* Laboratory of Structural Biology, The genomes of double-stranded (ds)RNA
viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment
IN eukaryotic and prokaryotic cells alike, the medium in
which genetic information is stored is double-stranded
(ds)1 DNA. From this repository, genes are copied in
the same medium (replication) or into different media
(transcription and translation). Viruses, more limited but
less constrained genetic entities than cells, exhibit greater
diversity in their replication cycles. All four alternative media, single-stranded (ss)RNA, ssDNA, dsDNA, and dsRNA,
are used by different virus families to encode their genomes (for review see Roizman and Palese, 1996 One such strategy that is widespread if not universal
among dsRNA viruses is their use of capsids as specialized
cytoplasmic compartments that genomes are confined to,
and are replicated and expressed from (Dryden et al.,
1993 The present study addresses the capsid structure and the
organization of encapsidated RNA in the L-A virus of
yeast, an excellent model system because of its relative
simplicity and well-known molecular biology. The L-A genome is a 4.6-kbp, single-segment molecule that encodes a
major capsid protein (Gag; 680 residues, 76 kD) and a minor capsid protein. The latter molecule is a fusion of Gag
with Pol, the RNA-dependent RNA polymerase (Pol; 868 residues, 100 kD) (Fujimura and Wickner, 1988a Motivated by the expanding generality of this unusual
capsid stoichiometry and architecture and the prospect of
gaining insight into the features that commend it as a compartment for the sequestration and replication of dsRNA,
we have pursued further cryoelectron microscopic studies
of the L-A capsid, extending the resolution to 16 Å. Using
these data, we have also examined the structural organization of the viral genome, both in the encapsidated state and as released from virions. Complementary information
was obtained by using dark-field scanning transmission
electron microscopy (STEM) (Wall and Hainfeld, 1986 Virus Particle Preparation
L-A virus was extracted from stationary phase cells of Saccharomyces cerevisiae strain TF229 [MATa His(3,4) Leu2 ski2-2 L-A-HN] grown in 1 liter of rich medium, and purified by CsCl equilibrium gradient centrifugation as described (Fujimura and Wickner, 1988b Cryoelectron Microscopy
Virions to be examined by cryoelectron microscopy were dialyzed against
buffer A for 8-12 h to remove CsCl. They were then diluted in buffer A
until (when examined by negative staining) a uniform, dense but not continuous, distribution of particles was observed. 5-µl drops of virus at this
concentration (typically, 2-5 mg/ml) were applied to holey carbon films on
400 mesh copper grids, and observed by cryoelectron microscopy, essentially as described (Booy et al., 1991 Table I.
Analysis of L-A Core Images
Image Analysis
Micrographs were digitized on a Perkin Elmer 1010MG microdensitometer (Norwalk, CT) at 12-µm intervals (×36,000 negatives) or 16-µm intervals (×46,000 negatives), corresponding to 3.13 or 3.30 Å per pixel, respectively. General image processing operations were carried out using
the PIC Software system (Trus et al., 1996 To obtain a distribution of particle orientations that uniformly cover
the asymmetric unit (Baker et al., 1989 To visualize the internal contents of full particles, we first calculated reconstructions from micrographs recorded at several different defocus values. The contribution of the capsid shell to each such image was obtained
by reprojecting the three-dimensional map in the corresponding orientation, after setting the internal densities to background (solvent) level. Finally, the projected capsid shell was subtracted from the raw image
(Baker et al., 1990 Scanning Transmission Electron Microscopy (STEM)
STEM was performed at the Brookhaven Biotechnology Resource (Upton, NY) (Wall, 1979 RNase III Assay
RNase protection assays were performed with three different samples:
dsRNA purified from full viral particles; freshly prepared full particles;
and viral particles disrupted by dilution to low ionic strength (25 mM
NaCl) and incubated for 45 min at room temperature. RNase III has an
exclusive specificity for dsRNA (Crouch, 1974 Cryoelectron Microscopy of Full and Empty Capsids
Fields of purified L-A particles are shown in Fig. 1, in micrographs recorded at several different defocus values. As
expected, the image contrast increases with defocus; however, L-A exhibits no conspicuous surface relief, even at
quite high defocus (Fig. 1 d). Empty particles are clearly
distinguished from full ones, and almost all capsids appear
intact (see below). We observed a small percentage of a
distinctive polymorphic variant, the "dimeric" capsid (e.g.,
Fig. 1 d, large arrowhead), it seems to be a fusion of two
capsids, both lacking one icosahedral cap. Whether they arise from pairing of two partially dissociated capsids or
are simply aberrant assembly products is not known. Filamentous material, presumably RNA released from virions, is usually visible (e.g., Fig. 1, small arrowheads). Some
full particles exhibit "swirl" patterns of curved striations
(e.g., Fig. 1 c, large arrowhead), we attribute this to the encapsidated RNA as they were never seen on empty
capsids. The spacing between these striations is 35-40 Å.
More often, full particles present punctate, nonsymmetric, motifs.
In this typical experiment, full and empty particles were
observed in a ratio of ~3:2. Since the sample was collected
as a compact band about one-third of the way up the CsCl
gradient, it originally contained only full particles. We infer that the empty particles were generated either during
dialysis (see Materials and Methods) or grid preparation.
Three-dimensional Structures of Full and Empty
Capsids at 16 Å Resolution
The presence of both kinds of particles in the same micrographs allowed us to calculate reconstructions that may be
compared directly, without concern about extraneous factors such as differences in defocus, ice thickness, radiation
damage, etc. The resolution achieved was 16 Å in both
cases. The outer diameter and average thickness of the
capsid shell were measured from spherically averaged radial density profiles (not shown) of maps whose magnification was carefully calibrated, as 430 and 46 Å, respectively.
However, the reconstructions reveal local variations in
shell thickness, which is maximally, ~54 Å (compare with
Fig. 2 b).
The molecular topography of the capsid is shown in stereo in Fig. 2 a, as viewed along a fivefold axis. The structure shown was calculated from images of full capsids and
its internal contents were erased computationally to expose the inner surface (Fig. 2 a). The placement of subunits on this surface lattice is indexed in Fig. 3 b. The penton is constructed of an inner ring of five elongated
subunits (A-subunits), surrounded by an outer ring of five
similar, partially intercalated, B-subunits. The nonequivalent bonding environments of the A- and B-subunits are
reflected in their subtly different morphologies (see below).
The empty capsid has an essentially identical structure,
particularly on the outer surface (data not shown). On the
inner surface, the match between full and empty capsids is
less perfect, but such differences are slight. Of these, the
most prominent are thin arms of density near the twofold
axes on the full capsid (Fig. 2 b, right, white arrows) that
connect the protein to the underlying genome-associated
density. These features imply that there is likely to be
close contact between the RNA and the capsid wall at
these sites, but (by comparison) not elsewhere.
Unlike the impression given at 26-Å resolution (Cheng
et al., 1994 In these maps, the capsids' interiors are icosahedrally
symmetric; although there is no reason to suppose that the
material in these regions actually conforms to this symmetry, which was imposed by the reconstruction procedure.
In the empty capsid, internal density is (as expected) at the
same level as the solvent outside. In the full capsid map,
the internal density gives the impression of three concentric shells 35-40 Å apart (Fig. 2 b, right). We do not interpret this to mean that the viral RNA is strictly confined to
three spherical shells. Rather, because the micrographs
contain a strong signal at these spacings, arising from the
distances between nearest-neighbor RNA filaments, the icosahedral density map calculated from them also contains strong features at the same spacings. The density of
these shells diminishes progressively towards the center,
which is probably a valid indication that proportionately
more RNA resides at higher radii.
Structures and Interactions of the Nonequivalent
A- and B-Subunits
The A- and B-subunits are very similar in morphology. On
the outer capsid surface, each subunit is shaped like a boomerang and subdivides into three domains, called A1, A2,
A3, and B1, B2, B3, respectively (Fig. 3 d). On the inner
surface, the two kinds of subunit are less alike, although
both present a Y-shaped motif (Fig. 3 e, right). This motif
is clearly visible in the spherical sections at inner radii
shown in Fig. 3 a (especially bottom row, third from left).
In the middle of the capsid wall is a hollow cavity (Fig. 2 b)
that separates the inner and outer surface domains. Within
a given penton (Gag decamer), neighboring A- and B-subunits can be aligned by a rotation of ~15° (see Fig. 3 e,
left), whence we conclude that they are oriented in parallel. The view from the inside (Fig. 3 e, right) supports this
conclusion.
The internal features of the Gag subunits are conveniently viewed in spherical sections through the density
map (Fig. 3 a). The protomer should be a Gag dimer. In
principle, there are three possible pairings of neighboring
A- and B-subunits. The pairing in which subunits interact
across the twofold axis (A3 to B3) involves only a small
contact surface. Consequently, we favor the two more
compact arrangements (Fig. 3 d, bottom), in which there
are more extensive interactions to stabilize the dimer.
From the spherical sections (Fig. 3 a), we can discern
likely boundaries between subunits, and the pattern of interactions that consolidates the surface lattice. A-subunits
cluster around the fivefold axis via A1-A1 interactions.
The corresponding region of the B-subunit (B1) makes
contacts with A1 and A2, further out from the fivefold
axis. Three B3 domains meet around the threefold axis,
whereas across the twofold axis, the contacts are head- to-head between A3 and B3. Interactions between twofold
and threefold axes take place deep in the lattice, from radii
at ~184 to 197 Å (whereas the protein shell extends from
radii 171 to 217 Å) via arclike densities (Fig. 3 a, top, far
right).
The thin arms of density linking the capsid with the underlying RNA (see above) are also visualized in the Fig. 3
a (bottom, far right) for the twofold axes, and they extend
from A-subunits.
Packing of dsRNA Inside the L-A Capsid
The protein shell, accounting for ~73% of the virion mass,
tends to dominate images of full particles, in which it is coprojected with the RNA. To obtain unimpeded views of
encapsidated genomes, we erased the shell contributions
from virion images, computationally (Booy et al., 1991 Resistance of Encapsidated RNA to RNase Digestion
The interstrand spacings measured above are considerably
larger than the values obtained for other dsDNA and
dsRNA viruses (Lepault et al., 1987
STEM Analysis of L-A dsRNA
We used dark-field electron microscopy to measure the
mass-per-unit length of L-A chromosomes, both after purification (e.g., Fig. 7 a) and as released from, but still
connected to, disrupted capsids (e.g., Fig. 7 b). The data
obtained for purified RNA molecules observe a quasi-Gaussian distribution with a mean of 2.8 ± 0.6 kD/nm
(standard deviation). For RNA filaments emanating from
capsids, the distribution (Fig. 7 b) is broader, displaced to
higher values, and is suggestive of multiple components.
The lower values (~30-40% of the data) match those obtained for purified molecules. The other measurements
are higher and appear to represent structures in which two
or more duplexes associate laterally to form multistranded filaments. We think it unlikely that their higher density reflects coating of the RNA with protein since these preparations contain no protein other than Gag in detectable
amounts. When a similar experiment was carried out with
particles that had been stored for a few days at 4°C (L-A
virions gradually lose their dsRNA upon storage, see
above), most of the measurements matched those obtained
for purified molecules.
The replication cycles of all known dsRNA viruses follow
the same pattern (Roizman and Palese, 1996 120-subunit Capsids
As noted in the Introduction, stoichiometry data indicate
that many dsRNA virus capsids contain 120 copies of at
least one protein. So far, no other kind of virus capsid has
this stoichiometry, which corresponds to the "forbidden"
T-number of two (Caspar and Klug, 1962 Capsid Structure and Control of Assembly
Our current three-dimensional map at 16-Å resolution depicts the 76-kD Gag monomer as an elongated molecule
with multiple domains coiled around a central cavity. The
structural differences seen between the nonequivalent
A- and B-subunits are minor, but their bonding environments are entirely different (Fig. 3 c). As shown by expression studies (Fujimura et al., 1992 Mass-per-unit-length Measurements Suggest that L-A
dsRNA Is A-form
Our STEM data on the mass-per-unit length of purified
L-A dsRNA yield a mean value of 2.8 kD/nm ( ± 0.6 SD; ± 0.08 SEM). Acknowledging the spread in these data,
they suggest an A-type duplex The L-A Capsid as a Molecular Sieve
One striking feature revealed by our capsid model is its
porosity; the capsid wall is perforated by many small holes.
While their sizes may be revised somewhat when higher
resolution data become available, current estimates suggest a strategically useful property. None of these holes is
large enough to pass an A-form duplex (~23-Å diam), but
the largest holes (triangular, average distance from apex to
base ~15 Å; Fig. 3 e) should readily allow passage of an ssRNA molecule. Thus the capsid appears to be configured to function as a molecular sieve: duplex RNA is retained
inside, but transcripts may exit. It would be expedient for
freshly synthesized transcript to be fed directly into an exit
port (Fig. 8), as this would forestall formation of secondary structure that might impede export. Since there are
only one to two copies of Pol per capsid, presumably no
more than two transcripts per capsid are being exported at
any one time. However, the other holes should facilitate
the infusion of nucleotides, of which a steady supply is required to sustain synthesis during transcription or replication. A further filtering property is that the holes are presumably small enough to exclude potentially degradative
enzymes.
Unusually Low Density of dsRNA Packing in L-A
In projection, encapsidated L-A genomes exhibit punctate
and/or swirl motifs (Fig. 4.). Presumably, these two classes
of motifs represent distinct views, although it is not clear
that the genome is packed in the same way in each particle. We infer that the genomes are loosely coiled, with
variable spacing between adjacent filaments. The diffraction peaks (Fig. 5) have average Bragg spacings of 36-40
Å, corresponding to a center-to-center spacing of 41-46 Å between filaments. The L-A motifs differ from those of dsDNA viruses such as phages T4 and RNA extending from capsids often has a uniform mass-per-unit length that is significantly higher than for a single
A-form duplex (Fig. 7), and appears to represent some
sort of higher order filament formed by two or more duplexes. We have not observed these thicker filaments in
preparations of isolated dsRNA, and see no obvious way
in which they might arise as an artifact of specimen preparation. It may be that within the confines of the capsid, i.e.,
under conditions of high packing density, some piecewise condensation of duplexes into higher order filaments takes
place. On isolation, i.e., at much lower density, the RNA
gradually relaxes to single duplexes.
Headful Replication and the Churning Model
of Transcription
L-A is also a helper virus for other dsRNA segments in
Saccharomyces cerevisiae (Wickner, 1996a In the second explanation, we speculate that the maximum viable packing density may be dictated by a requirement for long range mobility of the dsRNA in this transcriptionally active particle. The polymerase is thought to
reside on the inner surface, anchored by integration of its
Gag moiety into the surface lattice (Wickner, 1996a Is a similar mechanism operative in other dsRNA viruses? The consideration that dsRNA appears to be
packed more densely in the cores of both reovirus (Dryden et al., 1993 Received for publication 5 March 1997 and in revised form 30 June 1997.
We thank M. Simon (Brookhaven, Upton, NY) for help with STEM, T.S.
Baker (Purdue University, West Lafayette, IN) for reconstruction software, Dr. R.J. Crouch (National Institute of Child Health and Human Development) for a gift of RNase III, and D. Belnap, M.E. Cerritelli, J.F.
Conway, A. Zlotnick (National Institute of Arthritis, Musculoskeletal,
and Skin Diseases), and D.B. Furlong (Harvard, Cambridge, MA) for
stimulating discussions and other helpful input.
CTF, contrast transfer function;
ds, double-stranded;
ss, single-stranded;
STEM, scanning transmission electron microscopy;
TMV, tobacco mosaic virus.
Computational
Bioscience and Engineering Laboratory,
Department of Biology, Brookhaven National Laboratory, Upton, New York 11973
Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References
Abstract
the viral capsid. We have used
cryoelectron microscopy and three-dimensional image
reconstruction to study this compartment in the case of
L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution,
we distinguish multiple domains in the elongated Gag
subunits, whose nonequivalent packing is reflected in
subtly different morphologies of the two protomers.
Small holes, 10-15 Å across, perforate the capsid wall,
which functions as a molecular sieve, allowing the exit
of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes.
Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is
an A-form duplex, and that RNA filaments emanating
from disrupted virions often consist of two or more
closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered
inside full capsids, but it is packed relatively loosely; in
L-A, the center-to-center spacing between duplexes is
40-45 Å, compared with 25-30 Å in other double-stranded viruses. The looser packing of L-A RNA
allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the
polymerase immobilized on the inner capsid wall.
). Whereas
dsDNA and ssRNA molecules are abundant cellular components, dsRNA and ssDNA molecules are not. In these
circumstances, one might expect viruses of the latter kinds
to have developed special strategies for interfacing with
the metabolic resources of their host cells.
; Cheng et al., 1994
). Such capsids are selectively porous protein shells that the genome and replicase reside
within, nucleotides infuse into, and nascent strands (positive sense) are extruded from.
; Icho and
Wickner, 1989
) that is generated by ribosomal frameshifting (Dinman et al., 1991
; Tu et al., 1992
). The L-A capsid is
a spherical particle, ~430 Å in diam, composed of 120 copies of Gag (Esteban and Wickner, 1986
; Cheng et al.,
1994
), of which approximately two carry the Pol moiety
(Dinman and Wickner, 1992
; Ribas, J.C., and R.B. Wickner, unpublished results). Its surface lattice has the unusual property of being composed of 60 asymmetric
dimers (Cheng et al., 1994
), configured as an otherwise
conventional icosahedron of triangulation class T = 1 (Caspar and Klug, 1962
). Interestingly, 120-subunit capsids
have been detected in several other dsRNA viruses, including bacteriophage
6 (Mindich and Bamford, 1988
),
reovirus (Dryden et al., 1993
), orbivirus (Burroughs et al.,
1995
), aquareovirus (Shaw et al., 1996
), and rotavirus (Estes, 1996
), but thus far, in no other kind of virus.
;
Thomas et al., 1994
) to measure the mass per unit length
of L-A chromosomes extruding directly from capsids, and
after isolation.
Materials and Methods
), except that 12.5 ml CsCl
gradients were run at 157,000 g (44,000 rpm in a Beckman NVT65 rotor;
Beckman Instruments, Inc., Fullerton, CA) for 15 h at 4°C. The virus-containing fractions were dialyzed against buffer A (50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, pH 7.8) for 6 h, and stored at ~20
mg/ml at 4°C. All samples were used within 7 d of purification, as L-A virions tend to lose their dsRNA upon protracted storage.
; Booy, 1993
). Micrographs were recorded under minimal exposure conditions at nominal magnifications of ×36,000
or ×46,000 (~8 e
/Å2) on Kodak SO-163 films, using an electron microscope (EM400RT; Philips Technologies, Eindhoven, The Netherlands)
equipped with modified Gatan anticontaminator blades and a Gatan 626 cryoholder (Gatan, Warrendale, PA). In some experiments, bacteriophage
T4 was mixed with L-A, and the 40.5-Å axial spacing of its tail-sheath (Moody and Makowski, 1981
) was used as an internal magnification standard.
Micrographs were assessed for resolution and stigmation by optical diffraction of large areas, and their defocus values estimated from the positions
of the first zero of the contrast transfer function (CTF). For the micrographs chosen for analysis, the first zero was in the range of (15 Å)
1-(27
Å)
1 (Table I). Underfocus values (
) were calculated according to
= 1/
k2 , where
is the wavelength of the 100 keV electrons (0.037 Å), and k
is the spatial frequency of the first zero (Lepault and Leonard, 1985
).
Fig. 5.
Spectra calculated
from powder patterns of genome-revealing difference
images of L-A virions. The
positions and spread of the
peaks in these spectra give
statistically representative
estimates of the predominant
spacings between RNA filaments packed in L-A virus
capsids. Because the shape of
a spectrum depends on the defocus of the micrographs
from which it was calculated,
this analysis was repeated for
micrographs covering a range
of defocus values. In each
case, difference images were
calculated as shown in Fig. 4,
their diffraction patterns calculated and azimuthally averaged, and the powder pattern obtained by summing
over many such particles.
The various spectra are indexed according to the serial numbers
of the micrographs used. The closest-to-focus spectrum is at top,
and the furthest-from-focus one is at bottom (Table I). Each
spectrum shows a broad peak in the range of (47 Å)1 to (31 Å)
1 (gray vertical band). Also shown is the control spectrum calculated from empty particles in micrograph #7261. It shows a
much weaker peak in the same frequency range indicative of the
defocus value at which this micrograph was recorded, and confirms that the strong peak in the full particle spectrum reflects
scattering from the viral RNA. For reference, a line is shown at
(26 Å)
1, the average interduplex spacing for many other dsDNA and dsRNA viruses.
[View Larger Version of this Image (29K GIF file)]
Fig. 4.
Computer-filtered images of
encapsidated L-A genomes. Typical full
and empty particles are shown in a and e,
respectively. b and f show the same particles after low pass filtering at (28 Å)1. c
and g were obtained by reprojecting the
corresponding density maps in the appropriate viewing orientations, and reproduce the micrographs well, particularly in
terms of peripheral detail. d and h are
"difference images" calculated by subtracting c from a after nullifying the internal density of the "full" map, and g from
b, respectively. d reveals the encapsidated genome. The control h simply represents background noise, and confirms
that the shell is cleanly removed in this
procedure. The two particles shown are
from the highest defocus micrograph analyzed (2-µm defocus; first CTF zero at [27 Å]
1). i-l show two more examples each
of exposed genomes from four micrographs with different defocus values.
Their first CTF zeros at spacings of 15.5, 18.5, 21, and 23 Å, respectively. In each case, the density map used to calculate
the shell contribution to the virion image
was calculated from the micrograph in
question. These images illustrate the variable appearance of encapsidated genomes, and demonstrate that this variability does not arise from differences in
defocus. Bar, 100 Å.
[View Larger Version of this Image (182K GIF file)]
), running on Alpha workstations (Digital Equipment Co., Maynard, MA). Particles were extracted
and preprocessed using the automated procedure of Conway et al. (1993)
.
Particle orientations were determined by means of the Polar Fourier
Transform algorithm, an iterative, model-based procedure (Baker and
Cheng, 1996
). As a starting model, we used our earlier 26Å resolution
three-dimensional (3-D) map of empty L-A particles (Cheng et al., 1994
).
In all micrographs analyzed, reconstructions of empty and full particles were calculated separately, using Fourier-Bessel techniques (Crowther, 1971
; Fuller, 1987
; Baker et al., 1988
). Complete icosahedral (532) symmetry was imposed on the final density map. The contour level imposed for
surface rendering was based on 100% of expected volume, assuming a
protein density of 1.3 g/cm3. Resolution of the three-dimensional maps
was estimated in terms of a Fourier shell correlation procedure (Saxton
and Baumeister, 1982
; Conway et al., 1993
), and was, in each case, very
close to the first zero of the CTF. Data quality was also assessed by eigenvalue spectra (Fuller et al., 1996
).
), data from three micrographs
were combined. All three micrographs were well stigmated and had first
zeros at ~(15 Å)
1. Three independent reconstructions were first calculated and mutually scaled to the same magnification and density by matching their spherically averaged radial density profiles (Booy et al., 1994
). A
composite reconstruction including >80 particles (Table I) was then calculated, the orientation and alignment parameters of each particle refined, and a final map calculated. The resolution was 16 Å by the above
criterion.
; Booy et al., 1991
), thus providing an unobstructed view of the encapsidated dsRNA. As a control, the same procedure was applied
to images of empty particles. Diffraction patterns were calculated for all
difference images in each data set. These were then added together with
equal weighting (combining empty and full particles separately), and azimuthally averaged to obtain the respective one-dimensional powder patterns.
; Wall and Hainfeld, 1986
). Full particles, purified as
described above and stored briefly at a concentration of ~20 mg/ml, were
diluted 40-fold in buffer A, and a 5-µl drop was applied to a thin carbon
film. The grid was washed 10 times with 100 mM ammonium acetate, pH
6.5, frozen, and then dried (Wall, 1979
). To visualize isolated dsRNA, material purified by phenol extraction (Wickner, 1994
) at 2.4 mg/ml in diethyl pyrocarbonate-treated water was diluted 100 times with 100 mM
ammonium acetate and applied to a polylysine-coated carbon film,
washed with 20 mM ammonium acetate, and freeze-dried. Tobacco mosaic virus (TMV) at 50-100 µg/ml was included in all preparations as an internal mass standard. Digital micrographs (512 × 512 pixels) were recorded
using sampling steps of 20 or 10 Å/pixel. Mass determinations were carried
out as described (Newcomb et al., 1993
; Thomas et al., 1994
).
), and under our conditions,
is active only in the presence of 10 mM MgCl2. The reaction mixture (25 µl), containing 0.5 µg dsRNA and ~1 µg of RNase III in 50 mM Tris HCl,
pH 7.8, 150 mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 0 or 10 mM of
MgCl2, was incubated for 45 min at 37°C. The resulting products were
phenol extracted, ethanol precipitated, separated on a 1.5% agarose gel,
and then detected by ethidium bromide staining.
Results
Fig. 1.
Cryoelectron micrographs of purified L-A
virions at different defocus
values. (a) ~0.65 µm; (b)
~0.9 µm; (c) ~1.4 µm underfocus. One of the full particles exhibits a ringlike
"swirl" pattern (c, large arrowhead). (d) At ~2 µm underfocus; a "dimeric" capsid is shown (large arrowhead).
Filamentous material, presumably RNA, is observed in
all four micrographs (small
arrowheads). Bar, 1,000 Å.
[View Larger Version of this Image (129K GIF file)]
Fig. 2.
Three-dimensional density maps of L-A capsids at 16 Å resolution. (a) Stereo views of the
outer (upper panels) and inner
(lower panels) surfaces of the full
capsid, viewed along a fivefold
axis of symmetry. The internal
contents were computationally removed to expose the inner surface. (b) Transverse central sections taken from the maps of
empty (left) and full (right)
capsids, viewed along a twofold
axis. Darker shades represent
higher densities (corresponding to
protein and/or RNA). Black arrowheads, fivefold symmetry axes;
black lollipops, threefold axes;
and white arrowheads, twofold
axes. The two shells are virtually
identical. In the full capsid (right),
the closest contacts between the
inner surface of the protein shell
and the underlying RNA appear
to take place around the lateral
twofolds. The white rings just inside and outside the shell represent interference fringes arising
from phase contrast. Since the
holes in the capsid wall are too
small to admit an A-form duplex
(see Discussion), it seems likely
that the empty capsids lost their
RNA through a hole created by
the dislodging of one or a few Gag
subunits. With the averaging that
takes place in calculating a density
map, such a loss would not significantly affect the average density
at that lattice site, and thus may be reconciled with the essentially
identical structures visualized for
full and empty capsids. Bar, 50 Å.
[View Larger Version of this Image (121K GIF file)]
Fig. 3.
Structural organization of the two nonequivalent Gag monomers in the
L-A capsid. (a) Spherical sections through the density map of the L-A capsid, as
viewed along a twofold axis.
These sections correspond
to radii of 214, 207, 201, 194, 188, 181, 175, and 168 Å, respectively (left to right, and
top to bottom). Here, white
tones correspond to high
density, denoting protein
and/or RNA (i.e., the contrast has been reversed relative to Fig. 2 b). (b) Schematic diagram showing
arrangement of Gag subunits
in the L-A surface lattice, as
viewed along a fivefold axis
(compare with Fig. 2 a). The
A-subunits are green, and
the B-subunits orange. (c)
Diagram showing three pentons (each a Gag decamer)
clustered around a threefold
axis. Five- and twofold axes
are also marked. (d) The
outer crests of both subunits
may be subdivided into three domains: B1, B2, B3, and A1,
A2, A3, respectively. Two
possible modes of association
of neighboring A- and B-subunits into dimers are shown
in the lower part of the panel.
The one on the left appears
to have more extensive intersubunit contacts and therefore may be favored. (e) Closeup views down a twofold
axis from outside (left) and
inside (right). A- and B-subunits are marked in both cases (A and B, respectively).
On the left panel, the largest of the five kinds of holes that
traverse the capsid wall are
marked (arrows). On the
right panel, the Y-shaped
motifs seen on the subunits'
undersides are apparent. For
one A-subunit, this motif is
tinged with green and for a
B-subunit, with orange. Bars:
(a) 100 Å; (e) 25 Å.
[View Larger Version of this Image (80K GIF file)]
), the capsid wall is seen to be quite porous, perforated with many small holes (Figs. 2, a and b, and 3 e).
There are five classes of holes: those at the fivefold axes
(area = ~107 Å2); those surrounding the fivefold axes
(~165 Å2); those on the threefold axes (~118 Å2); relatively large Y-shaped holes surrounding the threefold axes (~244 Å2); and oblong holes between the A- and B-subunits (~110 Å2).
).
The resulting difference images show punctate motifs and/
or swirls; some examples are shown in Fig. 4. To estimate the interstrand spacings, we measured the positions of diffraction peaks in powder patterns calculated from many
such images (Fig. 5). Because phase-contrast imaging accentuates particular spatial frequencies, depending on the
defocus, this analysis was performed for micrographs that
cover a range of defocus values. All of these patterns exhibited broad peaks extending from ~(31 Å)
1 to ~(47
Å)
1. The position of the peak varied slightly with defocus
but was invariably between (36 Å)
1 and (40 Å)
1. As a
control, the same procedure was applied to empty capsids. The resulting profile (Fig. 5, dashes) has a small peak in
this range, this probably reflects noise amplified by the
phase-contrast at this defocus value. It is, however, much
weaker than the corresponding peak of the full particle
powder pattern, confirming that this peak primarily reflects scattering from the RNA.
; Booy et al., 1991
,
1992
; Dryden et al., 1993
). Accordingly, one might wonder
whether their RNA is partly extruded, with a concomitant
loosening up of the portion remaining inside the capsid.
The presence of empty capsids and free RNA in electron
micrographs is consistent with such a hypothesis. To test it,
we conducted accessibility experiments with RNase III, a
nuclease specific for dsRNA. The genomes of freshly prepared virions proved entirely resistant to the enzyme (Fig.
6); whereas in a positive control, isolated L-A RNA was
extensively digested (Fig. 6, lane 3). When virions were
preincubated in a low salt buffer, (known to destabilize
them) (Esteban and Wickner, 1986
), their RNA partially
degraded (Fig. 6, lane 8). These results imply that the
dsRNA is entirely sequestered inside freshly prepared virions. This conclusion is consistent with earlier STEM mass measurements that yielded masses for full L-A virions
consistent with their containing whole genomes (Cheng et
al., 1994
). RNA is gradually lost on storage, but release
from any given particle, once started, appears to proceed
quite rapidly. The conclusion that the L-A genome is indeed packed more loosely than in other viruses is supported by calculations of packing density (see Discussion).
Fig. 6.
RNase III accessibility for L-A dsRNA. Purified RNA (lanes 1-3),
dsRNA in intact full particles
(lanes 4-6), and dsRNA from disrupted full particles
(lanes 7 and 8) were treated
with RNase III as described
in Material and Methods, in
the absence (lanes 2, 5, and
7) or presence (lanes 3, 6,
and 8) of 10 mM MgCl2.
Lanes 1 and 4 are controls
for purified dsRNA and
dsRNA from intact full particles without RNase III treatment, respectively. On the left, molecular weight markers. After RNase treatment,
RNA was separated in a 1.5% agarose gel and detected by ethidium bromide staining. The digestion products are diffusely
spread out in lane 8, whereas in the other lanes the genome band
is either present at the appropriate position or absent, having
been digested to small fragments.
[View Larger Version of this Image (85K GIF file)]
Fig. 7.
STEM dark-field images of unstained L-A RNA molecules. (a) Freeze-dried preparation of purified dsRNA, with a
few tobacco mosaic virions (the rodlike particles), included as internal mass standards. At right is a histogram of mass-per-unit
length measurements made from these micrographs (n = 53). (b)
Purified L-A virions, with some free RNA; at right, the histogram
of mass-per-unit length data (n = 275). These measurements
were restricted to RNA segments visibly connected to capsids.
The arrows indicate densities corresponding to integral numbers
of strands per filament, assuming 2.8 kD/nm for an A-form RNA
duplex (Results). Black arrows mark even numbers of strands,
i.e., multiple duplexes. The precision of the measurements is insufficient to resolve the distribution into a set of well-defined peaks. In principle, these densities could be biased to higher values by residual salt deposits, as a consequence of insufficient grid
washing. However, in this case, one would expect greater variability in the data, and the standard deviation of the measurements made on TMV particles to be considerably higher. In fact,
the SDs were very similar, at 3.81% for the TMV in the experiments with purified RNA molecules, and 4.48% for those on
capsid-connected RNA filaments. Bar, 1,000 Å.
[View Larger Version of this Image (86K GIF file)]
Discussion
). First,
dsRNA is transcribed by a polymerase resident within the
virion. Next, (+) strand transcripts are extruded to serve
as (a) mRNA for the synthesis of viral proteins, (b) the
species that becomes encapsidated in progeny virions, and
(c) used as the template for synthesis of complementary
(
) strands in replication. Both transcription and replication take place inside the nucleocapsid, which is the complete virion for fungal dsRNA viruses or an inner core for
higher eukaryotic viruses like those of the Reoviridae family (e.g., reovirus [Harvey et al., 1981
; Dryden et al., 1993
];
rotavirus [Mansell and Patton, 1990
; Labbé et al., 1991
];
and orbivirus [Huismans et al., 1987
]). This model also applies to the prokaryotic dsRNA virus, bacteriophage
6
(Mindich and Bamford, 1988
). What features do these
various transcription-cum-replication chambers have in
common?
). It is of interest
to know whether this commonality extends to more detailed aspects of capsid architecture. Here, fewer data are
available, but we note a definite morphological similarity
between L-A capsid and the core of Bluetongue virus (Hewat et al., 1992
); this virus is also composed of 120 subunits (Burroughs et al., 1995
). Aquareovirus cores also
show 120 morphological subunits on their surface (Shaw et
al., 1996
). Although reovirus cores are more complex than
the L-A capsid, the presence of Y-shaped domains on
their inner surface (Dryden et al., 1993
) is a shared feature. Birnaviruses, whose genomes consist of two segments of dsRNA, seem to be an exception since they are
single-shelled T=13 particles with no internal core (Böttcher
et al., 1997
).
), Gag can selfassemble
into correctly sized capsids. This reaction should be controlled by some switching event that specifies the A and B
conformations (Johnson, 1996
). This switch may operate
at the level of dimer formation, yielding protomers of
which 60 equivalent copies would then self-assemble into a
conventional T = 1 icosahedron. The plausibility of this
model is supported indirectly by observations in other systems (e.g., the human growth hormone receptor [de Vos et
al., 1992
]), which attest that an asymmetric homodimer can be a stable state of assembly. Alternatively, A-subunits might associate into homo-pentamers, with the switch
accompanying the accretion of B-subunits to convert them
into decamers capable of forming the capsid pentons. The
fact that Gag-Pol appears to nucleate assembly of L-A in
vivo (Fujimura and Wickner, 1988b
), and there are only
1-2 copies of this protein per capsid, argues against the
pentamer-based model.
2.43 kD/nm, assuming
2.81 Å rise per base pair (Arnott et al., 1972
) and 682 D/
bp
and are less consistent with a B-form duplex
2.00
kD/nm, from 3.38-Å rise per bp (Lewin, 1990
). It has been
previously demonstrated by spectroscopy that packaged
6 dsRNA has an A-form structure (Bamford et al., 1993
).
However, x-ray diffraction studies of crystallized reovirus
dsRNA (for review see Nibert et al., 1996
) were indicative
of a double helix with 10 or 11 bp per turn and a 3-Å pitch,
this corresponds to a structure intermediate between A-form
(2.81-Å rise per bp) and B-form (3.38-Å rise per bp).
Fig. 8.
Hypothetical model of a transcriptionally active L-A
virion. The polymerase (Pol) is covalently attached to a Gag subunit, which we assume to be integrated into the icosahedral surface lattice, thus immobilizing Pol (Icho and Wickner, 1989). Pol
(and the COOH terminus of Gag) are assumed to reside on the
inner surface of the shell, to facilitate access to the substrate
dsRNA. The shape assigned to Pol in this diagram is arbitrary. In
this scenario, it is necessary for the genome to be propelled sequentially past Pol as transcription proceeds. Transcripts are hypothesized to be extruded directly through the largest holes in
the capsid shell (Discussion).
[View Larger Version of this Image (159K GIF file)]
(Lepault et al.,
1987
), T7 (Booy et al., 1992
) and herpes simplex virus
(Booy et al., 1991
), and other dsRNA viruses such as reovirus (Dryden et al., 1993
); these are relatively highly ordered and tightly packed, with center-to-center spacings of
25-30 Å. In part, the larger spacing in L-A reflects the fact
that A-form duplexes are wider than B-form duplexes. However, the average interduplex spacing may be calculated independently from the volume available (average
internal capsid diam = 348 Å) and genome length (1.287 µm, given 4.579 kbp, and 2.81 Å axial rise per bp). Assuming quasi-hexagonal close packing to a uniform density, a
spacing of ~40 Å is obtained. There is some flexibility in
this figure, arising from the center being less populated
with RNA, the packing not being perfectly crystalline, and
possibly also from some local compaction into multistranded filaments (see below). Nevertheless, this figure is
in good agreement with the spacings measured directly
from the powder patterns (Fig. 5). Its consistency supports
our inferences that (a) the dsRNA genome is fully internalized, and (b) its packing is much looser than in other
double-stranded viruses.
). These satellite viruses (M1, M2, etc.) and deletion mutants of L-A itself (such as X) contain more dsRNA segments than L-A,
but approximately the same number of base pairs per particle. This occurs by "headful replication," whereby one viral (+) strand is packaged, and replicates inside the particle until it is full. Subsequently synthesized (+) strands are
extruded from the particle (Esteban and Wickner, 1986
;
Fujimura et al., 1990
). As noted above, much more room
remains available in the packaged L-A capsid than in
other double-stranded viruses. We can think of two explanations. In one, the Gag gene has evolved recently to provide a more capacious capsid, but the genome as a whole
has not yet evolved to fully exploit this opportunity for expansion. The fact that the multisegmented satellites do not
package RNA more densely than L-A appears to argue
against this hypothesis.
,b). In
transcription (Fig. 8), large tracts of dsRNA must be propelled sequentially past this fixed point. In so doing, the
organization of the entire packaged genome must undergo
constant revision, or "churning" motion. (In contrast, the
tightly compressed genomes of dsDNA phages are thought
to be in a static vegetative state.) RNA polymerases are
known to be powerful translocases, capable of generating
forces in the range of 14 piconewtons (Yin et al., 1995
).
The ability of L-A Pol to function in replication and transcription depends on its being capable of overcoming the frictional drag imposed by the packaged genome. We infer
that this resistance increases with higher packing density,
whence the observed packing density may represent the
highest level of frictional resistance against which the enzyme can function effectively. This idea is testable by generating insertions in the L-A genome.
) and rotavirus (Prasad et al., 1996
) than in
L-A might suggest otherwise. However, these systems, in
which transcriptionally active particles have been visualized (Yeager, M., S.G. Weiner, and K.M. Coombs. 1996. Biophys. J. 70:116a.; Lawton et al., 1997
), are considerably
more complex; more proteins and many more RNA segments are involved, and precisely calibrated spacings for
their packaged genomes have yet to be reported. Transcription of reovirions is thought to require "activation"
by proteases, and concomitant conformational changes
(Powell et al., 1984
; Dryden et al., 1993
), which, we conjecture, may rearrange the core structure, so as to facilitate motion of the dsRNA during transcription. Nevertheless,
the same overall requirements apply. It will be fascinating
to learn more about possible solutions to this problem, and
the properties of protein shells that are not only closed
containers but also active metabolic compartments.
Footnotes
J.R. Castón's present address is Centro Nacional de Biotecnologia,
Consejo Superior de Investigaciones Cientificas, Universidad Autonoma
de Madrid, 28049 Madrid, Spain.
Abbreviations used in this paper
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