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Journal of Virology, March 1999, p. 2298-2308, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Reovirus Mutant tsA279 L2 Gene Is
Associated with Generation of a Spikeless Core Particle: Implications
for Capsid Assembly
Paul R.
Hazelton and
Kevin M.
Coombs*
Department of Medical Microbiology and
Infectious Diseases, University of Manitoba, Winnipeg, Manitoba,
Canada R3E 0W3
Received 12 August 1998/Accepted 23 November 1998
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ABSTRACT |
Previous studies which used intertypic reassortants of the
wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified
two ts lesions; one lesion, in the M2 gene segment, was
associated with defective transmembrane transport of restrictively
assembled virions (P. R. Hazelton and K. M. Coombs, Virology
207:46-58, 1995). In the present study we show that the second lesion,
in the L2 gene segment, which encodes the 
2 protein, is associated
with the accumulation of a core-like particle defective for the 2
pentameric spike. Physicochemical, biochemical, and immunological
studies showed that these structures were deficient for genomic
double-stranded RNA, the core spike protein 2, and the minor core
protein µ2. Core particles with the 2 spike structure accumulated
after temperature shift-down from a restrictive to a permissive
temperature in the presence of cycloheximide. These data suggest the
spike-deficient, core-like particle is an assembly intermediate in
reovirus morphogenesis. The existence of this naturally occurring
primary core structure suggests that the core proteins 1, 3, and
2 interact to initiate the process of virion capsid assembly through
a dodecahedral mechanism. The next step in the proposed capsid assembly
model would be the association of the minor core protein µ2, either
preceding or collateral to the condensation of the 2 pentameric
spike at the apices of the primary core structure. The assembly pathway
of the reovirus double capsid is further elaborated when these
observations are combined with structures identified in other studies.
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INTRODUCTION |
Virus assembly is an important late
step in viral replication that is poorly understood in animal viruses.
The mechanisms of viral assembly, release, extracellular transport,
attachment, penetration, and uncoating determine the size and shape of
the "packing crate" which must carry the viral genome between
replicative cycles (40, 70). This packing crate must be a
meta-stable structure to protect the genome during transport but also
capable of releasing the genome and necessary associated replicative
enzymes once it enters a new host cell. X-ray crystallography has
provided valuable insight into the overall structure and putative
protein interactions in a number of viruses at a macromolecular level (47, 82). Recent studies have elucidated structural
interactions of components such as the G-H loop of foot-and-mouth
disease virion capsid protein VP1 (24) and the hemagglutinin
of influenza virus expressed in a bacterial vector (46).
Electron cryomicroscopic studies have utilized image averaging and
various planes of focus to provide three-dimensional and, to a degree,
internal imaging of whole-virus structures (6, 75, 77, 86),
a limited number of subviral particles (28, 103),
reassortant viruses (85), capsids assembled from expressed
proteins (45), proteolytically degraded reovirus
intermediate structures (60), capsid-like structures
produced by engineered and expressed rotavirus capsid protein VP2
(54), and reovirus and rotavirus undergoing in vitro transcription (53, 104). These studies showed complete
structures and some assembly and disassembly intermediate structures,
permit the localization of some proteins, and may identify regions of interactions between some proteins (60) and nucleic acids
(77). Unfortunately, physical determinations of static
structures may not define the dynamic processes and pathways by which
viral proteins interact to assemble the packing crate needed to carry
the viral genomic cargo safely through a hostile environment to its
next address. For example, recent evidence suggests that the
conformation of flock house virus in solution may differ dramatically
from that predicted by X-ray crystallography (12).
An alternative approach to study macromolecular assembly processes is
to use assembly-defective systems. The assembly pathways of some
bacteriophages have been successfully studied in detail by using
conditionally lethal amber mutations of bacteriophage T4 (10, 11,
101) and P22 (78). However, the many different strategies for biochemical regulation and interaction with host cells,
genome organization, and assembly and structural designs employed by
the eucaryotic viruses have mitigated against similar success in
virus-eucaryote systems. The success in using bacteriophage conditionally lethal mutants (29, 30) to deduce procaryotic virus assembly pathways suggests that the Fields panel of conditionally lethal reovirus temperature-sensitive (ts) mutants (19,
32, 79) (reviewed in reference 21) may be
elegant tools for dissecting the assembly pathways of a eucaryotic virus.
The complete mammalian reovirus particle contains nonequivalent amounts
of eight different structural proteins assembled into a double-shelled
particle approximately 80 to 85 nm in diameter. The outer capsid is
made up of the major proteins µ1 and 3 (28) and the
minor protein 1 (91). The inner core structure, a protein shell approximately 60 nm in diameter, is composed of the major core
proteins 1 and 2, core spike protein 2, and minor core proteins 3 and µ2 and encases the 10 segments of double-stranded RNA (dsRNA) which comprise the genome (28). The segmented
nature of the viral genome allows for the generation of intertypic
reassortants which may be exploited to assign biologic functions to
individual genes and their protein products (26, 44, 57, 95,
105). (For recent reviews of the structures and properties of the
mammalian reoviruses, see references 68 and
71). The morphological variants produced at
restrictive temperatures have been reported for only a few of the
ts mutants of the Fields panel, either in thin-sectioned infected cells (33), by negative stain electron microscopy
of gradient fractions of cell extracts (62, 65), or by both
methods (23). Therefore, the identification of additional
assembly-defective ts mutants might help elucidate
eucaryotic virus assembly. The ts mutants of recombination
group A (32) contain one or more lesions in the
monocistronic M2 gene segment (67), which encodes the
reovirus µ1 protein, a major component of the outer shell of the
complete virion. Previous studies with the prototype clone tsA201 showed mild expression of the conditionally lethal
phenotype (32). This clone did not produce aberrant
particles when cultured under restrictive conditions (33).
However, recombination group A contains more than 20 different mutant
clones, all of which are believed to contain lesions in the M2 gene
segment (21, 32).
Using intertypic reassortant analysis, we previously reported that the
mutant clone tsA279 contains two ts mutations.
One lesion is in the M2 gene and is associated with strong expression of the ts phenotype at elevated temperatures due to a
blockade in the transmembrane transport of restrictively assembled
virions. The second lesion, associated with mild expression of the
ts phenotype at elevated temperatures, is in the L2 gene
segment (44), which encodes the core spike protein 2. In
this study we report a blockade in assembly as a second mechanism for
the expression of the ts phenotype by the mutant clone
tsA279. The blockade in assembly is associated with the
ts lesion in the L2 gene segment, and results in the
accumulation of primary core particles which are defective for proteins
2 and µ2. Correlating the previously reported assembly intermediates produced by other members of the Fields panel with the
pattern of assembly intermediates produced under restrictive growth
conditions by the mutant tsA279 further elaborates the assembly pathways of mammalian reoviruses.
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MATERIALS AND METHODS |
Cells, viruses, and immunologic reagents.
Mouse L929 cells,
reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and the ts
mutant clone tsA279, derived from T3D, are laboratory
stocks. tsA279 is a double ts mutant, with
lesions mapping to the L2 and M2 gene segments (proteins 2 and µ1,
respectively) (44). The panel of T1L×tsA279
reassortants, the plaque assay to determine virus titrations, and the
preparation of polyvalent rabbit anti-whole reovirus antiserum were as
previously described (44). Polyvalent rabbit anti µ2
antiserum was a gift kindly provided by E. Brown, University of Ottawa.
Hybridoma cell line 7F4, which produces a monoclonal antibody that
recognizes 2, was kindly provided by H. Virgin, Washington
University. Hybridomas were maintained and monoclonal antibody 7F4 was
purified as described previously (93).
Negative-stain-electron microscopy of infected cell
extracts.
Preliminary analyses of different negative stains and
stain conditions indicated that 1.2 mM phosphotungstic acid, pH 7.0 (PTA), gave the best overall quality of staining (43a).
Subconfluent L929 monolayers in 24-well culture dishes were infected
with either T1L, T3D, tsA279, or various
T1L×tsA279 reassortants at a multiplicity of infection
(MOI) of 5 PFU/cell. After attachment for 1 h at 4°C, the
infections were overlaid with prewarmed Joklik's modified minimal
essential medium (S-MEM) supplemented with 2.5% fetal calf
serum-2.5% VSP agammaglobulin neonatal bovine serum-2 mM L-glutamine and containing 20% preadapted medium and then
incubated for 36 h at either 32 or 40°C. In some experiments,
cycloheximide was added to the cultures at various times postattachment
to a final concentration of 100 µg/ml, and the incubation temperature was changed 15 min later to ensure that no permissively translated viral protein was available for assembly onto structures produced under
restrictive conditions (74); the infections were then cultured an additional 6 h (4). The cultures were
freeze-thawed three times, and the cell debris was cleared by
centrifugation for 10 s at 15,000 × g in an
Eppendorf model 5412 benchtop centrifuge. Then, 50-µl aliquots of the
supernatants were layered over 100-µl cushions of 30% potassium
tartrate (pH 7.2), and viral and subviral particles were pelleted by
centrifugation in a Beckman Airfuge (A100 rotor; 26 lb/in2,
1 h). Pellets were resuspended in 50 µl of 0.1% glutaraldehyde in S-MEM (pH 7.2), allowed to fix for 10 min at 4°C, centrifuged directly onto Formvar-coated, carbon-stabilized 400 mesh copper electron microscopy grids (Beckman Airfuge, EM-90 Rotor; 26 lb/in2, 30 min) as previously described (38),
and stained with 1.2 mM PTA. Samples were viewed and photographed at
machine magnifications of ×30,000 and ×70,000. The relative
proportions and types of particles produced in each infection were
determined by direct counting of all particles in each of five randomly
selected, nonadjacent grid squares from the four outer quadrants and
the central area of the grid (38). The infectious titer
produced by each culture was determined by standard plaque assay at
32°C as previously described (44). Statistical
probabilities were determined by using a 
2 test with the
Yates correction (41).
Immunoelectron microscopy of virions and assembly intermediate
structures.
Except where otherwise indicated, all preparative and
conjugation steps were conducted at 20°C. Gold probes (12 nm) were
prepared from chloroauric acid as described earlier (89) and
filtered through 0.2-µm pore filters to remove aggregates. Polyvalent
rabbit anti-reovirus immunoglobulin G (IgG) was purified by protein
A-Sepharose affinity chromatography essentially as described previously
(76). Briefly, antireovirus antisera was preabsorbed against
an L929 monolayer, heat inactivated at 56°C for 30 min, passed
through a protein A-Sepharose column (Sigma, St. Louis, Mo.), and
washed with 50 mM Tris (pH 8.0)-150 mM sodium chloride (wash buffer), and the bound IgG was then eluted directly into 0.5 M phosphate buffer
(pH 8.0) with elution buffer (100 mM sodium acetate, 150 mM sodium
chloride [pH 4.0]). UV absorbance was determined at 280 nm, and the
peak fractions were pooled. After extensive dialysis against 2 mM
sodium borate (pH 8.0), the pooled fractions were concentrated with a
Minicon B-15 concentrator (Millipore, Bedford, Mass.). The
concentration of IgG needed to stabilize 10 ml of gold probe was
determined by adsorption isotherm assay essentially as described
earlier (36), except that IgG was diluted in 2 mM sodium
borate (pH 8.0) for assay and conjugation to gold (48, 58,
94). After conjugation the probe was washed, resuspended in 10 mM
Tris (pH 8.0)-150 mM NaCl-1% bovine serum albumin (BSA)-0.1% Carbowax M20-0.2% low bloom gelatin (Sigma) (stabilizing buffer) as
described before (9), briefly sonicated at low energy to disrupt loose aggregates, filtered, and stored at 4°C until use (94). Viral lysates were prepared from T3D- and
tsA279-infected cells, cleared, and pelleted directly onto
Formvar-carbon-coated 400-mesh nickel electron microscope grids as
described above. The grids were washed successively in HEPES-buffered
MEM (pH 8.0), HEPES-buffered MEM with 1% BSA, and stabilization buffer
and then incubated in a humid chamber for 30 min at 20°C on 5-µl
aliquots of IgG labeled gold probes. The grids were washed with
successive changes of phosphate-buffered saline (pH 8.0) and stained
with PTA (81). The samples were evaluated and photographed
as described above.
Isolation and identification of labeled virus and intermediate
assembly structures.
L929 cells were infected with either
tsA279 or T3D at an MOI of 5 PFU/cell. After attachment for
1 h at 4°C, prewarmed supplemented S-MEM was added to provide
for a final concentration of 5 × 105 cells/ml, and
the infections were incubated in spinner cultures for 36 h at
40°C. Control infections were incubated at 32°C for an equivalent
number of replicative cycles (54 h). Viral proteins were labeled 3 h postattachment by the addition of
[35S]methionine-[35S]cysteine (NEN, Boston,
Mass.) to a final concentration of 25 µCi/ml. Infected cells were
harvested and extracted with 1,1,2-trichloro-1,2,2-trifluoroethane (Freon 113) as described earlier (35). Viral structures were then purified by buoyant density centrifugation on preformed 1.2- to
1.55-gm/ml cesium chloride (CsCl) gradients (7.5 h at 6°C at 35,000 rpm in an SW41 Rotor (Spinco, Palo Alto, Calif.), 300-µl fractions
were collected by bottom puncture, and the fraction density was
determined by refractive index (Abbe 3-L Refractometer; Bausch and
Lomb, Rochester, N.Y.). Aliquots from each fraction were evaluated for
the presence of labeled material with a model LS 5000CE Scintillation
Counter (Beckman Scientific, Palo Alto, Calif.), and for the presence
of viral material by electron microscopy after direct centrifugation as
described above. Aliquots of each fraction were diluted threefold in
sterile distilled water, precipitated at 
20°C overnight in 70%
ethanol-300 mM sodium acetate, and resuspended in 0.24 M Tris (pH
6.8)-1.5% dithiothreitol-1% sodium dodecyl sulfate (SDS)
(electrophoresis sample buffer) (51).
Protein separation with the Tris-glycine-urea
(TGU)-SDS-polyacrylamide gel system.
Proteins were separated in
the Laemmli discontinuous Tris-glycine buffering gel system
(51), modified by the inclusion of urea (TGU gel) as
previously described (20). Briefly, the TGU resolving gel
consisted of a 5 to 16% polyacrylamide exponential gradient covered
with a 4 to 5% polyacrylamide linear gradient, both containing 43%
urea. A 4% polyacrylamide step which did not contain urea was layered
over the upper gradient. The resolving gel was in 375 mM Tris (pH 8.8).
Gels were allowed to polymerize for between 6 and 10 h, a 3.75%
polyacrylamide stacking gel in 125 mM Tris (pH 6.8) was layered over
the resolving gel, and proteins were resolved by electrophoresis at
either 9 mA for 9.5 h or 7.5 mA for 16 h. The gels were fixed
and impregnated with Enlightning (DuPont, Boston, Mass.) and
fluorographed by exposure to X-ray film (Kodak X-AR, Rochester, N.Y.).
Multiple film exposures of the gels were made and scanned with an LKB
Ultroscan XL laser densitometer as described earlier (44),
and the amount of each protein present was determined. The densities of
major core protein 1 bands were used to standardize the relative
proportions of other viral proteins in each evaluated fraction.
Immunoblot analysis of protein composition of assembly
intermediate structures.
Proteins were separated by using the TGU
acrylamide gel system, and selected lanes were transferred to nylon
membranes (Immobilon; Millipore) by using a Bio-Rad Semi-Dry Transblot
transfer system (Bio-Rad, Richmond, Va.) at a 20-V constant voltage for
35 min. Nonspecific binding was blocked by washing the membranes in
TBS-T (10 mM Tris [pH 7.5]-100 mM NaCl-0.1% Tween 20 containing
5% skim milk proteins; the membranes were then reacted with a mixture of antireovirus antiserum and antireovirus µ2 antiserum in TBS-T for
90 min at room temperature and washed with fresh TBS-T, and the blots
were probed with goat anti-rabbit IgG conjugated to horseradish
peroxidase (Jackson ImmunoResearch Laboratories, West Grove, Pa.) in
TBS-T containing 0.1% BSA for 90 min at room temperature. After a
washing with TBS-T, the immune complexes were detected with diamino
benzoate (39). Primary and secondary antibodies were
stripped from the membranes by being washed in 100 mM
2-mercaptoethanol-2% SDS-62.5 mM Tris-HCl (pH 6.7) for 30 min at
50°C (1) and then reprobed with the anti-2 monoclonal
antibody 7F4 (93). After treatment with goat anti-mouse IgG
conjugated to horseradish peroxidase and a washing as above, the immune
complexes were detected by using the Amersham enhanced
chemiluminescence system (Amersham Life Sciences, Little Chalfont,
United Kingdom) in accordance with the manufacturer's instructions.
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RESULTS |
tsA279 selectively produces a novel subviral particle
at the restrictive temperature.
Previous experiments that used
thin-section electron microscopy to examine group A mutants suggested
there was no interference in assembly at 39°C in tsA201
infections (33) and that both core-like and top-component
particles were present in 39.5°C tsA279 cultures (43,
44). Thin-section electron microscopy provides insufficient
resolution to distinguish between structures with subtle differences.
In addition to difficulties in identifying particle type, analyses of
random cell sections may not include the population of particles
released by cell lysis. An ideal way to determine the nature of various
products is negative-stain electron microscopy because of the higher
resolution of this technique (13, 42, 64). However, when the
production of virally encoded products is reduced, as may occur in
restrictive mutant infections, total populations of intermediates may
be missed when determinations are made from gradient-purified
fractions. To determine the exact nature and quantity of the different
structures present, direct particle counting of negative-stained
whole-cell cytoplasmic extracts was conducted after direct
centrifugation onto an electron microscope grid (38, 63).
The relative proportions of structures produced by tsA279
and T3D at both restrictive and permissive temperatures, as determined from direct particle counts of whole-cell lysates, are shown in Table
1. Cells infected with tsA279
and grown at a restrictive temperature produced approximately 50-fold
fewer viral particles than those grown at permissive temperatures. The
reduction in particle yield is consistent with previous reports of
reduced viral protein production by this mutant under restrictive
growth conditions (44). The particle/PFU ratio did not
differ significantly between permissive and restrictive cultures of
both T3D and tsA279 and ranged from 9 to 14 particles per
PFU (data not shown). These values are similar to those in previous
reports (80).
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TABLE 1.
Distribution of species of particles produced by T3D and
tsA279 at permissive and
restrictive temperaturesa
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The predominant structural form produced at 32°C by both
tsA279 and T3D was the genome complete whole virion
(diameter of
approximately 80 nm) after both 36 and 72 h of
incubation (between
77 and 86% of the total particles produced) (Table
1). The next
most common type of particle produced was the top
component (6
to 19%, diameter of approximately 80 nm). Outer shell
structures
(diameter of approximately 90 nm) and core particles
(approximately
50 nm in diameter) each comprised less than 6% of the
particles
produced. When infected cells were incubated at a restrictive
temperature of 40°C, the distribution of particles produced was
different. The proportion of whole virions was reduced to approximately
50 to 60% of all observed particles for both parental T3D and
the
ts mutant. With T3D there was a trend toward increased
proportions
of top component and outer shells at the restrictive
temperature
at both 36 and 72 h postattachment. In addition, there
was a trend
toward an increased proportion of complete core particles,
which
contained both genome and the characteristic
2 apical spike,
at 72 h postinfection. The mutant produced similar proportions
of
top-component structures and about 10-fold fewer outer-shell
structures
than did T3D at a restrictive temperature. However,
the proportion of
core-like particles produced by the mutant clone
increased from less
than 2% at the permissive temperature to 18
and 31% at the
nonpermissive temperature for the two different
times tested
(
P < 0.0001 by
2 test with the Yates
correction) (Table
1).
Core-like particles produced by
tsA279 at a restrictive
temperature (diameter of approximately 50 nm) appeared to be defective
for the
2 pentameric spike structure (Fig.
1A), whereas core
particles produced
under permissive growth conditions for both
T3D and
tsA279
and at a restrictive temperature for T3D clearly
demonstrated the
presence of the
2 spike structure (data not
shown). In addition,
mutant core-like particles which were restrictively
assembled (cultured
and assembled under restrictive growth conditions)
were penetrated by
the negative stain, indicating that they were
deficient for genomic
dsRNA, while core particles produced under
permissive and restrictive
conditions by T3D and under permissive
conditions by
tsA279
were not penetrated by the negative stain,
indicating that these
particles contained genomic dsRNA. The identity
of the core-like
particles produced by
tsA279 under restrictive
culture
conditions was confirmed with polyclonal antireovirus
IgG conjugated to
a 12-nm colloidal gold probe (Fig.
1B, large
arrow). The distributions
of particles produced under the different
temperature conditions were
not significantly altered by input
MOIs of either 5 or 50 PFU/cell
(data not shown).
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FIG. 1.
Negative-stain and immunogold-labeled 2
spike-defective core particles produced at restrictive temperature. (A)
Restrictive temperature cytoplasmic extracts of
tsA279-infected cells were centrifuged directly onto
electron microscope grids and negatively stained with PTA as described
in Materials and Methods. (B) Same as in panel A except the grid
samples were immunolabeled (12-nm gold) before negative staining. The
core-like particles in both panels (large arrows) demonstrated apical
depressions where the 2 pentameric core spike structure would be
expected (small arrows). (C) Permissive-temperature cytoplasmic
extracts of tsA279-infected cells. (D)
Restrictive-temperature cytoplasmic extracts of T3D-infected cells.
Magnification, ×100,000. The bars represent 100 nm.
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The production of 2 deficient core particles maps to the
tsA279 L2 gene.
To determine which tsA279
gene(s) was associated with the spike-defective phenotype, we analyzed
the distribution of particles produced at the restrictive temperature
by various reassortant clones which segregated the mutant L2 and M2
gene segments (Table 2). Each of the six
clones that contain the mutant L2 gene produced significant amounts of
spike-defective particles, whereas each clone with the T1L L2 gene
produced insignificant amounts of spike-defective particles. All of the
remaining gene segments were randomly associated with respect to the
presence or absence of the spike.
tsA279 produces structures which do not contain the
2 core spike protein nor the minor core protein µ2.
The above
results indicated that the tsA279 L2 gene was associated
with the nonpermissive production of a 2 defective core-like structure. To determine whether the core-like particles contained 2,
but in a conformation which was not resolved, or were devoid of 2,
we purified them and analyzed their protein content. Restrictively assembled tsA279 assembly intermediate structures labeled
with [35S]methionine-[35S]cysteine were
isolated by using cesium chloride buoyant density centrifugation as
detailed in Materials and Methods. Scintillation counting of fraction
aliquots identified a single peak of activity at a density of 1.33 to
1.35 gm/ml (Fig. 2A).
Electron microscopic examination of this fraction revealed complete
virions at this density (Fig. 2B1). No individual peaks of
radioactivity could be distinguished in fractions of lower density.
Core-like particles which were deficient for both genome and the 2
spike structure were present at a density of 1.28 to 1.29 gm/ml (Fig.
2B2), and outer-capsid structures were isolated at a density range of
1.26 to 1.28 gm/ml (Fig. 2B3). No core-like particles were observed at
higher buoyant densities. It was not possible to cleanly separate the
2 protein-deficient core particles from the outer-shell structures by rate zonal centrifugation due to their similar sedimentation characteristics and the small amount of material present in the fractions after sucrose gradient centrifugation (data not shown).
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FIG. 2.
Purification of restrictively assembled core-like
particles. L929 cells were infected with tsA279, viral
proteins were labeled with
[35S]methionine-[35S]cysteine during
culture at 40°C, cell pellets were collected, the cytoplasmic
membranes were solubilized with desoxycholate, and the samples were
extracted two times with Freon 113. (A) Viral components from 6 × 108 cells were separated on preformed CsCl gradients,
fractionated, and the density of each fraction determined from the
refractive index; aliquots were then were counted for
radioactivity as described in Materials and Methods. , Radioactivity
in counts per minute/10 µl; +, fraction density in grams/cubic
centimeter. (B) Electron micrographs of representative particles found
in separate gradient fractions from the experiment shown in panel A. Panels: 1, whole virus (fraction 15); 2, 2 spike-defective core
particles (fraction 19); 3, outer-shell structures (gradient fraction
19); 4, separately prepared and purified T3D core particles prepared
from permissively assembled whole T3D virions. A small amount of top
component was present in each of fractions 17 through 20 (arrowhead,
panel 3). Note the apical cavity present in the spike-defective core
(small arrow, panel 2) (bar = 100 nm). (C) Fluorogram of a
TGU-SDS-PAGE gel loaded with equivalent scintillation counts of
permissively assembled T3D and tsA279 whole virions and from
the same samples shown in panel B. Note the absence of 2 in lanes 2 and 3, which are comprised primarily of core particles (lane 2) and
outer shells (lane 3), with a small amount of top component in each
fraction. T3, permissively assembled, purified T3D virions. Lanes 1 to
3 correspond to the samples represented in panel B and lane A shows
permissively assembled, purified tsA279 virions. (D)
Immunoblot of a TGU-SDS-PAGE gel run in parallel with and with the same
samples as shown in panel C, with the addition of lane 4 which
represents permissively assembled cores (same as panel B4). The gel was
probed simultaneously by both anti-whole reovirus antiserum and
anti-µ2 antiserum. Note the intense reaction of anti-µ2 in lane 4 and absence of reaction in the remaining lanes. The anti-whole reovirus
antiserum shows weak reaction to 2 in lane 4 at the concentration
used but no reaction to 2 in the remaining lanes. Similar results
were obtained in each of four other experiments.
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TGU-SDS-polyacrylamide gel electrophoresis (PAGE) protein analysis of
the above fractions indicated that both the spike-defective
cores and
the outer-shell structures contained little, if any,
protein
2 (Fig.
2C). Shorter exposures confirmed that these particles
were also devoid
of the
1 protein (data not shown). Parallel
TGU gels which were
immunoprobed with the anti-
2 monoclonal antibody
7F4 (
93)
confirmed the presence of
2 in intact core samples
and the absence
of
2 in the spike-less particle fractions (data
not shown). No
fractions which contained structures deficient
for the
2 protein
were identified electron microscopically or
by electrophoretic protein
profile in the permissive cultures
of either T3D or
tsA279.
To measure the copy numbers of various
proteins present in the
core-like particles, laser densitometric
scans of fluorograms
representing multiple samples were analyzed.
The number of cysteines
and methionines contributed by each structural
protein to whole virions
and core particles was determined by
multiplying the copy number of the
protein in the structure by
the number of cysteines and methionines
present in one copy of
the relevant protein. The copy number of
residues contributed
by each protein was then standardized to the
proportion of residues
contributed by the major core protein
1
(Table
3). The signal
for
1 was
determined for each fraction and used to determine
the predicted signal
for the remaining structural proteins in
that fraction. The actual
signal for each protein was then expressed
as a proportion of the
predicted signal (Table
4).
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TABLE 3.
Relative copy numbers and cysteine and methionine content
of the structural proteins in reovirus virions and core particles
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TABLE 4.
Relative amounts of proteins present in permissively and
restrictively assembled tsA279 virions and
intermediate structuresa
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When these densitometric calculations were applied to T3D and
tsA279 virions which had been permissively assembled
(cultured
and assembled under permissive growth conditions), there was
no
significant difference between the wild type and the mutant.
Restrictively
assembled T3D and
tsA279 virions also
contained essentially the
same number of copies of all structural
proteins as permissively
assembled virions (Table
4). However, there
were significant
differences in the relative amounts of core proteins
2 and µ2
in the restrictive mutant core-like particle fractions;
these
structures contained approximately 2% of the expected amount of
2, representing less than two copies per particle (Table
4).
These
fractions also contained outer-capsid proteins µ1/µ1c and
3,
which we assume to have been contributed by the contaminating
outer-shell
structures.
Gel analyses also indicated the minor protein µ2 produced by the
mutant
tsA279 at restrictive temperatures was either absent
from the
2-deficient core particles or migrated differently from
µ2 produced at the permissive temperature (Fig.
2C, arrow). Laser
densitometric scans indicated there were approximately normal
amounts
of the minor core protein
3 but that there were about
5% the
expected levels of µ2 (less than one copy per particle)
in the
core-like particles produced by the mutant at the nonpermissive
temperature. Western blot evaluation of parallel TGU gels with
polyclonal anti-µ2 antiserum confirmed that the core-like particles
were deficient for this protein (Fig.
2D).
To determine whether the spike-deficient particles may represent normal
assembly intermediates or "dead-end" products, we
next determined
the capacity of restrictively assembled spike-less
core particles to
accumulate spikes after temperature downshift
experiments. After
temperature downshift from 40 to 32°C in the
presence of
cycloheximide, the distribution of particles produced
by the mutant was
changed, with the proportion of core-like particles
produced at a
restrictive temperature being reduced from over
22 to below 2%, and
the proportion of core particles which contained
2 spikes increased
from below 1 to approximately 20% (Table
5).
There was little difference between
the proportion of other species
of particles produced after temperature
downshift. The proportion
of various particles that accumulated in the
presence of cycloheximide
after 6 h in each of the cultures
maintained at a constant temperature
did not differ dramatically from
the proportion of particles produced
in comparable
non-cycloheximide-treated cultures (Tables
1 and
5).
| |
DISCUSSION |
A mutant L2 gene (2 protein) is associated with the accumulation
of spike-deficient core particles.
The assembly pathways of simple
helical plant viruses have been extrapolated from disassembly and
reassembly studies of tobacco mosaic virus (15, 17, 34, 92).
While it has been possible to perform disassembly studies with complete
reovirus virions (68) and to assemble capsids of this
(102) and related viruses from expressed proteins (45,
91), sequential disassembly and reassembly has been marginally
successful when applied to this (5) or other moderately
complicated animal virus systems. Bacteriophage conditionally lethal
mutants have been successfully used to derive assembly pathways of
structurally complicated bacteriophage (29, 30). That
success suggested that the Fields panel of reovirus conditionally
lethal ts mutants (19, 32, 79) (reviewed in reference 21) could be used to determine the
assembly pathways of a eucaryotic virus.
Direct particle counting of cytoplasmic extracts from restrictive
tsA279-infected cultures confirmed the presence of and
provided
further insight into the nature of the core-like particles
observed
by thin-section electron microscopy (
44). First,
the proportion
of core particles produced by the mutant under
restrictive conditions
was significantly greater than the proportion of
cores produced
under any of the permissive conditions (Table
1).
Second, permissively
assembled core particles observed in cultures of
both T3D and
tsA279 and restrictively assembled core
particles from T3D demonstrated
the
2 apical spike and contained
genomic material. Third, the
core particles produced at restrictive
temperature were deficient
for both the
2 and µ2 proteins and
contained reduced levels of
genomic material. Finally, when culture
temperatures were shifted
from 40 to 32°C during culture and
maintained in the presence
of cycloheximide to ensure the
unavailability of permissibly transcribed
viral protein, the proportion
of spike-defective core-like particles
declined, while the proportion
of particles demonstrating spikes
increased. This is the first report
that core-like particles deficient
for core proteins
2 and µ2
(Table
4) may accumulate in the assembly
pathway. Analysis of a panel
of T1L×
tsA279 intertypic reassortants
indicated the
production of the
2 spike-deficient core particles
was associated
with the mutant L2 gene segment (Table
2).
The apparent inability to isolate comparable spike-less particles from
wild-type infected cell extracts, coupled with the
ability to readily
disassemble virions and create spiked core
particles (
22,
28,
59,
70), has led to the assumption
that the core particles usually
seen in infected cells by thin-section
electron microscopy are complete
core particles. This premise
is also supported by the accumulation of
core particles which
contain the
2 pentameric spike in restrictive
cultures from representatives
of recombination groups B (
65)
and G (
23,
65,
88), which
code for the core spike protein
2 and major outer capsid protein
3, respectively (
21,
67). Spike-less core particles have
been prepared in laboratory
settings from top component and whole
virus (
90,
96,
106).
The core particles generated by White
and Zweerink demonstrated apical
cavities after the removal of
the
2 pentameric structure by high-pH
treatment (
96), as do
those produced by treatment at high
temperature (
106). The core
particles produced by
tsA279 also demonstrated such cavities (Fig.
1A and 2B2,
small arrow). Similarities in particle morphology
of the
spike-deficient core particle produced by
tsA279 and the
spike-deficient core particles produced in vitro (
96,
106),
coupled with the protein profiles of previously prepared spikeless
cores (
96,
106), suggested that all other core proteins were
present. The unexpected observation that the µ2 protein was also
not
present in the core-like particles identified in this study
provides
further insight into the assembly pathway of
reovirus.
Assembly pathway of reovirus.
The accumulation of 2
spike-deficient cores in the restrictively grown tsA279
cultures implies that the assembly pathway is blocked as a result of
the altered 2 product. The identification of this structure in vivo
suggests that initiation of capsid assembly may involve interactions
between the core proteins 1, 3, and 2 to form a "primary
core particle" (Fig. 3B). This notion
is supported by coexpression studies that show that 1 and 2
together, but not alone, are minimally required for core capsid
assembly, and that coexpression of these two major proteins with any of the other core proteins resulted in incorporation of each protein into
a genome-deficient core-like particle (102). The inclusion of 3 in the primary core identified in the current study implies that this minor protein is incorporated into the structure at an early
point in assembly, while the concomitant lack of both 2 and µ2 in
the primary core suggests that neither protein is necessary for the
initiation of assembly.
View larger version (21K):
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|
FIG. 3.
The proposed model for the assembly of reovirus.
(A) Assembly of the dodecahedral base unit. Five dimers of the core
protein 1, ten units of 2, and one copy of 3 associate to form
the dodecahedral base structure. (B) Assembly of the primary core
particle. Twelve dodecahedral base units assemble to form the primary
capsid structure. (C) Assembly of the intermediate particle. Two copies
of µ2 associate with each apex of the primary core particles
immediately prior to or collateral with assembly of the twelve 2
pentameric spikes. The trimeric 1 attachment protein is most likely
coassembled with the 2 spike, with the amino termini of the 1
fibers interacting with the carboxy termini of the 2 spike proteins.
(D) The outer capsid proteins 3 and µ1 associate with each other
and then condense around the 2 pentameric spike to form the complete
virion.
|
|
Previous reports have identified l3, m3, s3, and s4 as the first viral
mRNAs present in reovirus-infected cultures (
52).
The
encoded proteins for three of these
µNS,
NS, and
3
are the
first viral proteins to associate with viral single-stranded RNA
(ssRNA) to form ssRNA-containing complexes (ssRCCs) in
restrictive
cultures of T3D and the dsRNA-negative
ts
mutants
tsC447,
tsD357,
and
tsE320,
which are defective for
2,
3, and
NS, respectively
(
3). Small quantities of
1 (encoded by L3) and
2 were
the
next constituents identified in the ssRCC complexes. The major
core
protein
2 was not identified in these complexes from T3D
or in the
different
ts mutants examined. These complexes were
not
examined for the presence of
3 nor µ2 (
3). The next
structures
identified in that study were dsRNA-containing complexes
(dsRCCs),
which were comprised of the proteins µNS,
NS,
3, and
2. Coprecipitation
studies with antibodies directed against the
major core proteins
2 and
1 also precipitated dsRCCs from
restrictive infections
with T3D (
3). The results from that
study are consistent with
previous reports of an ssRNA transcriptase
particle comprised
of the proteins
1,
2, µNS, and reduced
quantities of
2 (
66,
107,
108). Review of the data in
those reports does not preclude
the presence of µ2 or
3, which
comigrate with other proteins
in the gel system used. While the primary
core particle identified
in the present study differs from the dsRCCs
reported with other
ts mutants (
3) in that it
lacks the nonstructural proteins,
it bears some similarity to the
previously identified transcriptase
particle (
66). This
40-nm particle has been reported to contain
all core structural
proteins plus the nonstructural protein µ0
(now called µNS)
(
66).
These studies are consistent with a dodecahedral assembly model based
upon a five-sided apical complex which contains 1 central
copy of the
minor core protein
3, 5 dimers of the major core
protein
1 and 10 copies of
2, in which
3 interacts with the
1 amino termini
(
27,
68) to form the dodecahedral base unit
(Fig.
3A) from
which the primary core particle is subsequently
formed. Nonstructural
proteins would be excluded as base units
assemble, resulting in the
primary core particle identified in
the current studies (Fig.
3B).
Dodecahedral structures constructed
from penton base units have been
identified in adenovirus type
3 infections (
73).
Coexpression of the adenovirus 3 penton base
and fiber proteins also
produce dodecahedral particles comprised
of 12 penton and fiber base
units (
31,
83). Since each multimeric
penton base unit is
comprised of 5 penton protein monomers, these
particles are made up of
60 penton monomers, plus 12 trimeric
apical fibers, making them
analogous to miniature capsids (
83),
with a triangulation
lattice T=1 (
16,
50). Dodecahedral models
have also been
proposed for the assembly of small capsids with
triangulation lattice
numbers equal to 1, such as the picornaviruses
(reviewed in reference
2), bacteriophage
6 (
14), and the
yeast virus L-A (
18). Bacteriophage
6 and yeast virus L-A
are
other dsRNA viruses constructed from 120 copies of a major capsid
protein (
14,
18). The core particle of reovirus shares
similar
copy numbers of major core proteins and an apparent
triangulation
lattice value of 1 (
20,
68).
Subsequent assembly of the complete core particle is proposed to
involve association of the minor core protein µ2 with the
apices of
the primary core particle either immediately prior to
or collaterally
with
2 (Fig.
3C). Electron cryomicroscope reconstructions
of T1L
top-component cores further suggest that
2 and µ2 interact
with
3 at the apices of the primary core particle (
27). While
the precise location of the two minor proteins in the apical complex
have not been established, all three proteins have been associated
with
functions of the RNA-dependent RNA polymerase either separately
or in
different combinations (
72,
87,
106). The finding that
expressed
2 exists as a monomer (
61) suggests that
2
condenses
into pentamers during association with the primary core
particle.
Mutation(s) in the
2 protein could affect the appearance
of the
primary core particle in two ways: by either preventing
pentamerization
of the protein or blocking the interactions of the
protein and/or
its pentamer with the core to produce the complete core
particle.
In addition, the accumulation of outer-shell structures
comprised
of proteins µ1 and
3, but clearly lacking proteins
2
and
1
(Fig.
2), suggests that the mutant
2 protein or its
pentamer
is not able to interact with the outer capsid proteins when
assembled
at restrictive temperatures, in contrast to wild-type
2
protein,
which can interact with the outer capsid proteins (
28,
62).
The observation that temperature shiftdown experiments
(Table
5) lead to condensation of spikes onto the previously spike-less
restrictive particles is consistent with the proposed condensation
of
these proteins to form the complete core particle. In the proposed
dodecahedral assembly path for the reovirus primary core particle,
the
replication of the minus sense viral RNA to complete the genome
at this
stage could also result in a maturational event similar
to that
reported for
6 (
14), where the dodecahedral structure
of
the base unit would be displaced radially from the pentameric
faces
during assembly to provide a triangulation lattice T=1 icosahedral
structure.
The precise nature of the interactions of
1 with the
2 spike is
not clear (
27,
68). This protein was not examined during
previous coexpression and assembly studies. However, incorporation
of
the trimeric
1 attachment fiber with the
2 pentamer most
likely
occurs at the time the
2 spike condenses and associates
with the
primary core, with the amino terminus of the
1 fibers
interacting
with the carboxy terminus of the
2 proteins (
56,
60),
thereby forming an intermediate
particle.
The final stage of reovirus capsid assembly involves condensation of
the outer capsid around the core lattice (Fig.
3D). Previous
reports
had identified the presence of outer-shell structures
(L top component)
in restrictive cultures of
tsC447 that contained
2, µ1,
1, and
3 (
62). Electron cryomicroscope examination
of
whole virions and ISVPs have demonstrated interactions between
2 and
both µ1 and
3. The same study showed little contact between
µ1
and
3 and the core shell (
28). Those reports suggested
that
the
2 complex may serve as a nucleation site for condensation
of µ1 into the outer capsid lattice (
28). However, the
presence
of µ1/
3 outer-shell complexes that clearly lack both
2
and
1
in
tsA279 restrictive cultures indicates that these
proteins are
not required for condensation of an outer-capsid-like
structure.
Coprecipitation studies indicated that the outer capsid
proteins
µ1 and
3 form complexes in permissive cultures of T3D
(
55)
and
tsG453 (
88). The inability to
coprecipitate these outer
capsid proteins in restrictive cultures of
tsG453, combined with
the observation that the restrictive
product of
tsG453 infections
is a core-like particle
(
23,
88) rather than an intermediate
subviral particle,
indicate that the outer capsid proteins µ1
and
3 must interact
with each other before they can condense
to form the outer capsid
(
88).
In conclusion, investigations into the intermediate assembly structures
that accumulate in restrictive cultures of
tsA279,
a member
of the Fields panel of reovirus
ts mutants, have identified
a novel primary core capsid comprised of
1,
3, and
2. This
structure may represent the transitional stage between the
RNA-containing
complexes described by Antczak and Joklik (
3)
and the
2 spike-containing
core structure identified in restrictive
cultures of
tsB352 (
65)
and
tsG453
(
23,
65,
88). We propose that these complexes
combine
through a dodecahedral mechanism of assembly to form the
primary core
capsid as the first capsid structure in the reovirus
assembly pathway.
The remaining core proteins
2 and µ2 then combine
with the primary
core capsid, along with the
1 trimeric attachment
complex, to form
the more complete intermediate particle. Finally,
the outer capsid
proteins µ1 and
3 interact and coprecipitate
around this
intermediate particle to form the complete reovirus
capsid.
| |
ACKNOWLEDGMENTS |
We thank R. C. Brunham for his ongoing support of this work;
C. Power and J. N. Simonsen for critical review of this study; L. Petrycky-Cox for stock cell maintenance; and G. Dow, J. D. Berry,
M. Makarovsky, N. Keirstead, M. Patrick, G. Wong, and P. Yin for
helpful discussion.
This research was supported by grant MT-11630 from the Medical Research
Council of Canada.
| |
ADDENDUM IN PROOF |
Recent crystallography data of the bluetongue virus core structure
by Grimes et al. (J. M. Grimes, J. N. Burroughs, P. Gouet, J. M. Diprose, R. Malby, S. Zientara, P. P. C. Mertens, and D. I. Stuart,
Nature 395:470-478, 1998) suggest that the VP3 scaffold
assembles by a dodecahedral assembly mechanism.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada. Phone: (204) 789-3309. Fax: (204)
789-3926. E-mail: kcoombs{at}ms.umanitoba.ca.
| |
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Abstract
Introduction
