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Journal of Virology, April 2000, p. 3525-3536, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of Vaccinia Virus Intracellular Cores:
Implications for Viral Uncoating and Core Structure
Ketil
Pedersen,1,2
Eric J.
Snijder,3
Sibylle
Schleich,1
Norbert
Roos,2
Gareth
Griffiths,1 and
Jacomine Krijnse
Locker1,*
European Molecular Biology Laboratory, Cell
Biology Programme, 69117 Heidelberg, Germany1;
EM Unit for Biological Sciences, University of Oslo, Blindern,
N-0316 Oslo, Norway2; and Department of
Virology, Institute of Medical Microbiology, Leiden University,
2300 RC Leiden, The Netherlands3
Received 29 November 1999/Accepted 13 January 2000
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ABSTRACT |
The entry of vaccinia virus (VV) into the host cell results in the
delivery of the double-stranded DNA genome-containing core into the
cytoplasm. The core is disassembled, releasing the viral DNA in order
to initiate VV cytoplasmic transcription and DNA replication. Core
disassembly can be prevented using the VV early transcription
inhibitor actinomycin D (actD), since early VV protein synthesis is
required for core uncoating. In this study, VV intracellular cores were
accumulated in the presence of actD and isolated from infected cells.
The content of these cores was analyzed by negative staining EM and by
Western blotting using a collection of antibodies to VV core and
membrane proteins. By Western blot analyses, intracellular actD cores,
as well as cores prepared by NP-40-dithiothreitol treatment of
purified virions (NP-40/DTT cores), contained the core proteins p25
(encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small
amounts of the VV membrane proteins p32 (D8L) and p35
(H3L). While NP-40/DTT cores contained the major putative DNA-binding
protein p11 (F17R), actD cores entirely lacked this protein. Labeled
cryosections of cells infected for different periods of time in the
presence or absence of actD were subsequently used to follow the fate
of VV core proteins by EM. These EM images confirmed that p11 was lost
at the plasma membrane upon core penetration. The cores that
accumulated in the presence of actD were labeled with antibodies to 4a,
p39, p25, and DNA at all times examined. In the absence of the drug the
cores gradually lost their electron-dense inner part, concomitant
with the loss of p25 and DNA labeling. The remaining core shell still
labeled with antibodies to p39 and 4a/4b, implying that these proteins
are part of this structure. These combined data are discussed with
respect to the structure of VV as well as core disassembly.
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INTRODUCTION |
Vaccinia virus (VV), the prototype
of the poxvirus family, contains a double-stranded DNA genome of 190 kb
encoding over 200 proteins, of which about 100 make up the brick-shaped
particle (10). Poxviruses are unique in that both
transcription and DNA replication occur in the host cell cytoplasm
since the virus encodes for its own enzymes required for these two
processes (26).
Assembly of VV starts with the formation at 5 to 6 h postinfection
(p.i.) of typical crescent-shaped membranes modified by viral membrane
proteins. We and others have shown that these membranes are derived
from the smooth endoplasmic reticulum (ER)/intermediate compartment and
are composed of two tightly apposed cisternal membranes (32,
39, 46; see reference 12 for a different interpretation). Consistently, we have shown that a number of viral
membrane proteins are cotranslationally inserted into the rough ER and
transported to and retained in the intermediate compartment in infected
cells (21, 34). These crescents then form the completely
spherical immature viruses (IVs) composed of the two cisternal
membranes and an electron-dense central part that contains the viral
core proteins (5, 9, 38, 42). When these spherical IVs take
up the DNA, they undergo a complex series of morphological changes
resulting in the brick-shaped intracellular mature virus (IMV), the
first of the two infectious forms of VV. Morphologically, this
transition results in the formation of the (quasi-brick-shaped) core
structure, while at the biochemical level it coincides with the
cleavage of at least three of the major core proteins (19, 20, 27,
43, 44, 47).
The molecular details that underly the formation as well as the
structure of this quasi-brick-shaped core are poorly understood. Recombinant viruses have been generated in which the expression of
specific core proteins is regulated by inducible promoters (29,
45, 48). The information obtained from such genetic studies has,
however, been limited since in the absence of specific core proteins,
assembly was either blocked at the IV stage (29, 48) or
generated IMVs that appeared morphologically indistinguishable from
wild-type virus (45).
Other approaches to study the core structure have included a systematic
disassembly of intact, purified IMV. Treatment of IMVs with a mixture
of the detergent NP-40 and a reducing agent has been used to chemically
prepare VV cores (8, 37). In fact, VV proteins are
classically defined as core proteins when they remain associated with
pelletable particles after treatment of the IMV with detergent and
reducing agents. The major components of such cores are the gene
products of A10L (4a), A3L (4b), A4L (p39), L4R (p25), and F17R (p11)
as well as the viral genome and the enzymes required for early
transcription (15).
A final approach aimed at understanding the core structure has been to
study intermediates of uncoating upon VV infection. Although VV entry
is poorly understood (see Discussion), it is generally accepted that
its final result is the delivery of the viral core (seemingly
indistinguishable from chemically prepared cores) into the cytoplasm
(6). Studies by Sarov and Joklik (36) and by
Holowczak (13) have shown that several core uncoating intermediates, characterized by different sedimentation properties, different protein content, as well as different morphological appearances, can be distinguished in infected cells. However, these
studies were performed before the VV genome was sequenced, and many of
the proteins associated with these uncoating intermediates remain
therefore unidentified. Other studies have shown that when the
synthesis of VV early mRNAs and/or early proteins is blocked, a
specific uncoating intermediate accumulates in infected cells (13,
17, 35). In this intermediate the viral genome is still protected
from DNase digestion, suggesting that its release from the core
requires the synthesis of at least one early viral protein (17,
30).
In this study, we have analyzed cores isolated from infected cells in
the presence of actinomycin D (actD), an inhibitor of VV early
transcription, and compared them to cores that had been prepared by
treatment of the virus with NP-40 and dithiothreitol (NP-40/DTT cores).
Moreover, in a detailed time course we have analyzed the fate of
several typical core proteins in infected cells by electron microscopy
(EM). The combined results have implications not only for VV uncoating
but also for the structure of the IMV.
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MATERIALS AND METHODS |
Preparation of virus stocks.
HeLa cells (CCL2) were obtained
from the American Type Culture Collection and grown in Dulbecco's
modified Eagle medium (DMEM) containing penicillin and streptomycin and
5% heat-inactivated fetal calf serum (FCS). VV (Western Reserve
strain) was propagated in HeLa cells as described before
(15). The virus was semipurified by pelleting through a 36%
(wt/vol) sucrose cushion in a Beckman SW40-Ti rotor at 24,000 rpm and
4°C for 30 min. Virus pellets were resuspended in 10 mM Tris-HCl (pH
9.0), aliquoted, and stored at 
80°C. 35S-labeled IMV
was prepared as described by Krijnse Locker and Griffiths
(22). To prepare virus whose DNA genome was labeled with
[3H]thymidine, cells were infected at a multiplicity of
infection (MOI) of 3 to 5. After 2 h, 0.5% FCS and 25 µCi of
[3H]thymidine (Amersham) per ml were added to the
inoculum, and the virus was isolated 3 days later. All virus stocks
were plaque titrated on HeLa cells and were typically between
108 and 5 × 108 PFU per ml. By measuring
the optical density at 260 nm (OD260) of a diluted virus
preparation, the amount of particles per milliliter was calculated
(15). By dividing this number by the PFU per milliliter as
measured by plaque assay, the particle/PFU ratio was determined as 50. The incorporated amount of radioactivity was determined by spotting an
aliquot of the virus preparation on filter paper, followed by
trichloroacetic acid precipitation and liquid scintillation counting.
Antisera.
The antibodies to p16 (A14L), p8 (A13L), p21
(A17L), p11 (F17R), and anticore have been described before (15,
22, 34). Antibodies generously provided by colleagues were as
follows: p14 (A27L [31]) and p39 (A4L
[24]) (from M. Esteban); p25 (L4R) and 4a/4b (A10L/A3L
[43, 44]) (from D. Hruby); and p32 (D8L
[28]) (from E. Niles). The antibody recognizing p35
(H3L [4]) was made using a peptide corresponding to
amino acids 31 to 43 of the H3L gene. Rabbits were immunized as
described by Cudmore et al. (5). The anti-DNA immunoglobulin
M (IgM) monoclonal antibody was from Boehringer GmbH (Mannheim, Germany).
Purification of viral and subviral structures from infected
cells.
HeLa cells were grown to 80% confluency in 6-cm-diameter
dishes (approximately 1.5 × 106 cells). The virus was
thawed and sonicated at 35 kHz for 1 min at room temperature in a water
bath sonicator. Usually, cells were infected with an MOI of between 50 and 100. Inocula were prepared in serum-free DMEM and were put on the
cells for various periods of time (between 0.5 and 3 h), depending
on the experiment. When actD (Sigma) was used, the drug was added
together with the virus. At 3 h p.i., the inoculum was removed,
and the cells were washed once with phosphate-buffered saline (PBS),
and DMEM containing 0.5% FCS and 3 µg of actD was added. To isolate
disassembled forms of VV, the cells were washed twice with PBS and put
on ice. Subsequently, they were scraped from the dish in PBS and
pelleted by low-speed centrifugation. Cell pellets were resuspended in
500 µl of 10 mM Tris-HCl (pH 9.0), and the cells were broken with a
Dounce homogenizer (10 to 15 strokes). After removal of the nuclear
fraction (by centrifugation in a Eppendorf microcentrifuge at 2,500 rpm for 5 min), the cytoplasmic fraction was directly loaded onto a linear
12 to 26% (wt/wt) sucrose gradient in 10 mM Tris-HCl (pH 9.0).
Directly prior to loading, samples were sonicated for 1 min at 35 kHz
in a water bath sonicator. The gradient was run in a Beckman SW40-Ti
rotor at 15,000 rpm and 4°C for 25 min. Gradients were pumped out
from the top using a peristaltic pump and fraction collector. The
volume of the gradient (14 ml) was divided into 23 to 25 equal
fractions. In some sedimentation analyses, NP-40/DTT cores were
included for comparison.
ELISA on gradient fractions using an anticore antiserum.
To
identify the position of the core peak in gradients loaded with
unlabeled virus preparations, we developed an enzyme-linked immunosorbent assay (ELISA) using the anticore antibody.
Twenty-five-microliter samples of gradient fractions were diluted twice
in 10 mM Tris-HCl (pH 9.0). An ELISA plate was coated with these
samples for 30 min at 37°C. After blocking with 0.5% gelatin in PBS,
a 1:1,000 dilution of the anticore antiserum was added to the wells.
After incubation with goat anti-rabbit coupled to horseradish
peroxidase (HRP) (Bio-Rad) as second antibody, the ELISA was developed
using a 3,3',5,5'-tetramethylbenzidine-HRP detection kit (Pierce, KMF Laborchemie Handels GmbH, St. Augustin, Germany) and enhanced chemiluminescence (ECL) according to the manufacturer's instructions, and the OD450 of the wells was determined in an ELISA plate reader.
Immunolabeling of particles from entry experiments and
preparation of cryosections.
The presence of various VV proteins
on the surface of viral or subviral structures from cell lysates or
gradient fractions was analyzed using a panel of VV antibodies. Samples
(5 to 10 µl) were put on carbon-coated copper grids and incubated for
15 min at room temperature. After blocking with 10% FCS in PBS, a standard immunogold labeling procedure (9) was carried out, followed by negative staining using 0.3% uranyl acetate or 2% ammonium molybdate. For cryosections, HeLa cells grown to 70% confluency in 6-cm-diameter dishes were infected at an MOI of 200 as
described elsewhere (22), using semipurified virus prepared as described above. The cells were fixed at the indicated times after
infection by adding an equal volume of fixative to the medium, and
cryosections were prepared and labeled essentially as described by van
der Meer et al. (41).
Western blot of particles from entry experiments.
Using a
Pierce bicinchoninic acid protein assay reagent, the protein
concentration of the particles, of purified IMV and of NP-40/DTT cores
was determined according to the instructions of the manufacturer. About
800 ng of protein was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% gel,
transferred to nitrocellulose, and detected as described elsewhere
(9).
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RESULTS |
Biochemical isolation of natural cores.
To study intracellular
cores, we designed a method to isolate them from infected cells. For
this, we prepared IMV in which either the DNA or proteins had been
labeled by using [3H]thymidine or
[35S]methionine/cysteine, respectively. The cells were
broken in a hypotonic buffer by using a Dounce homogenizer, and only
mild sonification was used to dissolve aggregates. Subsequently, the samples were loaded onto a sucrose gradient and analyzed by biochemical and EM methods.
Distributions of the various types of radiolabel in sucrose gradients
from a typical experiment are shown in Fig. 1A and
B. A large amount of the labeled virus
and/or disassembly intermediates remained at the top of the gradient,
possibly due in part to aggregation with cellular membranes. Although
intact virus particles and cores were seen by EM in fractions from the
central part of the gradient, the separation of these two structures
and the reproducibility of these experiments were not satisfactory.
Therefore, we decided to let disassembly intermediates accumulate by
using the drug actD, a transcription inhibitor which is known to block
VV uncoating (17, 25).
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FIG. 1.
Separation of viral and subviral components from
VV-infected cells on sucrose gradients. HeLa cells were infected at an
MOI of 50 to 100 with virus that had been metabolically prelabeled with
[3H]thymidine (A) or
[35S]methionine/cysteine (B) or was unlabeled (C). Cells
were either infected for 3 h in the absence of actD (AcD) or
infected in the presence of 3 µg of actD per ml (+AcD), after which
the infection was continued for 3 h in the presence of the drug.
Cell lysates were prepared as described in Materials and Methods and
separated on 12 to 26% (wt/vol) sucrose gradients. When radioactively
labeled virus was used (A and B), the location of viral and subviral
components in the gradient was determined by liquid scintillation
counting. When unlabeled virus was used (C), the location of the viral
cores in the gradient was determined by ELISA using the anticore
antibody (see Materials and Methods).
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Radiolabeled virus was added to HeLa cells in the presence or absence
of 3 µg of actD per ml. The cells infected in the absence
of the drug
were harvested at the end of the absorption period,
while those
infected in the presence of actD were incubated for
another 3 h in
the presence of the drug before being harvested
and analyzed. The use
of actD had a remarkable effect. When either
[
3H]thymidine- or
[
35S]methionine/cysteine-labeled virus was used, the
large amount
of label at the top of the gradient was no longer seen
(Fig.
1A
and B). Instead, two clear peaks were detected in the central
region of the gradients. Comparison with the sedimentation behavior
of
intact IMV and artificially prepared NP-40/DTT cores revealed
that the
material in these two peaks cosedimented with complete
virus particles
(around fraction 15) and core structures (around
fraction 10),
respectively (data not shown). The relatively long
incubation times in
the presence of actD (3 h of absorption and
3 h of infection) were
required to resolve the distinct (virus-
and core-containing) peaks.
Shorter or longer times of infection
in the absence of actD did not
affect the pattern (no detectable
peaks with the bulk of the material
remaining at the top of the
gradient) obtained after 3 h of
absorption, while shorter incubations
in the presence of actD did not
result in discrete core and virion
peaks (not
shown).
To confirm the nature of the peaks, we developed an ELISA using a
rabbit antiserum that had been raised against cores prepared
by
NP-40/DTT stripping of purified IMV. By Western blotting this
antibody
recognizes predominantly a 65-kDa band representing the
mature forms of
4a and 4b (A10L and A3L, respectively) and weakly
p11 (F17R) (Fig.
2), but no other viral proteins. For
simplicity,
this antibody will further be referred to as anticore
antibody.
Undenatured material from gradient fractions was used as
antigen
to coat the wells of an ELISA plate. As expected, the ELISA
produced
a strong signal only in the wells coated with material from
the
putative core peak (Fig.
1C), not in wells containing intact virus
in which the core antigens should not be accessible to the antiserum.
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FIG. 2.
Western blot showing that the anticore antibody
predominantly recognizes a 65-kDa band. Proteins of purified IMV were
separated by SDS-PAGE on a 10% gel and blotted onto nitrocellulose.
Core proteins were detected with antibodies to 4a (anti-A10L), 4b
(anti-A3L), p11 (anti-F17R), p25 (anti-L4R), and p39 (anti-A4L),
followed by anti-rabbit coupled to HRP and ECL. The two leftmost lanes
were probed with the anticore antibody. Upon short exposure (the
leftmost lane), the antibody detects a 65-kDa band only. Since the
cleaved forms of 4a and 4b cannot be resolved by SDS-PAGE, it is not
clear whether the antibody reacts with the mature form of 4a or 4b.
Upon longer exposure (second lane from the left), however, the antibody
does detect the uncleaved forms of 4a and 4b (of which a variable
fraction is always present in purified IMV preparations) as well as the
11-kDa F17R gene product.
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Characterization of core structures by immunolabeling.
Postnuclear supernatants (PNS) from infected cells treated with actD or
gradient fractions were analyzed by negative staining EM. In total PNS
we detected only two distinct structures, which appeared to represent
intact virus particles and cores (Fig.
3). Later examination of the two peaks in
the various gradients revealed some cross-contamination, but in general
the material in the virus and core peaks was relatively pure.
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FIG. 3.
Negative staining EM and immunolabeling of intact IMVs
and actD cores. (A) Labeling with anti-p14. The upper left shows a
heavily labeled IMV; in the lower right an unlabeled core is evident.
(B and C) labeling with anticore, showing an unlabeled IMV (B) and a
labeled actD core (C). Bars = 100 nm.
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To characterize the two structures in more detail, we used a set of
specific antisera against typical VV core and membrane
proteins to
label the surface of virus and cores, respectively,
and evaluated
labeling by negative staining EM. In this approach,
only antigens on
the surface of the particle have access to antibodies.
The results are
summarized in Table
1, while Fig.
3 shows
examples
of such labeling by negative staining EM. We detected intact
IMVs
heavily labeled for the surface protein p14 but no labeling with
this antibody on intracellular cores (Fig.
3A). Conversely, the
anticore antibody detected abundant labeling on the cores (Fig.
3C),
while the IMVs were unlabeled (Fig.
3B).
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TABLE 1.
Quantitation of immunolabeling with antibodies to VV core
and membrane proteins on purified IMVs and isolated
intracellular cores
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Intact IMVs labeled as expected; the surface labeled significantly with
antibodies to the membrane proteins p8 (A13L [
34])
and
p32 (D8L [
40]) and showed moderate levels of labeling
with
antibodies to the cleaved N terminus of p21 (A17L) (Table
1).
No
p16 (A14L) labeling was detected, consistent with previous
data showing
that this membrane protein is part of the inner of
the two VV membranes
(
32,
34). Poor labeling was also detected
with antibodies to
the IMV membrane protein p35 (H3L [
4]),
suggesting
that the epitope recognized by our antibody may not
be exposed on the
surface of the IMV. Finally, as expected, no
labeling was found on
intact IMVs with antibodies to core proteins
(Table
1). On the VV
cores, no labeling of the membrane proteins
p21, p8, and p16 was
detected. Surprisingly, low but significant
labeling was observed for
the putative membrane proteins p32 and
p35 (Table
1). The core proteins
p39 and 4a/4b (labeled with
anticore) were detected on the surface of
the cores, but no labeling
was seen with antibodies to p11 (F17R) and
p25 (L4R), suggesting
that these proteins might be buried inside the
core (see
below).
Characterization of viral and subviral structures by Western
blotting.
To investigate the protein content of the isolated core
fractions, we performed an SDS-PAGE analysis of the gradient fractions from Fig. 1B in which 35S-labeled virus was used. Again,
when analyzing the fractions of cells infected in the absence of actD,
we could detect no clear pattern. In the presence of actD, however, a
number of viral proteins were specifically concentrated in the core
peak compared to the virus-containing fractions (Fig.
4). Among those was the characteristic doublet of 65 kDa, most likely representing the major core proteins 4a
and 4b. Four other abundant proteins were clearly present in the core
peak fractions, one migrating at about 40 kDa (most likely p39) and
three with molecular masses between 15 and 28 kDa. Surprisingly, a
11-kDa band, that most likely corresponded to the major core protein
p11, was depleted from the core fraction while clearly present in the
virus-containing peak (Fig. 4; see below).
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FIG. 4.
35S-labeled pattern of subviral components
isolated from VV-infected cells separated on sucrose gradients.
Gradient fractions from Fig. 1B were separated by SDS-PAGE on a 15%
gel, which was then processed for autoradiography. The upper panel
represents gradient fractions from untreated (-ActD) cells, while the
lower panel is from treated (+ActD) cells. The top and bottom of the
gradients, as well as positions of the core and IMV peak, are
indicated. In each case, fractions with even and odd numbers were
pooled such that, e.g., fraction 1 denotes the combined radioactive
pattern of fractions 1 and 2. M, 14C-labeled markers of 14, 30, 45, and 69 kDa.
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To more firmly establish the protein content of the subviral
structures, we subjected them to Western blotting using a variety
of
antibodies to membrane and core proteins of the IMV. The protein
pattern was compared to those of pure IMV preparations and of
NP-40/DTT
cores. The latter cores behaved as expected, with a
total absence of
the typical membrane proteins p16 and p21, while
p39, 4a, and 4b as
well as p25 and p11 were clearly present (Fig.
5). The subviral particles isolated from
infected cells also contained
the typical core proteins 4a, 4b, p39,
and p25 but not p16 and
p21 (Fig.
5). Consistent with the SDS-PAGE
analysis, these structures
appeared to lack the major putative
DNA-binding protein p11 (
18).
Finally, in agreement with the
negative staining results, small
amounts of the IMV membrane proteins
p35 and p32 were found on
both the NP-40/DTT and actD cores (Fig.
5;
see Discussion).
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FIG. 5.
Detection by Western blotting of several VV core and
membrane proteins in purified IMV (Virus), NP-40/DTT cores, and actD
cores. In each lane of an SDS-15% polyacrylamide gel, 800 ng of
purified IMV, NP-40/DTT cores, or actD cores was loaded and after
electrophoresis blotted onto nitrocellulose. VV core and membrane
proteins were detected by Western blotting using ECL.
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Characterization of VV cores on cryosections.
The experiments
described above suggested that the cores that had accumulated in the
presence of actD were devoid of one of the most abundant core proteins.
This could be explained in three possible ways: p11 was (i) lost upon
VV entry, (ii) lost from the core intracellularly upon exposure to the
cytoplasm, or (iii) artifactually lost during the isolation procedure.
To address this issue, we immunolabeled cryosections prepared from
cells infected for different periods of time in either the presence or
absence of actD. Using antibodies to selected core proteins, labeling
was quantified on IMVs that had remained outside at the plasma membrane
and on intracellular cores. The data (Table
2) show several surprising results.
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TABLE 2.
Quantitation of immunolabeling on IMVs and intracellular
cores with antibodies to VV core proteins and to DNA
on cryosections
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First, we observed that, in agreement with the Western blot results,
the intracellular cores were entirely devoid of p11 labeling
(Fig.
6B
to D). Consistently, on extracellular
IMVs the labeling
for p11 appeared to be associated more with the viral
membranes
than with the core (Fig.
6B and C). Many images suggested
that
p11 separated from the core at the plasma membrane (Fig.
6B to
D),
leaving what appeared to be p11-positive viral membranes at
the cell
surface (Fig.
6D). The images shown in Fig.
6 also revealed
a number of
striking features of the entry process and the fate
of the viral core.
First, we found that many of the virions at
the plasma membrane looked
significantly different from normal
IMVs (Fig.
6C,
7B and
C, and 9A). Although the significance of
these morphological changes is not clear at present, by negative
staining EM of our purified virus preparations such particles
were
never observed, making it unlikely that they represented
damaged
virions contained in the inoculum (not shown). Second,
Fig.
6B and D
show what we believe to be putative entry intermediates,
supporting the
idea that the viral membranes as well as the nonmembrane
protein p11
remain outside while the core crosses the membrane.
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FIG. 6.
P11 is absent from intracellular cores. The
cryosections, all from HeLa cells infected for 30 min in the absence of
actD, show intracellular cores (large arrowheads) that are devoid of
p11 labeling; extracellular IMVs (I) are labeled. Note that the bulk of
the labeling on the IMVs is excluded from the interior, nucleoid
region. (B to D) Putative entry intermediates in which the cores are
separated from the outer membranes and p11 remains extracellularly. The
small arrowhead in panel B shows a possible connection between the
viral membranes and the core. (B and C) Membrane fragments (small
arrows) at the plasma membrane that label for p11; (B and D) such
fragments adjacent to an incoming core; (D) double labeled for p11
(10-nm gold; small arrowheads) and p16 (5-nm gold; small arrows),
showing a viral membrane fragment (star) labeled for both markers that
is outside the cell. Bars = 100 nm.
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FIG. 7.
Labeling of intracellular cores with the anticore
antibody. In all images, the intracellular cores are indicated by large
arrowheads. (A) From 60-min infection in the presence of actD, showing
labeling of two cores and four extracellular virions (I). The small
arrowhead shows an extracellular membrane fragment labeled for
anticore. Note that in the IMVs the labeling is excluded from the
central nucleoid region (arrow in panel A). (B and C) From 90-min
infection in the absence of actD. Small arrowheads show extracellular
virions that have dramatically changed their shape upon encountering
the plasma membrane. The intracellular cores are often close to the ER
(B, G, and I). (D and E) From 120- and 60-min infections, respectively,
in the presence of actD. (F through I) At 90 min postinfection in the
absence of the drug, showing the appearance of full (D and E) versus
electron-lucent (F to I) cores that all label with anticore. The images
in panels F and G also show what appears to be an opening in the core
shell. Bars = 100 nm.
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In cells treated with actD, the typical core proteins p39, p25, 4a/4b
(labeled using the anticore antibody) as well as the
DNA (labeled with
a monoclonal antibody to DNA) remained mostly
associated with
the cores (Table
2). In general anti-p39 and
anticore tended to label
primarily the outer rim of the cores
(Fig.
7A,
7E to H, and
8), while anti-DNA and p25 labeled mostly
the inner, central part (Fig.
9A to E and
10A to D). In some sections
this inner
part occasionally revealed what appeared to be the
viral nucleoid (Fig.
9B to E and 10A to D). Cores that accumulated
in the absence or
presence of actD often tended to be located
close to the ER or,
occasionally, the nuclear envelope (Fig.
7B,
7G,
7I,
8D,
9H, and
10D).
View larger version (119K):
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[in a new window]
|
FIG. 8.
Localization of p39 to intracellular cores in the
absence of actD. (A, C, and D) Cells infected for 120 min; (B) after a
60-minute infection. Panels A through D clearly show that p39 is mostly
localized to the surface of the outer shell of the cores. Panel D
clearly shows electron-dense material surrounding the core shell, which
we believe to be a spike-like structure (5, 33), with which
the p39 labeling is predominantly associated. (B) Rare image of a
putative core uncoating intermediate. The core in panel C appears to
have two compartments (arrowheads) separated by a piece of core shell,
giving the impression of this core being bilobed. The core in panel D
is close to the nuclear envelope (N, nucleus). Bars = 100 nm.
|
|
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[in this window]
[in a new window]
|
FIG. 9.
P25 labeling of intracellular cores and
IMVs. (A and E) From cells at 30 min after infection; (B) 60 min of
infection (all in the absence of actD) (C) 180 min in the presence of
actD; (D and F through I) from cells infected for 90 min in the absence
of the drug. (A through E) typical p25 localization to intracellular
cores, in which the inner electron-dense part is heavily labeled. In
the extracellular virions (I) in panels A and B, p25 also mostly labels
the most central nucleoid part. At later times of infection in the
absence of actD (F to I), the intracellular cores are devoid of p25
labeling, while this labeling is not lost in the presence of actD (C).
Some of the images in panels F through I show that while the innermost
part as well as the p25 labeling of these cores is lost, some
stain-excluding material that lines the core shell persists. The arrow
in panel F denotes a structure that seems to have left the adjacent
core (large arrowhead) and which is labeled for p25. The core in panel
H is close to ER membranes. Bars = 100 nm.
|
|
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[in this window]
[in a new window]
|
FIG. 10.
Cryosections labeled with anti-DNA. (A to C and E) From
cells infected for 30 min; (D and E) from cells at 90 min postinfection
(all in the absence of actD). Anti-DNA labels mostly the central part
of the cores (arrowheads) as well as of the IMV (I) in panel A. (D and
E) Cores devoid of labeling for anti-DNA. In panel C, the nucleus (N)
also labels for DNA. Bars = 100 nm.
|
|
In the absence of actD, labeling for p25, p39, 4a/4b (labeled with
anticore) and DNA was gradually lost from the core (Table
2). Instead,
at the later time points, we often observed labeling
for these antigens
in the cytoplasm, but we do not know whether
this labeling was
associated with any particular (cellular or
viral) structure (not
shown). Quantitation showed that the labeling
for DNA and p25 was lost
more rapidly from the core structure
than that for p39 and anticore.
These quantitative data appeared
consistent with our morphological
observations, whereas the actD
cores always retained a full appearance;
in the absence of the
drug, the cores appeared to gradually lose their
electron-dense,
nucleoid material. Such electron-lucent cores still
labeled for
anticore and p39 (Fig.
7F to I and 8) but were devoid of
p25 and
anti-DNA labeling (Fig.
9F to I, 10D, and 10E), suggesting that
under uncoating conditions these latter components were able to
dissociate from the remaining (p39 and anticore-positive) core
structure. These cores that had lost the DNA and p25 also revealed
several interesting characteristics. First, anticore labeled
mainly
the membrane-like shell of the core (Fig.
7E to H), while the
anti-p39 labeling tended to be some distance away from this structure,
being more associated with what we believe to be spike-like protrusions
of the core (
5,
33) (Fig.
8A, C, and D). Second, the empty
core shells often appeared to have acquired what looked like an
opening
on one side of the core (Fig.
7F to H and 9I). Finally,
although p25
and DNA had left these cores, some electron-dense
material lining the
inner part of the core shell was still apparent
(Fig.
7F and H, 8A and
D, 9F to I, and 10E show clear examples);
we did not analyze the
composition of this
material.
| |
DISCUSSION |
In this study we performed a detailed analysis of VV
intracellular cores. Since we were unable to repeat the results
obtained by Sarov and Joklik (35) and Holowczak
(13), in which distinct uncoating intermediates were
separated on sucrose gradients, we accumulated cores in the presence of
actD and compared these to chemically prepared cores. Uncoating beyond
the actD-sensitive step was nevertheless studied at the ultrastructural level.
The composition of intracellular cores.
Our data shed more
light on the structure of the intracellular VV core. VV cores with a
fuller appearance and empty cores seen in this study were described
before, obtained either by systematic chemically disintegration of the
IMVs (8, 14) or by separating uncoating intermediates upon
viral infection (13). Our combined results strongly suggest
that p39 and 4a/4b may be part of the outer shell of the core, while
p25 and the genome are located in the inner part. This was demonstrated
by negative staining EM, showing that p39 and 4a/4b were exposed on the
surface of the core, while p25 and DNA (not shown) were not. Moreover,
in cryosections, antibodies to p25 and DNA labeled mostly the central part, while p39 and 4a labeled the rims of the cores. In addition, when
the central part of the cores was lost, the electron-lucent cores were
devoid of p25 and DNA, but the remaining shell labeled with p39 and
4a/4b. Since p25 has been proposed to be a double-stranded and
single-stranded DNA (and RNA)-binding protein (2, 47), its location inside the core, close to DNA, appears logical.
We have shown before that p39 is located between the core and the viral
membranes and have suggested that the protein may
be part of the
spike-like structure on the core surface (
5,
33). The
present data clearly corroborate these previous findings,
showing that
p39 is also on the surface of intracellular cores,
while inspection of
some sections suggested their localization
to the core spikes. Although
we have not attempted to quantify
our Western blot data, this technique
suggested that the actD
cores contained less p39 than NP-40/DTT cores.
This may imply
that p39, like p11, is either partially lost at the
plasma membrane
or lost upon isolation of the actD core from infected
cells. This
latter explanation appears most plausible considering the
EM data;
no labeling for p39 was seen at the plasma membrane (except in
extracellular virions), while quantitation showed that the early
cores
(60 min after infection) labeled as much for anti-p39 as
did the
extracellular
IMVs.
We were unfortunately unable to detect any of the proteins involved in
early transcription either by Western blotting or by
EM, most likely
because their abundance was too low. Our data
therefore allows no
conclusions as to their distribution in the
VV
cores.
Core uncoating, VV entry, and role of p11.
The present data
show that the abundant core protein p11 did not enter with the core but
remained outside, apparently together with the viral membranes. From
its sequence p11 is not predicted to be a membrane protein, and
accordingly it does not behave like a membrane protein during assembly
(15). Its loss at the plasma membrane is thus most easily
explained by a tight interaction with viral membrane proteins. We
believe that the loss of p11 is compatible with our recent results on
VV entry (J. Krijnse Locker et al., submitted for publication and
unpublished data). The accepted view is that VV infects cells via a
fusion process at the plasma membrane (1, 3, 7). Although we
have also observed images similar to those obtained by Chang and Metz
(3) and Armstrong et al. (1) suggesting
continuity of the viral membranes with the cell surface, none of the
viral membrane proteins that we have analyzed are implanted in the
plasma membrane, arguing against fusion. Instead, while attached to the
plasma membrane, both the IMV and the extracellular enveloped viral
membranes appear to separate from the core. The core then penetrates
into the cytoplasm in a manner that is not understood, leaving viral
membrane remnants behind, attached to the plasma membrane but not
merged with it. Thus, while the loss of the core protein p11 from the
IMV (as well as the extracellular enveloped virion [not shown])
during entry appears incompatible with viral membrane fusion, its loss at the cell surface does fit into an elusive nonfusion process of entry.
While the mechanism of VV entry is still under investigation, our data
also help define the role of this putative DNA-binding
protein
(
18) in the VV life cycle. Although a functional p11
is
essential for IMV assembly (
48), we observed that the
protein
is not colocalized with the DNA-containing nucleoid (as is the
other DNA-binding protein p25) but is localized to the space between
the viral outer membrane and the core. Our data therefore raise
the
possibility that in addition to its role in assembly, p11
may perform
some function during the entry process as
well.
Does the intracellular core contain a membrane?
Cores
accumulated in actD-treated cells or prepared after NP-40/DTT treatment
contained small amounts of the IMV membrane proteins p32 and p35. This
raises the question of whether the core contains any lipid bilayer with
which these proteins could associate. On cryosections, the outer shell
of the core indeed very much appears as a membrane, in agreement with
observations made by Dales (6) and Holowczak
(13). Moreover, in a separate study on the structure of the
IMV, we have acquired extensive data strongly suggesting that the newly
assembled core is indeed surrounded by a membrane that is continuous
with the IMV outer cisternal membranes (G. Griffiths et al.,
unpublished data). To determine whether the intracellular core
contained lipids, we ran gradients of isolated actD cores comprising
lipids prelabeled with [3H]choline. The results were,
however, inconclusive (not shown). An option to consider is that the
putative core membrane is rapidly lost during entry. Indeed, studies by
Joklik (16) strongly suggested that viral phospholipids
dissociated immediately from the incoming particle. After penetration,
the organization of the proteins around the previously present bilayer
remains, giving the impression of a membrane, while trace amounts of
membrane proteins like p32 and p35 remain attached to it. Such a
mechanism is not unprecedented in virology; during rotavirus assembly,
the virion transiently acquires a membrane derived from the ER.
Subsequently, the membrane is lost, leaving the major membrane protein
VP7 behind in the lipid-free membrane structure that surrounds the
mature particle (reviewed in reference 11).
| |
ACKNOWLEDGMENTS |
E.J.S. was supported by an EMBL travel grant from the Netherlands
Organization for Scientific Research. Part of this work was supported
by an EU TMR grant (ERB4061PL970064) to G.G., K.P., and N.R. and by
Norwegian National Research Council grant 131033/300 to N.R. and K.P.
The following people are kindly acknowledged for providing us with the
antibodies: Mariano Esteban, Ed Niles, and Denis Hruby.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: European
Molecular Biology Laboratory, Cell Biology Programme, Meyerhofstrasse
1, 69117 Heidelberg, Germany. Phone: 49 6221 387244. Fax: 49 6221 387306. E-mail: Krijnse{at}EMBL-Heidelberg.DE.
| |
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Journal of Virology, April 2000, p. 3525-3536, Vol. 74, No. 8
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Abstract
Introduction
