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Journal of Virology, September 1998, p. 7551-7556, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Poliovirus Empty Capsid Specifically Recognizes the
Poliovirus Receptor and Undergoes Some, but Not All, of the
Transitions Associated with Cell Entry
Ravi
Basavappa,1,*
Alicia
Gómez-Yafal,1,
and
James M.
Hogle1,2
Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts
02115,1 and
Committee for Higher Degrees
in Biophysics, Harvard University, Cambridge, Massachusetts
021382
Received 9 March 1998/Accepted 27 May 1998
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ABSTRACT |
Experimental results presented here demonstrate that the poliovirus
empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with
mature virus for binding sites on cells. Hence, induced changes in the
structure and/or stability of the particle by RNA encapsidation and
virus maturation are not necessary for recognition by receptor. In
mature virus, heat-induced rearrangements mimic those induced by
receptor at physiological temperatures in several important respects,
namely, expulsion of VP4 and externalization of the VP1 N-terminal arm.
It is shown here that in the empty capsid the VP1 N-terminal arm is
externalized but the VP4 portion of VP0 is not. Thus, these two
hallmark rearrangements associated with cell entry can be uncoupled.
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TEXT |
That the first step in cell entry by
poliovirus is attachment to specific receptors on the cell surface has
been well established (16, 17, 31). However, the structural
features in the poliovirus particle necessary for receptor recognition
and the ensuing structural changes that allow the viral RNA to enter
the host cell cytoplasm are not well understood. According to a widely
accepted model, binding to receptor at physiological temperatures
induces a specific set of structural changes that give rise to the cell
entry intermediate termed the 135S particle. The changes that
characterize the conversion to the 135S particle, in addition to the
shift in sedimentation coefficient from 160S to 135S, include a
transition from the native (N) antigenic state to the heated (H)
antigenic state, an increase in hydrophobicity, and an enhancement in
protease sensitivity (6, 8, 11, 14, 25, 26, 28). Two
dramatic specific changes are also known to occur, namely,
externalization of the N-terminal arm of the VP1 polypeptide and
expulsion of the entire VP4 polypeptide (14, 36), both of
which are internal in the native poliovirus (15). Later in
infection the second type of particle, the 80S particle, accumulates,
with a coordinate loss of internalized 135S particles (14).
The 80S particle does not contain genomic RNA. The 80S particle may be
the empty protein shell which remains after the RNA is released from
the 135S particle.
The 135S particle has been considered a required cell entry
intermediate since it is the major type of internalized virus-derived particle observed soon after the start of infection (10,
24). However, cold-adapted mutants of poliovirus that do not
accumulate 135S particles have been reported recently (9).
It is not clear whether these mutants bypass the 135S stage entirely or
whether the kinetics of cell entry have changed such that the 135S
stage is no longer rate limiting. In the former case, the genuine cell entry intermediate may arise from subtle transitions akin to the breathing motions previously described (22), and the 135S
particle may represent an exaggerated form of these changes
(9). In either case, understanding the mechanism that leads
to the 135S particle should provide clues about the transitions
necessary for cell entry.
Examination of the crystal structure of the poliovirus native empty
capsid (3) indicates that the native empty capsid can be
used as a unique probe in investigating receptor recognition as well as
the mechanism of conversion to the 135S particle. The native empty
capsid is a putative assembly intermediate that contains the full
complement of capsid proteins but not the genomic RNA (20,
29). The native empty capsid is in an immature form, in that the
polypeptide VP0 has not been cleaved to form the VP4 and VP2
polypeptides present in the mature virus (18, 19). This
particle is considered to be in the native state because it has the
same N antigenic surface as mature virus.
Comparison of the crystal structures of the native empty capsid
(henceforth referred to simply as the empty capsid) and the mature
virus reveals that their outer surfaces and the bulk of their shells
are very similar (3). The primary difference is the presence
of three amino acid residues at the C terminus of VP3 in the empty
capsid (3). These are not observed in the mature virus
structure and may be cleaved off during virus maturation. In contrast,
the inner surfaces of the protein shells are radically different. One
set of major differences in the inner surface is due to the very
different disposition of the 10 residues on either side of the VP0
scissile bond in the empty capsid and the corresponding residues in the
mature virus. These segments must undergo large-scale rearrangements in
the transition from empty capsid to mature virus. The other set of
major differences arises from the disorder in the N-terminal arm of VP1
in the empty capsid. This arm is ordered in the mature virus and makes
numerous intraprotomer, intrapentamer, and interpentamer contacts. The
absence of these contacts in the empty capsid may explain, at least
partially, the empty capsid's greater lability. Here, we exploited the
similarities and differences between the empty capsid and the mature
virus to arrive at a better understanding of the structural
requirements for receptor recognition and the subsequent conformational
changes.
Preparation of virions and empty capsids.
HeLa cells in
suspension culture were maintained in Joklik's modified minimal
essential medium supplemented with 10% bovine calf serum, 0.3 g
of glutamine per liter, 0.5 g of pluronic acid per liter, 3.5 g of glucose per liter, and nonessential amino acids (Gibco). L cells
in monolayers were maintained in Eagle's minimal essential medium
containing Earle's salt solutions (BME) supplemented with 10% fetal
bovine serum. Both mature virus and empty capsid particles were
prepared from HeLa cells infected with the Mahoney strain of type 1 poliovirus.
Unlabeled mature virus particles were propagated in suspension culture
as described elsewhere (22) except that after attachment the
cells were resuspended in the supplemented medium indicated above.
Labeled empty capsids were grown by the infection procedure described
previously (3). Labeled mature virus particles were grown by
a procedure similar to that for the empty capsids except that
guanidine-HCl was not added. Labeled mature virus and labeled empty
capsids for the cell binding studies were prepared from a single
radiolabeled infected culture which was divided into equal volumes
3.5 h after the start of infection. Guanidine-HCl was added to a
concentration of 0.20 mM to the volume to be used for empty capsid
preparation. Guanidine-HCl inhibits poliovirus RNA polymerase and thus
favors accumulation of empty capsids. Mature virus particles were
purified by isopycnic centrifugation in a CsCl gradient, as described
previously (1). Empty capsid particles were purified by
isopycnic centrifugation in a Nycodenz gradient followed by rate zonal
centrifugation in a 15 to 30% sucrose gradient (3).
Particle protein concentration was quantitated by bicinchoninic acid
protein assay (Pierce) and Bradford protein assay (Bio-Rad). Mature
virus concentration was also determined by absorbance at 260 nm and an
extinction coefficient of 7.7 ml · mg
1 · cm1.
Empty capsid attachment to cells in a receptor-dependent
fashion.
The similar exterior surfaces and dissimilar interior
surfaces of the empty capsids and the mature virus prompted us to test whether the empty capsid possesses the structural features necessary for specific attachment to cells. These tests consisted of (i) saturation in binding to cells, (ii) differential binding to cells with
and without receptor, and (iii) competition with mature poliovirus for
receptor binding sites. In all of these experiments, the behavior of
the empty capsid was compared to that of mature virus.
Cell attachment was assayed by adding varying concentrations of
radiolabeled particles (empty capsid or virion) to 1 ml of
washed cells
at a density of 1.3 × 10
7 cells in phosphate-buffered
saline (PBS) and incubating for 30
min at room temperature with
constant gentle mixing. After incubation,
the cells were pelleted by
centrifugation at 16,000 ×
g for 5
min in a
microcentrifuge. The supernatant and a 200-µl wash of
the cells with
PBS were combined and counted for radiolabel. The
washed cells were
resuspended in 600 µl of PBS and transferred
to scintillation vials,
along with 600-µl washes of the tubes,
and counted for radiolabel.
The binding curves from these experiments (Fig.
1A) indicate that the empty capsid is
able to bind to HeLa cells and does
so in a manner very similar to that
of the mature virus. In both
cases, at low concentrations of input
particles the binding curve
possesses a saturable component. However,
at higher concentrations
of input particles, the binding does not
plateau. The presence
of only a partial saturability in the binding of
poliovirus to
susceptible cells has been reported previously with
poliovirus
(
23). The lower-affinity mode may represent
nonspecific, adventitious
binding or weak binding to an as yet
unidentified class of cell
surface molecules. The level of binding
observed with the empty
capsid sample cannot be due to contamination of
the sample with
mature poliovirus. The purification protocol used for
empty capsid
preparation yields samples that, when analyzed by sodium
dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis, have
undetectable
levels of VP2 and therefore negligible amounts of mature
virus.
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FIG. 1.
Binding of empty capsids and mature virions to HeLa
cells. Various amounts of labeled particles (empty capsids or mature
virus) were added to a constant number of HeLa cells in suspension.
After incubation, the cells were pelleted and the radioactivity
copelleting with the cells was measured. (A) Saturation binding curve.
(B) Scatchard plot of the data in panel A. As a rough approximation,
for each of the binding curves two lines have been fitted by the linear
least-squares method. The solid and broken lines are the best-fit lines
for the empty capsid and mature virus, respectively. The heavy lines
represent a high-affinity mode of binding, whereas the light lines
represent a low-affinity mode of binding.
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The two modes of binding are even more evident when the data are
replotted in the Scatchard format (Fig.
1B). At lower concentrations
of
particles, empty capsids and mature virions bind cells with
similar
high affinities. At higher particle concentrations, a
lower-affinity
mode of binding predominates. It is difficult to
determine the number
of high-affinity binding sites based on conventional
Scatchard plot
analysis (
21). A rough estimate is 5,000 to 6,000
binding
sites per cell. Since the cross-sectional area of 6,000
capsids is very
much less than the surface area of a HeLa cell,
the saturability of
this binding is not due simply to blanketing
of the cell surface with
the particles. This is in approximate
agreement with the estimated
value of 3,000 binding sites per
HeLa cells (
27).
Preferential binding to poliovirus-susceptible cells.
Whether
the empty capsid binds preferentially to cells containing poliovirus
receptor was determined by performing attachment assays with HeLa cells
(which possess receptor) and L cells (which do not possess receptor
[30]). In the attachment assay, the number of
particles added was approximately that needed to saturate the
high-affinity HeLa cell binding sites. This comparison reveals that the
empty capsid displays the same discrimination as mature virus in
binding to cells with or without poliovirus receptor (Table
1).
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TABLE 1.
Discrimination in binding of empty capsid and poliovirus
to cells with poliovirus receptor (HeLa cells) and cells without
poliovirus receptor (L cells)
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Competition with mature virus for binding sites.
In
experiments to determine if empty capsids and mature virions compete
for the same cell binding sites, a constant amount of radiolabeled
empty capsids and varying amounts of unlabeled mature virions were
added in a cell binding assay at a concentration of radiolabeled
particles that would result in the saturation of high-affinity binding
sites. Attachment was assayed as in the binding curve experiments. As a
control, unlabeled mature virus was allowed to compete with labeled
mature virus. The results of these experiments show that unlabeled
mature virus and empty capsid compete for the same binding sites on the
cell surface (Fig. 2). Moreover, the
empty capsid competes as efficiently with mature virus as mature virus
competes with itself. This provides compelling evidence that the empty
capsid is able to bind to the poliovirus receptor and that it does so
with an affinity similar to that of mature poliovirus.
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FIG. 2.
Competition for cell surface virus receptors by
radioactively labeled particles (empty capsids or mature virions) and
unlabeled mature virions. Competition for cell-surface poliovirus
receptor was assayed by determining the amount of labeled particle
attaching to HeLa cells as the concentration of competing unlabeled
virus was increased. The effect of increasing amounts of unlabeled
virus is presented by plotting the amount of label copelleting with the
cells (normalized to the value obtained with no unlabeled virus added)
versus the amount of competing unlabeled virus added.
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The above set of experiments demonstrates the ability of empty capsids
to attach to cells in a receptor-specific manner. These
results
indicate that the presence of the three additional amino
acid residues
at the C terminus of VP3 (which occur in or near
the proposed receptor
binding site [
32,
35]) does not interfere
with
binding. Moreover, these results demonstrate that the encapsidation
of
RNA, the cleavage of VP0, and the consequent reorganization
of the
inner network and increase in stability of the mature particle
are not
required either to potentiate formation of the virus-receptor
complex
or to stabilize the complex once formed. Whether or not
the empty
capsid would be internalized by the cell is an intriguing
question.
However, the appropriate experiment would be quite difficult
to perform
since the empty capsid is very heat labile and would
undergo conversion
to a nonnative form under the conditions necessary
for poliovirus
internalization.
Structural changes in the empty capsid that mimic those in the
mature virion.
Next, we determined whether the empty capsid is
able to undergo the same type of structural rearrangements which
transform the mature virus to the 135S or 80S particle. In these
transitions, the N-terminal arm of VP1 is externalized and the entire
VP4 polypeptide is expelled (14). Given that in the empty
capsid the VP1 N-terminal arm is disordered (but on the inside surface
of the protein shell) and VP4 is still covalently attached to VP2, we
did not know what the fate of these segments would be during the
conversion of the empty capsid to an H antigenic form. The
accessibility of these two segments in the heated empty capsid
particles was assayed by protease sensitivity and
immunoprecipitability. The assays regarding the VP1 N terminus are
based on previous demonstrations that the native-to-135S particle
conversion exposes a V8 protease-sensitive site at or near residue 31 of VP1 and renders the particle immunoprecipitable by antibodies
against peptides corresponding to the N-terminal region of VP1
(14). The assay for exposure of the VP4 portion of VP0 is
based on demonstrations that the breathing motions of the mature virus
at physiological temperatures expose VP4 and allow anti-VP4 antibody to
immunoprecipitate the particle (22).
The 135S and 80S particles can be prepared in vitro by heating the
particles under the appropriate conditions. The 135S particles
were
prepared by heating purified mature virus in 20 mM Tris-2
mM
CaCl
2 for 3 min at 50°C, conditions that result in the
virtually
complete conversion of native virion to 135S particle
(
7,
37).
The 80S particles were prepared by heating purified
mature virus
at 56°C for 10 min. These conditions produce virtually
complete
conversion of native particle to 80S particle (
4,
14). Native
empty capsids were converted to an H antigenic form
by heating
at 40°C for 1 h. Such incubation completely converts
the empty
capsid to the H antigenic state, as is evidenced by the
particle's
loss of alkaline dissociability into pentamers (
2,
34).
Sensitivity to V8 protease activity.
Experiments with V8
protease (which cleaves preferentially at Asp and Glu residues) have
demonstrated that the VP1 capsid protein is resistant to cleavage when
in the mature virus form but is susceptible to cleavage when in the
135S or 80S form (14). The sensitivities to V8 protease of
the heated and unheated empty capsid were compared to those of native
and heated (80S) virus to determine if a similar transition in the VP1
N-terminal arm occurs in the empty capsid upon heating.
The V8 protease (Boehringer Mannheim) has optimum activity at pH 7.8. Since the native empty capsids are unstable at alkaline
pH, a
suboptimum pH of 7.5 (in PBS) was used for all digests.
The amount of
V8 and length of digestion time were adjusted to
provide a clear signal
in the control digest of 80S particles.
Approximately 5 µg of V8
protease was added to each 10 µg of particle
protein and incubated
for 2 h at room temperature.
Cleavage of VP1 by V8 was assayed by Western blot analysis with
antibodies anti-pep1 and anti-pep9, which were raised against
synthetic
peptides corresponding to residues 24 to 40 and 270
to 287 of VP1,
respectively (
5). Samples were electrophoresed
in an
SDS-12.5% polyacrylamide gel with 2% cross-linking, and
the
polypeptides were transferred to nitrocellulose membranes
with the
Phast transfer system (Pharmacia). The membranes were
blocked by
incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl,
0.05% Tween
20) and 3% dry milk (Carnation) for 30 min at room
temperature. This
was followed by incubation with antiserum (1:2,000
dilution in TBST) at
room temperature for 30 min. Anti-rabbit
immunoglobulin G-alkaline
phosphatase conjugate (Vector Laboratories)
(1:5,000 dilution in TBST)
was the secondary antibody. The incubation
was at room temperature for
30 min. The bands were visualized
with BCIP
(5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium
(Promega)
according to the manufacturer's directions.
The results of the V8 sensitivity experiments demonstrate that upon
heating of the empty capsid, the N-terminal arm of VP1
becomes
susceptible to cleavage by V8 protease (Fig.
3) and thus
must be externalized.
Moreover, the extent to which the VP1 N-terminal
arm is externalized is
the same in both the empty capsid and the
mature virus, since in both
cases Western blot analysis with anti-pep9
(recognizing residues 270 to
287 of VP1) yields the same cleavage
pattern as with anti-pep1 (which
recognizes residues 24 to 40
of VP1, a sequence that spans the V8
cleavage site).
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FIG. 3.
V8 protease sensitivity of heated and unheated empty
capsids, native virus, and 80S particles. Native virus, 80S particles,
unheated empty capsids, and heated empty capsids were treated with V8
protease. The capsid proteins were separated by SDS-polyacrylamide gel
electrophoresis. Fragments containing the antigenic sequence of
interest were visualized specifically by immunoblotting and staining
with the procedures outlined in the text. As controls, particles not
treated with V8 protease were analyzed in parallel. (A) Western blot
analysis with polyclonal antibodies binding the pep1 region of VP1
(residues 24 to 40). (B) Western blot analysis with polyclonal
antibodies binding the pep9 region of VP1 (residues 270 to 287). PV,
poliovirus; EC, empty capsid.
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Immunoprecipitation of unheated and heated empty capsids.
The
conformation of the exposed VP1 arm and the fate of the VP4 portion of
VP0 in heated empty capsids were assayed by immunoprecipitation with
polyclonal anti-VP4 antibody raised against synthetic VP4 peptide
(22); polyclonal anti-pep0 and anti-pep1 antibodies raised
against synthetic peptides corresponding to residues 7 to 24 and 24 to
40 of VP1, respectively (5); and monoclonal anti-pep1
antibody. The monoclonal anti-pep1 antibody has been shown previously
to be more reactive with the 135S particle than with the 80S particle
(14). This is due presumably to minor differences in the
exposure or conformation of the amino terminus of VP1. Antibody binding
was quantitated by incubation at room temperature for 60 min of
[3H]leucine-labeled particles (~10,000 cpm) with serial
fivefold dilutions of antisera in PBS-0.1% egg albumin (PBSeA). Then,
50 µl of a 10% solution of protein A-Sepharose (Sigma) in PBSeA and 750 µl of PBSeA-0.05% Nonidet P-40 were added to the samples. The
samples then were incubated for 120 min at 4°C with continuous gentle
mixing. The immunocomplexes were collected by centrifugation at
16,000 × g for 10 min. The radioactivity remaining in
the supernatant was removed and counted for radiolabel. The bound
radioactivity was released by boiling the pellets in PBS-2% SDS-2%
-mercaptoethanol and then counted together with a 200-µl wash for
radiolabel. Percent precipitation was calculated as the ratio of bound
counts per minute to total (bound and unbound) counts per minute. The
pentamers used for the positive control in the anti-VP4
immunoprecipitation experiment were generated by treating native empty
capsids with high pH. Specifically, an equal volume of 0.1 M Tris was
added to empty capsids in 25% sucrose in PBS, and the mixture was
incubated on ice for 10 min. The pH was then neutralized by adding 4 volumes of 10× PBS.
The results of the immunoprecipitation experiments show that the
polyclonal antibodies raised against peptides corresponding
to segments
in the N-terminal arm of VP1 bind to the heated empty
capsid but not to
the native empty capsid (Fig.
4A and B).
This
corroborates the finding of V8 sensitivity experiments that
heat-induced
conversion of the particle externalizes this arm. However,
the
heated empty capsid is not bound by a monoclonal anti-pep1 antibody
that does bind the externalized arms in the 135S and 80S particles
(Fig.
4C). Thus, the externalized arm in the heated empty capsid
evidently adopts a different conformation than that in either
the 135S
or 80S particle.
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FIG. 4.
Immunoprecipitation titrations of heated and unheated
empty capsids. Titrations were with the following antibodies (Ab). (A)
Polyclonal antibodies binding the pep0 region of VP1 (residues 7 to
24). (B) Polyclonal antibodies binding the pep1 region of VP1 (residues
24 to 40). (C) Monoclonal antibody raised against pep1. (D) Polyclonal
antibodies binding VP4. In each panel, the percent immunoprecipitated
versus the log of the dilution is plotted. Symbols:  , empty capsids;
 , heated empty capsids;  , pentamers;  , mature virus;  ,
135S;  , 80S. The dashed line in panel D represents the background
level for pentamer immunoprecipitation determined as described in the
text. The reactivity of the undiluted anti-pep1 and anti-pep0
polyclonal antibodies toward unheated empty capsids may be due to
instability of the empty capsids in serum concentration. The high
background in control experiments using pentamers is due to
adventitious binding of pentamers to the protein A-Sepharose beads.
Mock experiments in which no anti-VP4 antibody was added resulted in
precipitation of the pentamers approximately equal to the background
level observed in panel D.
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The immunoprecipitation experiment with polyclonal antibodies against
VP4 showed no reactivity with heated empty capsid (Fig.
4D). The lack
of reactivity with the heated empty capsid is unlikely
to be due simply
to the inaccessibility of a partially exposed
VP4 segment. Previous
experiments showed that VP4, when even partially
exposed during the
normal "breathing motion" of the mature virus,
can be bound by the
anti-VP4 antibody (
22). The lack of reactivity
is most
easily explained by the VP4 portion of VP0 not being externalized
at
all, perhaps as a consequence of it still being covalently
attached to
the VP2 portion of VP0. However, it is also possible
that this region
is externalized transiently and then later completely
internalized
again during the transition to the 80S equivalent
of the empty capsid.
In either case, the ability of the N terminus
of VP1 to be externalized
without the permanent coexternalization
of VP4 is significant since it
indicates that these two hallmarks
of the transition to the cell entry
intermediate to some degree
can be uncoupled, in contrast to what was
previously believed
(
14).
The immunoprecipitation results, when interpreted in the context of the
empty capsid structure, provide hints regarding the
mechanism for
conversion of the native mature virus to the cell
entry intermediate.
First, that the extreme N-terminal region
of VP1 is externalized in the
heated empty capsid even though
it is completely disordered in the
native empty capsid suggests
that this region is not an essential part
of the conversion mechanism.
Rather, the extreme N-terminal region of
VP1 seems to be a passive
component in the process. This idea is
reinforced by the observation
that this region contains one of the most
poorly conserved sequences
in picornaviruses (
33). Thus, the
arm is unlikely to make any
highly sequence-specific interactions in
the virion as part of
the conversion mechanism. A passive role in
externalization also
makes mechanistic sense if the capsid pores
thought to open in
the receptor-heat-induced transition are situated,
as proposed,
near the quasi-threefold axis near the center of each
protomer
(
13). Much of the N-terminal arm in the mature
virus is positioned
near this region (Fig.
5). The N-terminal arm in the empty
capsid
must also be somewhere in this region because it is tethered
nearby
to the beta-barrel core of VP1. Thus, if the pores do open by
disruption of the intraprotomer contacts at the quasi-threefold
axes,
then the N-terminal arms might be externalized by virtue
of their
proximity to these pores. Second, VP4 may also be externalized
primarily due to its location. In the mature virus, this polypeptide
runs underneath the postulated pores. Third, the experimental
results
presented here suggest that the mechanism for conversion
to the cell
entry intermediate does not rely on the rearrangements
which occur in
the inner surface of the capsid during the final
stages of mature virus
assembly. Instead, the mechanism seems
to be contained entirely within
the surface features of the capsid
and the beta-barrels which comprise
the bulk of the shell.
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FIG. 5.
View of the poliovirus protomer. The view is from inside
the particle looking out. The N-terminal arm of VP1 (residues 6 to 10 and 20 to 69) and all of VP4 are represented as blue and green ribbons,
respectively. Residues 1 to 5 and 11 to 19 are disordered and therefore
are not shown. The remaining portions of the protomer are represented
by the surfaces, with blue, yellow, and red corresponding to VP1, VP2,
and VP3, respectively. The sphere indicates the position of the VP0
scissile bond in the empty capsid. The quasi-threefold axis relating
VP1, VP2, and VP3 in the protomer is located near the VP0 scissile bond
and is roughly perpendicular to the page.
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ACKNOWLEDGMENTS |
We thank Marie Chow for helpful discussions and for the antibodies
used in these experiments. We thank Alane Taratuska for technical
assistance.
This work was supported by NIH grant AI20566 (to J.M.H.). R.B. was the
recipient of an NIH postdoctoral fellowship (AI08780-03).
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FOOTNOTES |
*
Corresponding author. Present address: Department of
Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (716) 273-4799. Fax:
(716) 275-6007. E-mail:
ravi_basavappa{at}urmc.rochester.edu.
Present address: Therion Biologics Corp., Cambridge, MA
02142.
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REFERENCES |
| 1.
|
Baltimore, D.,
M. Girard, and J. E. Darnell.
1966.
Aspects of the synthesis of poliovirus RNA and the formation of virus particle.
Virology
29:179-189[Medline].
|
| 2.
| Basavappa, R., and J. M. Hogle. Unpublished
data.
|
| 3.
|
Basavappa, R.,
R. Syed,
O. Flore,
J. P. Icenogle,
D. J. Filman, and J. M. Hogle.
1994.
Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 Å resolution.
Protein Sci.
3:1651-1669[Medline].
|
| 4.
|
Breindl, M.
1971.
The structure of heated virus particles.
J. Gen. Virol.
11:147-156[Abstract/Free Full Text].
|
| 5.
|
Chow, M.,
R. Yabrov,
J. Bittle,
J. Hogle, and D. Baltimore.
1985.
Synthetic peptides from four separate regions of the poliovirus type 1 capsid protein VP1 induce neutralizing antibodies.
Proc. Natl. Acad. Sci. USA
82:910-914[Abstract/Free Full Text].
|
| 6.
|
Crowell, R. L., and L. Philipson.
1971.
Specific alteration of coxsackievirus B3 eluted from HeLa cells.
J. Virol.
8:509-515[Abstract/Free Full Text].
|
| 7.
|
Curry, S.,
M. Chow, and J. M. Hogle.
1996.
The poliovirus 135S particle is infectious.
J. Virol.
70:7125-7131[Abstract/Free Full Text].
|
| 8.
|
De Sena, J., and B. Mandel.
1977.
Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membrane-modified particle.
Virology
78:554-566[Medline].
|
| 9.
|
Dove, A. W., and V. R. Racaniello.
1997.
Cold-adapted poliovirus mutants bypass a postentry replication block.
J. Virol.
71:4728-4735[Abstract].
|
| 10.
|
Everaert, L.,
R. Vrijsen, and A. Boeye.
1989.
Eclipse products of poliovirus after cold-synchronized infection of HeLa cells.
Virology
171:76-82[Medline].
|
| 11.
|
Fenwick, M. L., and M. J. Wall.
1973.
Factors determining the site of synthesis of poliovirus proteins: the early attachment of virus particles to endoplasmic membranes.
J. Cell Sci.
13:403-413[Abstract/Free Full Text].
|
| 12.
|
Filman, D. J.,
R. Syed,
M. Chow,
A. J. Macadam,
P. D. Minor, and J. M. Hogle.
1989.
Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus.
EMBO J.
8:1567-1579[Medline].
|
| 13.
|
Flore, O.,
C. E. Fricks,
D. J. Filman, and J. M. Hogle.
1990.
Conformational changes in poliovirus assembly and cell entry.
Semin. Virol.
1:429-438.
|
| 14.
|
Fricks, C. E., and J. M. Hogle.
1990.
Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding.
J. Virol.
64:1934-1945[Abstract/Free Full Text].
|
| 15.
|
Hogle, J. M.,
M. Chow, and D. J. Filman.
1985.
Three-dimensional structure of poliovirus at 2.9A resolution.
Science
229:1358-1365[Abstract/Free Full Text].
|
| 16.
|
Holland, J. J.
1961.
Receptor affinities as major determinants of enterovirus tissue tropisms in humans.
Virology
15:312-326[Medline].
|
| 17.
|
Holland, J. J., and B. H. Hoyer.
1962.
Early stages of enterovirus infection.
Cold Spring Harbor Symp. Quant. Biol.
27:101-112[Abstract/Free Full Text].
|
| 18.
|
Holland, J. J., and E. D. Kiehn.
1968.
Specific cleavage of viral proteins as step in the synthesis and maturation of enteroviruses.
Proc. Natl. Acad. Sci. USA
60:1015-1022[Free Full Text].
|
| 19.
|
Jacobson, M. F.,
J. Asso, and D. Baltimore.
1970.
Further evidence on the formation of poliovirus proteins.
J. Mol. Biol.
49:657-669[Medline].
|
| 20.
|
Jacobson, M. F., and D. Baltimore.
1968.
Morphogenesis of poliovirus. I. Association of viral RNA with coat protein.
J. Mol. Biol.
33:369-378[Medline].
|
| 21.
|
Klotz, I. M.
1982.
Number of receptor sites from Scatchard graphs: facts and fantasies.
Science
217:1247-1249[Abstract/Free Full Text].
|
| 22.
|
Li, Q.,
A. Gomez Yafal,
Y. M.-H. Lee,
J. Hogle, and M. Chow.
1994.
Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature.
J. Virol.
68:3965-3970[Abstract/Free Full Text].
|
| 23.
|
Lonberg-Holm, K.
1981.
Attachment of animal virus to cells: an introduction, p. 1-20.
In
K. Lonberg-Holm, and L. Philipson (ed.), Virus receptors, receptors and recognition, series B, vol. 8, part 2. Chapman and Hall, Ltd., London, England.
|
| 24.
|
Lonberg-Holm, K.,
L. B. Gosser, and J. C. Kauer.
1975.
Early alteration of poliovirus infected cells and its specific inhibition.
J. Gen. Virol.
27:329-342[Abstract/Free Full Text].
|
| 25.
|
Lonberg-Holm, K.,
L. B. Gosser, and E. J. Shimshick.
1976.
Interaction of liposomes with subviral particles of poliovirus type 2 and rhinovirus type 2.
J. Virol.
19:746-749[Abstract/Free Full Text].
|
| 26.
|
Lonberg-Holm, K., and B. D. Korant.
1972.
Early interaction of rhinovirus with host cells.
J. Virol.
9:29-40[Abstract/Free Full Text].
|
| 27.
|
Longberg-Holm, K., and L. Philipson.
1974.
Early interaction between animal viruses and cells, p. 24-26.
In
J. L. Melnick (ed.), Monographs in virology, vol. 9. S. Karger, Basel, Switzerland.
|
| 28.
|
Madshus, I. H.,
S. Olsnes, and K. Sandvig.
1984.
Requirements for entry of poliovirus into cells at low pH.
EMBO J.
3:1945-1950[Medline].
|
| 29.
|
Maizel, J. V.,
B. A. Phillips, and D. F. Summers.
1967.
Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1.
Virology
32:692-699[Medline].
|
| 30.
|
McLaren, L. L.,
J. J. Holland, and J. T. Syverton.
1959.
The mammalian cell-virus relationship. I. Attachment of poliovirus to cultivated cells of primate and non-primate origin.
J. Exp. Med.
109:475-485[Abstract].
|
| 31.
|
Mendelsohn, C. L.,
E. Wimmer, and V. R. Racaniello.
1989.
Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.
Cell
56:855-865[Medline].
|
| 32.
|
Olson, N. H.,
P. R. Kolatkar,
M. A. Oliveira,
R. H. Cheng,
J. M. Greve,
A. McClelland,
T. S. Baker, and M. G. Rossmann.
1993.
Structure of a human rhinovirus complexed with its receptor molecule.
Proc. Natl. Acad. Sci. USA
90:507-511[Abstract/Free Full Text].
|
| 33.
|
Palmenberg, A. C.
1989.
Sequence alignments of picornaviral capsid proteins, p. 211-241.
In
B. L. Semler, and E. Ehrenfeld (ed.), Molecular aspects of picornavirus infection and detection. American Society for Microbiology, Washington, D.C.
|
| 34.
|
Rombaut, B.,
R. Vrijsen, and A. Boeye.
1989.
Denaturation of poliovirus procapsids.
Arch. Virol.
106:213-220[Medline].
|
| 35.
|
Rossmann, M. G.,
E. Arnold,
J. W. Erickson,
E. A. Frankenberger,
J. P. Griffith,
H.-J. Hecht,
J. E. Johnson,
G. Kamer,
M. Luo,
A. G. Mosser,
R. R. Rueckert,
B. Sherry, and G. Vriend.
1985.
Structure of a common cold virus and functional relationship to other picornaviruses.
Nature (London)
317:145-153[Medline].
|
| 36.
|
Rotbart, H. A., and K. Kirkegaard.
1992.
Picornavirus pathogenesis: viral access, attachment and entry into susceptible cells.
Semin. Virol.
3:483-499.
|
| 37.
|
Wetz, K., and T. Kucinski.
1991.
Influence of different ionic and pH environments on structural alterations of poliovirus and their possible relation to virus uncoating.
J. Gen. Virol.
72:2541-2544[Abstract/Free Full Text].
|
Journal of Virology, September 1998, p. 7551-7556, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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