shell proteins. Yet
due to the unavailability of the crystal structures of empty capsids,
the interactions of RNA/protein (both TEC and shell protein) cannot be
discerned directly in either case. Moreover, at atomic resolution, the
RNA densities cannot be optimally resolved due to the lack of
icosahedral symmetry at such resolution. Conversely, the high quality
of cryo-EM data particularly in the resolution range of 10-30 Å facilitates resolving dsRNA genome densities and RNA-protein
interactions by imaging both full and empty capsids for
three-dimensional structure comparison. Previous 100-kV cryo-EM studies
of CPV at ~25 Å resolution effectively illustrated the overall
structural features of the capsids but failed to reveal sufficient
structural information on genome organization or dsRNA-protein
interactions (11, 12). Improvements in imaging by using a 400-kV
cryo-EM in the current study have allowed us to substantially improve
the resolution of full and empty CPV structures to 13 Å, thus enabling
the direct visualization of the enclosed TEC and RNA genome structures
in greater detail. We show that the dsRNA genome is organized into
layers consisting of continuous coils of dsRNA bundles spaced ~27 Å apart and surrounding the TEC densities, which is consistent with a
cone-shaped spooling organization of dsRNA duplexes spiraling around
each TEC. These observations form the basis of our proposed mechanism
that may explain the highly efficient and well orchestrated endogenous transcription of dsRNA viruses.
Purification and Cryo-EM of CPV--
The full and empty
particles were purified from the gut tissues of infected fifth-instar
larvae of Dendrolimus spectabilis by differential
centrifugation as described previously (11). The full and empty capsids
were imaged together to permit a direct structural comparison of the
particles recorded under the same condition. Focal pair micrographs of
CPV capsids embedded in vitreous ice were taken in a 400-kV JEOL 4000 electron cryo-microscope at ×50,000 magnification with an electron
dose of about 12 electrons/Å2 for each micrograph (17).
The micrographs were digitized on a Zeiss microdensitometer (Z/I
Imaging, Huntsville, AL) at a step size of 2.8 Å/pixel, and individual
virus particles were extracted as images of 300 × 300 pixels.
Data Processing and Three-dimensional Reconstruction--
The
determination of the center and the orientation parameters of each
boxed out particle and subsequent refinement were carried out using
procedures based on Fourier common lines as described previously
(18-20). The particles from the far-from-focus micrographs were used
as an aid in the determination of the parameters of the particles in
the close-to-focus micrographs (18). Prior to the merging of particle
images for three-dimensional reconstruction using Fourier-Bessel
synthesis, the Fourier transform values of individual images were
corrected for the contrast transfer function (9).
Visualization and Modeling--
The visualization of the
particle structure and modeling of the RNA were carried out using Iris
Explorer (NAG, Downers Grove, IL) with custom-designed modules. All
maps were displayed as shaded surface representation at a contouring
level of 1.5 S.D. above the mean density (1.5 Three-dimensional Structural Comparisons between Full and Empty
Capsids--
Empty and full particles are visually distinguishable in
the cryo-EM micrographs (Fig. 1). We
merged particle images from a set of close-to-focus micrographs with
different defocus values in order to obtain an even data sampling
across a wide range of spatial frequencies. The reconstructions for the
full and empty capsids were computed from 1100 and 774 individual
particle images, respectively. The effective resolution of the empty
capsid reconstruction is determined to be 13 Å based on the criterion
of the Fourier shell correlation coefficient between two independent
reconstructions being larger than 0.5. The map of the full particles
was determined to a higher resolution and subsequently Fourier-filtered
to the same resolution (13 Å) as that of the empty particles for
structure comparison. Both the full and empty capsids show an almost
identical icosahedral shell, each decorated by 12 characteristic
turrets at icosahedral vertices, 120 square-shaped large protrusions, and 120 globular small protrusions (Fig.
2, A and B). The
turret has an open upper cavity above a star-shaped constriction (Fig. 2, A and B, arrows). These structural
features, although grossly similar to those revealed at 25 Å previously (11, 12), are now resolved with much greater detail,
enabling the subsequent identification of protein-protein and
RNA-protein interactions and structural comparison of the empty and
full capsids.
Densities Attributed to the dsRNA Genome and TEC--
The central
slice of the full capsid shows strong internal densities organized into
roughly spherical layers (Fig. 2C), which are not present in
the central slice of the empty capsid (Fig. 2D). As the only
chemical difference between the empty and the full capsid is the
presence/absence of the RNA genome (11), we conclude that these layers
of densities correspond to the RNA genome in the full capsid. Each
mushroom-shaped density attached to the internal surface at the 5-fold
axis of the empty capsid represents the contribution of one TEC (Fig.
2D) (11).
The RNA layer densities closer to the capsid layer are more ordered
than those close to the center of the capsid (Fig. 2C). The
three layers closest to the capsid have continuous and well organized
densities. In addition, the RNA layers are not perfectly spherical but
rather angular, having a shape similar to that of the surrounding
capsid layer, except those below the turret where the RNA densities
appear to be displaced inward. This displacement is most obvious in the
third layer, possibly due to the presence of the bulk of the TEC. Thus,
it seems that both the shell protein and TEC are important in
maintaining the layer structure of the RNA genome, preventing it from
expanding freely, which is energetically more favorable in the absence
of protein constraints.
The difference between the density distributions in the full and empty
capsids can also be illustrated by comparing their radial density
distributions (Fig. 2E). The protein density distributions of the two capsids are almost identical as indicated by the good match
of the spherically averaged radial density distributions beyond radius
230 Å, corresponding to the region of the capsid shell and spikes
(Fig. 2E). However, only the full capsid has density peaks
regularly spaced ~27 Å apart within radius 230 Å, which correspond
to densities of the RNA genome. The 27-Å spacing indicates that the
CPV genome is more densely packed than that of BTV (7) but less densely
packed than those of bacteriophages, which exhibit a spacing of 22-24
Å (22).
Previously, crystallography studies on the cores of animal reovirus and
bluetongue virus have resolved their structures to atomic resolution
(7, 15). However, due to the unavailability of the empty capsid
structures at the same resolution, the genomic RNA densities and their
interactions with viral proteins could not be unambiguously resolved.
In this study, with both full and empty CPV capsids reconstructed to 13 Å, a difference map between the full and empty capsids representing
only the contribution from the RNA density can thus be computed to
provide a clear demarcation of RNA-protein interactions and shed
further insights into the dsRNA organization (Fig.
3). The RNA densities are organized into concentric dodecahedral cages (Fig. 3A). The inner surface
of the protein capsid imposes a high level of icosahedral order on nearby dsRNA by making numerous contacts on the first layer of RNA
density around the 5- and 2-fold axes (Fig. 3B). Therefore, due to their interactions with the enclosing capsid shell, the outer
layers of the RNA densities are arranged with a certain level of
icosahedral symmetry, at least to the current resolution range of the
reconstructions, even though no sequence repeats or homologies are
present among the different segments of the RNA genome.
Around each pentagonal opening of the second shell of the dodecahedral
shaped RNA density are two roughly continuous coils of RNA density
bundles with an inter-bundle spacing of about 27 Å (Fig.
3D). In the first layer, similarly separated bundles of densities are also visible when displayed at a higher density contour
level (not shown). The average diameter of each density bundle is about
20 Å, which is the same as the diameter of dsRNA duplex, suggesting
that each density bundle revealed in these layers represents a dsRNA
duplex. The TEC density is situated at the center of the dsRNA density
coils and interacts with the surrounding RNA densities (Fig.
3E). It is conceivable that the coils in different RNA
density layers surrounding the same TEC represent different portions of
the same dsRNA segment. The linker joining these neighboring coils is
invisible due to icosahedral symmetrization imposed during
three-dimensional reconstruction.
Proteins Involved in mRNA Transcription and
Post-transcriptional Processing--
To closely examine the
protein-RNA and protein-protein interactions contributing to the RNA
transcription and processing steps, we segmented out regions around the
icosahedral 5-fold axis encompassing the TEC, the turret, and its
associated capsid shell densities from the empty and full maps. The TEC
densities revealed in the empty capsid are weaker than the capsid shell
densities. But when displayed at a lower density threshold (0.5
RDRP has the highest level of conservation among proteins of
dsRNA viruses, including CPV and the bacteriophage
Studies of the turret protein of the animal reovirus core suggested
that a central channel is used for mRNA release (15), whereas
research on rotavirus implied one of the five peripheral channels as a
possible candidate for the release pathway (4). Below the CPV turret,
there is a small pore that can either be plugged or opened (26). This
pore is closed in both the full and empty CPV capsids that are not
undergoing active transcription. Our result of the RNA binding cleft on
the TEC near the 5-fold axis (see above) lends support for a release
pathway along the 5-fold axis, using the pore that can switch from an
open to a sealed state under different chemical conditions (26). It is conceivable that this pore is closed when the CPV capsid is at a
quiescent state and would become open by a conformational switch leading to the release of mRNA.
Model of RNA Organization and Implications for Transcription
Mechanism--
CPV has a dsRNA genome of about 25 kb, consisting of 10 dsRNA segments with length ranging from 0.94 to 4.19 kb
(27-32).2 As estimated from
our reconstruction, the entire genome must be packaged within a volume
of ~6 × 107 Å3, corresponding to a
concentration of RNA of ~500 mg/ml within the core. It is possible
that at this concentration the RNA is packed in a liquid crystalline
state and thus can slide over each other with little constraint in the
presence of positively charged small molecules such as spermidine or
cations (34). The above-observed structure of RNA layers and the
interactions between TEC and dsRNA have led us to propose a model of
CPV genome organization (Fig. 5A), which extends from those
proposed for the genomes in other Reoviridae members (7, 34). In this
model, each dsRNA molecule spirals around the TEC down toward the
center of the capsid like a layered cone-shaped spool with an
inter-layer distance of 27 Å. The outmost layers are composed of
multiple coils of RNA duplex with an inter-duplex spacing of 27 Å,
although close to the center the dsRNA strand is in a less organized
form. The strand is confined both laterally and radially by the steric
hindrance due to the presence of adjacent RNA cones. Both ends of the
dsRNA strand are attached to the TEC, as suggested by the observations
that the segmented dsRNA functionally operates as circular templates during transcription (10, 35). The anchoring of both terminals to the
capsid-associated TEC make the genome super-coiled and circular, which
may provide additional stability. Given a dsRNA chain density of 3 Å/bp (36) and the average length of the dsRNA segment being 2.5 kb,
spatial confinement would force the dsRNA duplex to wind about 2-3
times around each TEC at the outmost layer. In this regard, the RNA
densities in the outer layers indeed show two strong bundles of
densities surrounding each TEC (Fig. 3D).
In spite of extensive studying on endogenous transcription mechanisms
of segmented dsRNA viruses, a detailed description of the dynamic
process during transcription is currently unavailable. A generally
accepted conceptual mechanism is the so-called "moving template"
model of transcription that depicts a simultaneous movement of both
product and template RNA during transcription (37). Recently, an
"endless tape" model was proposed to abstract the successive
transcription nature of circular RNA segments of rice dwarf virus and
CPV passing through each TEC repeatedly (38). The partially ordered RNA
genome observed in the full CPV is conducive to endogenous RNA
transcription, because it is well organized around the TEC yet has
extra space between adjacent layers (the 27-Å spacing is larger than
the 22-Å spacing required for close packing of dsRNA) necessary for
the loosening, expansion, and orchestrated movement of the genomic RNA
segments during transcription. Obviously, an appropriate spatial
arrangement of the RNA genome, TEC and turret is essential for mRNA
synthesis and processing, with the protein shell serving as a scaffold
to ensure a proper spatial arrangement of the various components.
Previous biochemical and structural studies suggest that nascent
mRNA is transcribed from the dsRNA strands by TEC and then capped
by the turret protein on its exit pathway through the turret into host
cell cytoplasm (11, 39-42). By incorporating the new structural
information presented in this study, we propose a detailed stepwise
transcription mechanism that allows the maintenance of the local
ordering of the genome during active transcription (Fig. 5,
B-D). At a quiescent state, the RNA genome is likely to be
organized with its 3' end of the minus strand attached to the TEC,
forming an initiation complex (Fig. 5B). Upon initiation of
transcription, the helicase domain of the capsid shell protein unwinds
the RNA duplex so that the minus strand (the parental strand) can be
inserted into the small RNA-binding cleft on the TEC (Fig.
4F). The minus strand rejoins with the plus strand to form a
duplex at the exit point near the RNA-binding domain of the RDRP (Fig.
5C). During the elongation process (Fig. 5D), TEC
provides the structural framework for guiding the movement of the
re-joined dsRNA duplex toward the center of the capsid, coupled with
the capping (39) and subsequent release of the newly synthesized
mRNA strand from the turret. The ATP consumption during mRNA
synthesis in CPV is greater than that calculated from ATP integrated
into mRNA (33), suggesting that ATP may provide the energy needed
for the movement of both template and nascent transcripts during this
process. Due to space limitations near the center of the capsid, the
stretched dsRNA strands are forced to coil upward. The clashing force
with the neighboring dsRNA molecules and with the other end of itself
would lead to the upward coiling of the dsRNA eventually into a spiral shape (Fig. 5D). Upon completion of this round of
transcription, the RNA molecule resumes the coiled spiral shape and
enters into a new cycle, leading to a continuous, well orchestrated,
and highly efficient RNA transcription process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) unless otherwise
indicated. The densities of the full and empty capsid reconstructions
were first scaled by matching the peaks corresponding to the capsid
shells in the spherically averaged density plots (see Fig.
2E) and subtracted from one another to generate a
difference map representing the viral RNA densities. The
atomic structure of the
6 RDRP monomer A (21) was visualized as
ribbons using WebLab ViewerPro (Accelrys Inc., San Diego). The
electron density map of the
6 RDRP was generated by
Gaussian-filtering its atomic structure to 10 Å using PDBto2MrcDensity (20) and oriented such that its RNA-binding cleft faces the interior surface of the capsid and roughly overlaps with the icosahedral 5-fold axis. This density was then symmetrized by
imposing 5-fold symmetry.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Typical area of the close-to-focus
(A, underfocus 0.8 µm) and
far-from-focus micrograph (B, underfocus 3.3 µm) of a focal pair of 400-kV electron micrographs
of ice-embedded CPV capsids. The filled and open
arrows indicate a full and an empty CPV capsid,
respectively.
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Fig. 2.
Structural comparisons of the full and
empty capsids at 13-Å resolution. A and B,
shaded surface views of the reconstructions of the full (A)
and empty (B) capsid along 2-fold axes. The
arrows point to a star-shaped constriction in the turret.
C and D, central sections (~55-Å thick) from
the maps of the full (C) and empty (D) capsid.
Unless otherwise indicated, the maps in this and subsequent figures are
color-coded according to particle radius such that the 12 turrets are
in pink, the protrusions on the capsid layer in
aquamarine, and the capsid layer in green. The
internal densities (within a radius of 245 Å) in the full capsid are
shown in red, and those in the empty capsid, attributed to
the TEC, are shown in purple. C, the TEC
densities extracted from the empty capsid are superimposed on the full
capsid to reveal their relative radial locations. E, radial
density distributions of the empty and full CPV reconstructions. The
mass densities in the three-dimensional maps of the full (solid
line) and empty (dotted line) capsids are spherically
averaged and plotted as a function of particle radius. The radial
positions of structural components in full and empty capsids are
indicated.
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Fig. 3.
Comparison of the densities inside the empty
and full capsids. The upper (A-C) and
lower (D-F) panels are shaded
surface representations of the spherical cutaways of the capsids at
radii 218-249 and 176-218 Å, respectively, which roughly correspond
to the first and second layers of RNA densities. A, first
layer of RNA genome obtained from the difference map of the full and
empty capsids. B, superposition of A on the
radial cutaway of the empty capsid, revealing interactions of the RNA
genome and the capsid shell protein (yellow). C,
radial cutaway view of the empty capsid between radii 218 and 249 Å.
D, radial cutaway view of the difference map between radii
176 and 218 Å showing the second RNA layer. The arrows
indicate the 27-Å separation between adjacent density bundles.
E, superposition of D on the radial cutaway of
the empty capsid, revealing interactions of the RNA genome and TEC.
F, radial cutaway of the empty capsid between radii 176-218
Å shown at a relatively higher contour level.
),
they appear tethered to the inner surface of the capsid shell (Fig.
4A). The weaker TEC density is
expected as only one transcriptase is present below each 5-fold vertex,
which was smeared out by 5-fold averaging imposed during
three-dimensional reconstruction. From the side view of the densities
surrounding the TEC, it is clear that the bulk of TEC is located below
the turret between the first and third RNA layer (Fig. 4B).
The genomic dsRNA molecules interact extensively with both the TEC and
the capsid shell as can be identified from the side view of the full
map (Fig. 4B).
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Fig. 4.
Structures of proteins involved in
transcription. A and B, side views of the
region encompassing the turret and its underlying TEC associated with
intertwining RNA densities from empty (A) and full
(B) particles. C and D,
three-dimensional structure of the 6 RDRP determined by x-ray
crystallography (21) shown either at atomic resolution using ribbons
(C) or Gaussian-filtered to 10-Å resolution and rendered as
shaded surface representation, with the RNA-binding cleft indicated by
an arrow (D). E, the 10-Å density map
of
6 RDRP after aligning its RNA-binding cleft aligned along a
5-fold axis and imposing 5-fold symmetry. The 5-fold axis is
perpendicular to the page. The arrow points to a central
hole apparent after symmetrization. F, densities attributed
to CPV TEC computationally extracted from the three-dimensional map
with the 5-fold axis perpendicular to the page. The arrow
points to a prominent central hole along the 5-fold axis. G,
superposition of the CPV TEC and
6 RDRP as shown in D.
The central hole of CPV TEC is aligned with the RNA-binding cleft,
which is indicated by the arrow. H, superposition
of E and F. Note their central holes match
precisely (arrow). I, slightly tilted side view
of the superposition of
6 RDRP as shown in E on the
turret-TEC region of the empty CPV. J, the same as
I but the unsymmetrized
6 RDRP density as in D
is shown. The maps were all displayed using a density contour level of
1.5
except that in A, which is displayed at 0.5
.
6 (14, 21, 23,
24). A recently determined atomic structure of the bacteriophage
6
RDRP has revealed it as a globular right-hand-shaped protein composed
of finger, thumb, and palm domains (Fig. 4C), with an
overall fold similar to other RNA transcriptase from viruses such as
the hepatitis C virus (21, 25). Therefore, we expect the CPV RDRP, the
major protein constituent of TEC, to be globular also. Although the TEC
densities are smeared out and thus difficult to interpret, some of its
structural features may provide interesting clues about the functional
organization of TEC inside the capsid. First, arm-like tethers are seen
connecting the body of TEC to the capsid shell (Fig. 4, A
and F), suggesting that either a large conformational change
of the transcriptase or binding of additional proteins is necessary to
form the arms from the globular transcriptase structure. Second, there
is a hole with a diameter of about 8 Å in the TEC densities along the
5-fold axis on its side facing the capsid shell (Fig. 4F).
Interestingly, when we oriented the
6 RDRP (Fig. 4, C and
D) with its RNA-binding cleft facing the capsid shell along
the 5-fold axis and imposed 5-fold symmetrization on it, a hole of
similar size can also be seen on the symmetrized density along the
5-fold axis facing the capsid shell (Fig. 4, E,
H, and I). Therefore, we propose that the
location of the hole on the TEC density corresponds to the RNA-binding
cleft of the CPV TEC. With such an orientation, the C-terminal thumb
domain of the RDRP is the closest to the capsid shell, making it a
probable candidate to interact with the protruding densities of the
capsid shell protein (Fig. 4J).
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Fig. 5.
Models of RNA genome organization
(A) and transcription mechanism
(B-D). A, each RNA molecule is
modeled to spool around a TEC below the icosahedral vertex toward the
center of the capsid in a cone shape. Because icosahedral symmetry
(frame) was imposed during data processing, the densities attributed to
different RNA molecules below the 12 vertices of the icosahedron are
indistinguishable and are modeled identically. Only one coil within
each layer is depicted for simplicity. B-D, schematic
illustration of our proposed RNA transcription mechanism. The plus and
minus strands of the genomic RNA are shown in blue and
pink, respectively. B, at the quiescent state,
the RNA genome is anchored to the TEC, and the 3' end of the minus
strand is positioned in the RNA-binding cleft of the TEC, forming an
initiation complex. C, at the initial stage of the
transcription process, the 3' end of the minus strand is driven passing
through the channel of the TEC and re-associates with the plus strand,
whereas the newly synthesized mRNA is simultaneously capped by
methyltransferase at the inner surface of the turret complex. The
small arrows along the genomic RNA depict directions of the
continuous movement of the RNA template chains. D, during
elongation process, the RDRP keeps on adding NTP to the 3' termini of
the mRNA; the capped mRNA is being released through the central
channel of the turret simultaneously during elongation. The rejoined
parental strands are propelled toward the center of the capsid, where
steric hindrance posed by neighboring RNA segments forces it to coil
upward and ultimately leads to a spiral shape. Upon completion of one
round of transcription, new cycles of transcription
repeat.
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ACKNOWLEDGEMENTS |
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We acknowledge the use of the electron cryomicroscopy facility at the National Center for Macromolecular Imaging directed by W. Chiu (supported by National Institutes of Health Grant P41RR02250). We thank X. Yu for technical assistance in specimen preparation, H. Zhang for preliminary data processing, K. Kuo for encouragement, and P. Lo for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI46420 and CA94809, Welch Foundation Grant AU-1492, the American Heart Association Grant 0240216N, and the National Natural Science Foundation of China Grants 10274106 and 30070169 (to J. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Pew Scholar in Biomedical Sciences and a Basil O'Connor
Starter Scholar of the March of Dimes Birth Defects Foundation
supported by Grant 5-FY99-852. To whom correspondence should be
addressed. Tel.: 713-500-5358; Fax: 713-500-0730; E-mail:
Z.H.Zhou@uth.tmc.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M205964200
2 S. Rao, K. Hagiwara, S. W. Scott, and G. R. Carner, GenBankTM accession number AF323784.
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ABBREVIATIONS |
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The abbreviations used are: CPV, cytoplasmic polyhedrosis virus; TEC, transcription enzyme complex; dsRNA, double-stranded RNA; RDRP, RNA-dependent RNA polymerase.
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REFERENCES |
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