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Journal of Virology, February 2000, p. 1342-1354, Vol. 74, No. 3
Laboratory of Structural Biology, National
Institute of Arthritis, Musculoskeletal and Skin
Diseases,1 and Computational Bioscience
and Engineering Laboratory, Center for Information
Technology,3 National Institutes of Health,
Bethesda, Maryland 20892; Department of Biological Chemistry
and Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 021152; and Committee on
Higher Degrees in Biophysics, Harvard University, Cambridge,
Massachusetts 021384
Received 4 August 1999/Accepted 19 October 1999
Upon interacting with its receptor, poliovirus undergoes
conformational changes that are implicated in cell entry, including the
externalization of the viral protein VP4 and the N terminus of VP1. We
have determined the structures of native virions and of two putative
cell entry intermediates, the 135S and 80S particles, at ~22-Å
resolution by cryo-electron microscopy. The 135S and 80S particles are
both ~4% larger than the virion. Pseudoatomic models were
constructed by adjusting the beta-barrel domains of the three capsid
proteins VP1, VP2, and VP3 from their known positions in the virion to
fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These
movements create gaps between adjacent subunits. The gaps at the sites
where VP1, VP2, and VP3 subunits meet are plausible candidates for the
emergence of VP4 and the N terminus of VP1. The implications of these
observations are discussed for models in which the externalized
components form a transmembrane pore through which viral RNA enters the
infected cell.
Poliovirus, like related
picornaviruses, is a small nonenveloped virus consisting of a
plus-sense RNA genome enclosed within a protein shell or capsid
(reviewed in reference 53). The capsid consists of
60 copies each of four proteins (VP1, VP2, VP3, and VP4) arranged on an
icosahedral lattice. VP1, VP2, and VP3 have similar wedge-shaped cores,
each an eight-stranded beta-barrel (the strands are designated B, I, D,
G, C, H, E, and F), but each protein has unique loops connecting the
strands and unique N and C termini (31). VP4 is small,
myristylated (15), and has an extended structure.
The wedge-shaped cores of the subunits form the closed protein shell of
the virion, with five copies of VP1 packing around the fivefold axes
and VP2 and VP3 alternating around the threefold axes of the particle.
The virion is stabilized by interactions among the wedge-shaped cores
and by a network Despite extensive chemical and molecular characterization, the
mechanism of picornavirus cell entry is known only in outline (14,
48, 49, 51-53, 59). In order to initiate a productive infection,
the viral RNA must be externalized, cross a membrane, and be delivered
to the cytoplasm. Infection begins with binding of the virion to a
receptor that is a member of the immunoglobulin superfamily (37,
45), whereupon the virion undergoes an irreversible rearrangement
to form the 135S, or A, particle. The 135S particle has a lower
sedimentation coefficient (135S versus 160S for poliovirus [52]) and altered antigenic and proteolytic
properties. This conformational change results in the externalization
of VP4 and the N terminus of VP1 To explore these transitions and their implications for cell entry, we
have examined the 160S, 135S, and 80S particles by cryo-electron
microscopy (cryo-EM), taking advantage of the property that similar if
not identical particles are produced by heating 160S virions at
~50°C in hypotonic medium containing divalent cations (20,
58). We determined their structures at ~22-Å resolution by
three-dimensional (3-D) image reconstruction. These maps were used to
obtain pseudoatomic models of the altered particles by appropriate
rigid-body movements of VP1, VP2, and VP3 from their positions in the
virion (cf. reference 55), which are known from the
high-resolution crystal structure (31). We proceeded on the
reasonable assumption that the core domains of the capsid proteins
remain unchanged in all three forms of the virus. This made it possible
to interpret the cryo-EM reconstructions in much greater detail than
their nominal resolution would imply. The resulting models are
consistent with a multistep model for cell entry involving RNA
translocation through a membrane channel formed by the externalized N
termini of VP1.
Preparation of viral particles.
Virus was grown and purified
by differential centrifugation and CsCl density gradient fractionation
as described previously (20, 54). Altered particles (135S
and 80S) were prepared by heating purified virions for 3 min at 50°C
(135S) or 10 min at 55°C (80S) in hypotonic buffers containing
Ca2+ (20 mM Tris, 2 mM CaCl2 [pH 7.5])
(20; C. N. Hiremath, unpublished data).
EM.
Samples of 160S, 135S, and 80S particles at 0.1 to 0.6 mg of protein/ml were frozen and imaged as described previously
(9, 63). Size calibration standards were mixed with some
specimens before freezing (see below). Philips EM400, CM120, and CM200
electron microscopes (FEI, Mahwah, N.J.), all equipped with Gatan
(Pleasanton, Calif.) 626 cryoholders, were used to record micrographs
at magnifications of ×38,000 to 60,000. Some fields were imaged twice
at different focal settings (63). The micrographs were
screened by optical diffraction to assess defocus, drift, and
stigmation and by eye for suitable distributions of particles.
3-D reconstruction.
Micrographs were scanned on
microdensitometers: Perkin-Elmer (Norwalk, Conn.) 1010MG at 18 to 24 µm/step or Zeiss (Englewood, Colo.) SCAI at 7 µm/step (Zeiss scans
were computationally reduced to 14 or 21 µm/step). Particle images
were extracted, processed, and normalized as described previously
(8, 18). Icosahedral reconstructions were determined by
standard procedures (26). For the 135S and 80S data, initial
models for model-based orientation and origin determination
(3) were determined by common-line methods (11,
26). The initial model for the 160S reconstruction was the 135S
reconstruction. Resolution was estimated by use of a reliability index
(62) computed by comparing structure factors from two
reconstructions, each calculated from half of the data. Eigen values
(19) were Correction of CTF.
The final reconstructions were computed
from images that were corrected for contrast transfer function (CTF)
effects as described by Zlotnick et al. (63) but modified as
follows. Fourier amplitudes between spatial frequencies of 0 and the
first CTF minimum were multiplied by a factor intended to restore 50%
of the difference between the theoretical and the actual
(CTF-attenuated) amplitudes. This correction gave a 160S reconstruction
in which a single contour level optimally fit the atomic structure. To
avoid overcorrection of low amplitudes and division-by-zero errors, the
divisor was set to 0.1 if the amplitude of the combined (sum of
multiple images in a focal series) or uncombined theoretical CTF Size calibration.
The crystal structure of the 160S capsid
(31) was used as a size standard. The 160S reconstruction
was calibrated against it both by fitting the atomic coordinates into
variously sized reconstructions and assessing each fit by an
R-factor or phase residual (see below), and by computing a
22-Å-resolution density map of the 160S capsid from the coordinates
and scaling it to the 160S reconstruction by 3-D cross-correlation
(13). The two methods gave consistent results.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Tectonic Model of Virus Structural
Transitions: the Putative Cell Entry States of Poliovirus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
on the inner surface of the protein shellthat is
formed by VP4 and the N-terminal extensions of VP1, VP2, and VP3. The
high-resolution structure of an empty capsid assembly intermediate
shows that formation of the internal network is dependent on a late
proteolytic cleavage of the capsid protein precursor, VP0, to yield VP4
and VP2 (5). This "maturation cleavage" is associated
with the encapsidation of the viral RNA and is required for virion
stability. Unfortunately, the viral RNA (which lacks the icosahedral
symmetry of the protein coat) is not visible in the high-resolution
structure of the virus, and its role in stabilizing the virus structure
is unknown.
components of the stabilizing network
on the inner surface of the 160S capsid. The externalized N terminus of
VP1 is predicted to form an amphipathic helix and has been shown to
allow the 135S particle to attach to synthetic membranes in vitro
(24). This suggests that the maturation cleavage of VP0 may
serve to trap the virion in a metastable state that is primed to
undergo receptor-mediated conformational changes that are necessary for
subsequent virus-cell interactions (14, 59). Analogous roles
have been proposed for the maturation cleavages of the glycoproteins of
influenza virus and other enveloped viruses (12). Later, the
135S particle releases its RNA in converting to the 80S (or H)
particlethe putative end state. Although several lines of evidence
support the proposition that the 135S particle is indeed a cell entry
intermediate (20, 24, 28, 51), its status as such has been
questioned (22, 48, 49) (see Discussion).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
10 for the 160S, 135S, and 80S reconstructions, indicating an adequate diversity of views.
at
spatial frequency 
was less than 0.1 maximum amplitude. Focal pairs
were combined for the 160S (1.1- and 1.5-µm underfocus) and 135S
(1.3- and 1.9-µm underfocus) images as part of the correction process
(63). 80S images (1.2-µm underfocus) were also corrected,
although focal pairs were not available.
Atomic modeling. Alpha-carbon atomic models of poliovirus capsid proteins VP1, VP2, and VP3 (31) were fitted individually as rigid bodies to the 160S, 135S, and 80S reconstructions, using the program FRODO (33). Prior to fitting the 135S and 80S reconstructions, VP4 and the N terminus of VP1 were removed from the model. Other extended loops and termini were removed if their structures clearly depended on contact with another subunit. In the truncated models, the fivefold symmetric beta-tube formed by the intertwined N termini of VP3 (residues 1 to 12) was treated as a separate object.
Automated rigid-body refinements were carried out by a modification of protocols developed for crystallographic refinement (32). A box of density, which generously enclosed a single protomer, was extracted from each reconstruction. 3-D Fourier transformation of the box, treating it as if it were periodic, yielded a set of complex-valued pseudostructure factors (~Fo) that served as a standard for the rigid-body refinement. The phases of this standard remained constant throughout refinement and were unaffected by the atomic model. Model-based electron density values for all lattice points in the "protomer box" were calculated by using a standard five-term summed-Gaussian approximation for the density attributable to each atom within the protomer box. To compensate for the lack of RNA in the model, density values in the RNA and solvent areas of the reconstructions were identified by using inner and outer radial masks (Table 1) and were corrected to match the solvent area's average prior to refinement. In 10-Å buffer zones around the mask edges, there was a linear transition between modified and unmodified density values. The model-based pseudo-diffraction data were scaled linearly to the reference Fourier coefficients in shells of resolution. Refinements evaluated the fit of the model to the map with either the standard crystallographic R-factor or an amplitude-independent scoring function such as <
>, the mean
|F|-weighted absolute phase difference (see Table 1 for definitions). Between iterations, coordinates for the individual proteins were allowed to move as rigid bodies translationally and
rotationally.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Structural analysis. (i) EM. Poliovirus 160S, 135S, and 80S particles appear roundish when viewed in either negative stain or vitreous ice (Fig. 1A to C), although in some views straight edges are seen, particularly for the 135S and 80S particles. Negative staining typically showed differences in internal stain penetration for the three particles: 160S, little or none; 135S, partial; and 80S, heavy. Density maps of the 160S, 135S, and 80S particles (Fig. 2D and 3A) were computed from 123, 120, and 262 images, respectively. Their resolutions are 22, 22, and 23 Å, respectively. The sizes of the 135S and 80S reconstructions were calibrated against the known 160S structure (31) (Fig. 1D and E).
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(ii) Pseudoatomic modeling. The 135S and 80S reconstructions were used to derive pseudoatomic models of these particles by adjusting the positions and orientations of the beta-barrel domains of VP1, VP2, and VP3 (Fig. 4) from their locations in the 160S virion. At the current resolution of the reconstructions, the density does not provide sufficient constraints to allow refinement of the internal structures of the capsid protein subunits. Therefore, the core structures were fitted as rigid bodies into the cryo-EM maps by a combination of visual adjustment and quantitative refinement. To assess the reliability of this procedure, the three core domains, as well as VP4 and nontruncated VP1, VP2, and VP3, were fitted to the 160S reconstruction. The quality of each fit was assessed in terms of crystallographic parameters and by tabulating the number of short-range contacts (indicating possible collisions between subunits) and medium-range contacts (indicating favorable packing interactions between subunits) (Table 1). These statistics, along with visual assessment of the fit of the models to the density (Fig. 5), indicate that the rigid-body approximation is justified.
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(iii) Comparison of Cryo-EM and X-ray structures of the 160S
virion.
Initial 160S reconstructions (not shown) agreed well with
the crystal structure on the outer surface of the virion but less well
in other parts of the capsidmost notably within the protein shell and
at the inner surface, where there appeared to be missing protein
density. To address this problem, our correction of the cryo-EM images
for effects of the CTF was adjusted to include a correction of
low-resolution terms. This measure largely rectified the discrepancies.
For consistency, a similar CTF correction was applied to both the 135S
and 80S data.
(iv) Fit of X-ray coordinates into cryo-EM map. By visual criteria, the 160S atomic model fits snugly into the cryo-EM reconstruction (Fig. 5A). In both structures, the thicknesses of the protein shells are similar, and solvent bubbles are seen at fivefold and threefold axes (Fig. 2E and 5A). Only a few residues (e.g., 50 to 54 and 60 to 64 of VP4) lie outside the capsid boundaries. As expected, the full coordinate set fits the reconstruction significantly better than the truncated version (Table 1).
(v) RNA structure. Poliovirus RNA is not icosahedrally ordered and hence was not observed in the crystal structure at high resolution. In contrast, density corresponding to the RNA is observed in the reconstruction (Fig. 3A). Although icosahedral symmetry is (spuriously) imposed on the RNA, the cryo-EM map does retain valid information about its radial density distribution and its points of contact with the surrounding capsid. RNA is quite densely packed throughout the interior of the virion. The outermost parts of RNA follow the inner surface of the capsid, resulting in apparent invaginations in the RNA at the fivefold axes where the capsid protein protrudes inwards. In a rendering of the inner surface in which densities were trimmed at a radius of 109 Å (Fig. 2E, top), RNA and protein densities appear to merge around each fivefold axis and in finger-like regions that extend towards threefold axes. Unfortunately, it is not possible to describe the protein RNA interactions in any more detail due to the artificially imposed symmetry, the limited resolution, and the presence of a negative density ripple (which is caused by series termination) between the protein shell and the RNA.
The 135S particle. (i) Gross features. Although the 135S particle is generally similar to the virion in morphology, it differs in several respects (Fig. 2 and 3). Its mesa is broader and has a more rounded profile. Its propeller has a triangular plateau that fits between the blade tips instead of three bumps at the center, and its blade tips are relatively swollen. The capsid is thinner and more angular. Small bumps are found on the inner surface adjacent to the twofold axes. In cross-section, the bubble on the fivefold axis is flanked by dense regions on the inner and outer edges, including strong density for the VP3 beta-tube on the inner side of the bubble. The bubble at the threefold axis is weaker than before on its inner surface, presumably due to externalization of VP4 and rearrangements of the N terminus of VP2. The 135S capsid is (4 ± 1)% larger than the virion at the twofold and fivefold axes but is only (1 ± 1)% larger on the threefold axis.
In a central section of the 160S reconstruction (Fig. 3A, top left), strong finger-like features extend outward from either side of the vertical twofold axis. They represent a slice through twofold-related EF loops of VP2the propeller blade tips. In the corresponding 135S
sections (Fig. 3A), these features are fainter and the angle between
them is wider, reflecting a shift of the EF loops relative to the
section plane.
The overall distribution of RNA is changed significantly in the 135S
particle, compared to the virion (cf. Fig. 3A). The RNA has moved
closer to the inner capsid edge near the fivefold axis, and
invaginations in the RNA distribution are seen at twofold instead of
fivefold axes.
(ii) Pseudoatomic model. Nearly all parts of the truncated VP1, VP2, and VP3 subunits fit comfortably within the 135S envelope (Fig. 5B). The R-factor and phase difference between the model and reconstruction were lower than in the 160S controls, but the number of close contacts was higher (Table 1). The modeling indicated root mean square (r.m.s.) shifts in subunit positions of 6.1 to 8.7 Å (Table 1). To a first approximation, the 160S-to-135S transition can be described as follows. The wide ends of all three beta-barrels move outwards while the narrow ends (the ends near the fivefold and threefold axes) undergo much smaller shifts (Fig. 5D). VP1 and VP2 also undergo significant rotations about their principal axes. These concerted movements increase the angle that VP1 makes with VP2 and VP3 and cause the protomer (Fig. 2B and C) to appear flatter in profile (Fig. 5B), accounting for the more angular appearance of the 135S particle (Fig. 3A) and (together with the loss of VP4) contributing to the observed thinning of the shell.
According to the pseudoatomic models, the 160S-to-135S transition is accompanied by the opening of gaps between core domains. One such gap is located between adjacent VP1 subunits (Fig. 6B). Like an opening umbrella, the wide ends of the VP1 beta-barrels move apart to leave gaps between adjacent copies. These gaps extend to the base of the canyon and end near the point at which the VP1, VP2, and VP3 subunits of a single protomer meet. This gap lies immediately above the point where the N-terminal extension of VP1 joins the B strand of the VP1 beta-barrel, and is wide enough to allow the extrusion of the N-terminal extension of VP1 and perhaps VP4. Density that is not accounted for by the truncated model extends above this gap, filling the creases between VP1 beta-barrels and connecting to additional unoccupied density at the top of the fivefold mesa (Fig. 5B). A second gap, which extends from the twofold axis towards the threefold axis, disrupts the contacts that VP2 normally makes with symmetry-related copies of VP2 and VP3 from neighboring pentamers (Fig. 2B and C and 6B). Additional unexplained density is present at the base of the canyon and at inward protrusions beneath the inner surface of VP2 near the twofold axis.
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The 80S particle. (i) Gross features. Apart from its lack of RNA, the 80S particle is generally similar to the 135S particle (Fig. 2 and 3), both in dimensions and appearance. Surface renderings bring out some differences. The arms of the fivefold mesa appear less substantial than those of the 135S capsid, and the canyon around the mesa is less pronounced. The threefold propeller has a distinctive swirl with a small peak at the threefold axis, and the blades are smaller than in the 135S particle. The inner surface of the capsid has inward protrusions visible at the twofold and fivefold axes.
(ii) Pseudoatomic model. In their modeled positions, the truncated VP1, VP2, and VP3 subunits (Fig. 4) fit well into the 80S reconstruction (Fig. 5C and Table 1). In the 135S-to-80S transition, the shifts of VP2 and VP3 which occur in the 160S-to-135S transition are reversed, but incompletely (Fig. 5C and D). Again, the wide end of each subunit moves more than the narrow end. In contrast, the VP1 subunit shifts only slightly from its position and orientation in the 135S particle. The largest difference in VP1 involves a rotation that causes the wider end of the beta-barrel to move slightly inward. Nevertheless, both ends of the VP1 beta-barrel remain farther from the particle center in the 80S state than in the 160S virion.
As a consequence of these movements, the gaps between adjacent VP1 subunits become smaller, and the gaps between neighboring pentamers (Fig. 2B and C) are almost entirely repaired (Fig. 6C). At the same time, the drop of the wider ends of the VP2 and VP3 beta-barrels to a lower radius opens a new gap at the interface that they share with VP1, which fails to drop significantly. In the 80S particle this gap lies immediately above the point where the N terminus of VP1 leaves the beta-barrel. There is unfilled density at the base of the canyon above this gap (Fig. 5C). Other unfilled density is found on top of the fivefold mesa and near the twofold axis beneath the inner surface of VP2 (Fig. 5C). As in the 135S model, the excluded portions of the subunits (Fig. 4) probably occupy the unfilled density. For example, likely candidates for the unfilled density at the base of the canyon are the N terminus of VP1, the GH loop of VP1, the C terminus of VP1, and the C terminus of VP3. The unfilled density on the inner surface of VP2 is also present in the 135S particle and may be occupied by the N terminus of VP2.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison of cryo-EM and X-ray renditions of the virion. The 160S reconstruction matches well with the crystal structure (31), by both visual appraisal (Fig. 2A and D and 5A) and quantitative criteria (Table 1). To obtain an optimal match between the cryo-EM and X-ray structures, we found it necessary to restore the low-resolution terms of the reconstruction, which are attenuated in the phase-contrast imaging mode (46) used in cryo-EM. Since attenuation of low frequencies is essentially an "edge sharpening," it may have little adverse effect when the goal is simply to define molecular envelopes. However, restoration of these frequencies appears to be more important when the goal is to depict the distribution of density throughout the molecular volume, as in the present study. Thus, our initial 160S reconstructions (calculated without restoring the low-resolution terms) agreed well with the crystal structure on the outer surface, but to achieve a satisfactory fit elsewhere, it was necessary to effect a 50% restoration of the low-resolution terms. However, it is important to note that the pseudoatomic modeling process was sufficiently robust for the outcome to be essentially unaffected by this CTF correction.
Sedimentation coefficient change: 160S to 135S. If, apart from the 5% mass loss due to the exit of VP4, the change in sedimentation coefficient were due solely to a change in particle size, we would expect a radial expansion of 10 to 12%. This calculation is based on the Svedberg and Stokes-Einstein equations for spherical particles and employed published values for the sedimentation coefficients, the virion mass (8.43 MDa), partial specific volume (0.685 ml/g), percent RNA (31.6), and VP4 subunit mass (7.5 kDa) (31, 52, 53). The observed expansion is significantly smaller at 4%. This discrepancy implies that other factors contribute to the observed change in sedimentation coefficient. These factors may include a small increase in partial specific volume (up to 3%) or additional changes in the diffusion coefficient due to (i) the nonuniform expansion (Fig. 3), (ii) increased drag from the exposed N terminus of VP1, or (iii) increased flexibility (that would allow the particles to deform in the gradient).
Plate tectonic model of the 135S and 80S transitions. Our modeling experiments with the 135S and 80S density maps revealed rigid-body displacements of up to 8.7 Å for the VP1, VP2, and VP3 core domains (Table 1) as well as changes in their orientations (Fig. 5D). Similar fits for the 160S reconstruction (Fig. 5A and Table 1) showed only small shifts (0.5 to 2.2 Å) and provided a significance baseline for the shifts detected in the 135S and 80S models. By this standard, their movements of 3.7 to 8.7 Å (Table 1) are highly significant. Further confidence in this conclusion is given by the fact that the quality of fit of the 135S and 80S models is similar to that of the 160S complete fit and slightly better than the 160S truncated fit (Table 1). Given the scale of these movements, it follows that the intersubunit interactions undergo major revision in the transitions to the 135S and 80S states.
In the models, gaps are generated between the core domains. Although not evident in the reconstructions at ~22-Å resolution, the gaps are large enough in the case of the 135S particle to allow passage of an extended polypeptide chain. As such, they may provide conduits for externalization of VP4 and the N termini of VP1.Externalization of internal components. Viral capsids are dynamic structures, and the exposure of internal components (such as VP4 and the N terminus of VP1) during conformational transitions is not unique to poliovirus. Indeed, exposure of the N-terminal extensions of capsid protein subunits occurs upon expansion of simple plant viruses (30). Similarly, during maturation of some bacteriophage (34) and herpesvirus (4) procapsids, internal scaffold proteins are expelled, creating space for the incoming DNA. In the radical conformational change that accompanies maturation and stabilization of the phage T4 capsid, epitopes are transferred between the inner and outer surfaces (36, 56).
In addition to irreversible changes, virus particles undergo reversible alterations as well. Studies with poliovirus have demonstrated that VP4 and the N-terminal extension of VP1 are transiently and reversibly exposed when the virus is incubated at physiological temperature (40). More recent studies with limited proteolysis and mass spectrometry have shown that Flock House virus and rhinovirus undergo similar apparently reversible conformational changes that externalize normally internal components (10, 39).How do VP4 and the N termini of VP1 exit the particle? The observation that irreversible exposure of the VP1 N terminus in 135S particles correlates with their ability to attach to membranes suggests that this peptide may insert into the cell membrane during entry (24). It has been proposed that VP4, the N terminus of VP1, and the viral RNA all exit the particle through a channel at the fivefold axes (29, 47, 53). This model is attractive in that five copies of the VP1 N terminus would be exposed around the axis, poised for membrane insertion and pore formation. However, two observations from the 135S reconstruction argue strongly against the fivefold axis as the exit site for the N terminus of VP1. (i) The plug formed by the N termini of VP3 is present in the 135S particle. (ii) There is insufficient density and, perhaps more importantly, insufficient room on the inner surface of the fivefold mesa to accommodate five copies of the N terminus of VP1 without invading the well-defined bubble.
The data presented are consistent with an alternative model in which the N terminus of VP1 exits near the base of the canyon (23, 38) and follows the surface to the top of the fivefold mesa. Thus, in our pseudoatomic models, VP1 undergoes an umbrella-like movement in which the loops at the narrow end of VP1 serve as pivot points for a radial swing of the wide end of the subunit. This movement opens gaps in the interfaces between fivefold-related copies of VP1 and between VP1 and fivefold-related copies of VP3 at the base of the canyon. The nature of these changes is consistent with mutational data that implicate the interfaces disrupted by these gaps and the loops of VP1 near the fivefold axis as determinants of particle stability and of the ability to undergo the 160S-to-135S transition (16, 17, 23, 41-43, 60). In principle, egress of the N terminus of VP1 from a point at the base of the canyon might pose problems for membrane insertion, and in particular for coinsertion of several copies in close enough proximity to form a pore. However, when the model is superimposed on the 135S reconstruction there is unfilled density in the crease between neighboring VP1 beta-barrels (Fig. 6B) that may correspond to an extended segment of the N terminus of VP1 as it follows the surface of the fivefold mesa toward the top. The well-characterized expansions of two T=3 plant viruses, tomato bushy stunt virus (50) and cowpea chlorotic mottle virus (55), results in disruption of analogous interfaces. In the case of tomato bushy stunt virus the holes opened up by the disruption of the analogous interfaces have been suggested to be the sites of extrusion of portions of the N-terminal extensions of the capsid subunits. Note, however, that in contrast to the expanded forms of the plant viruses, where the holes are sufficiently large to be visualized at low resolution, the gaps between VP1 subunits are barely large enough to allow passage of an extended polypeptide chain. This suggests that at some point during the 160S-to-135S transition the holes are larger. Indeed, the observation that the N terminus of VP1 and portions of VP4 are transiently and reversibly externalized when viruses are incubated at physiological temperatures (40) is consistent with this concept.How does RNA exit the virion? The reconstructions do not directly indicate the manner of RNA exit from the 135S particle, as we see no holes in the symmetrical 135S or 80S reconstruction (Fig. 2). This implies that during the 135S-to-80S transition the particle contains holes of sufficient size for RNA to exit. A model in which holes are opened by dislodging protein subunits from the 135S or 80S particle is deemed improbable in light of the resistance of the 135S particle to RNase (20) and the limited sensitivity of the 80S particle to proteases (25). In principle, the holes might be created by symmetric expansion, producing multiple equivalent holes (as in the expansion of tomato bushy stunt virus [50] and cowpea chlorotic mottle virus [55]). Alternatively, an individual hole could result from an asymmetric local expansion. In either case there must be a factor that determines which symmetry equivalent hole is used or which symmetry equivalent site undergoes local expansion. Otherwise the RNA would be extruded through multiple sites and could not be fully released. The factor may be structural (e.g., some portion of the RNA or perhaps the VPg protein which is covalently linked to the 5' end of the RNA) or kinetic (once expansion or release is initiated at one site, subsequent release is fast). The proximity of the membrane could provide the symmetry-breaking factor. However, the ability of the RNA to be released by heating in vitro argues that membrane proximity need not play an essential role in determining the exit portal.
The role of the receptor in cell entry. There is a wealth of biochemical and genetic data that suggests that the interaction between poliovirus and its receptor induces conformational changes in the virus that are essential for cell entry (reviewed in reference 14). Thus, the receptor serves as both a "hook" that increases the local concentration of virions at the surface of the cell and the "unzipper" that initiates uncoating (49) by using some of the energy of receptor binding to facilitate conformational transitions (59). Interestingly, cells lacking the receptor but expressing the high-affinity Fc receptor can be infected by complexes of the virus with certain monoclonal antibodies (1). Clearly, the Fc receptor provides the hook that brings the virus-antibody complexes to the cell surface, but what provides the unzipper? A clue may come from the observation that only certain monoclonal antibodies are capable of facilitating infection. Either these antibodies facilitate a step that is downstream of binding or the antibodies which fail to facilitate infection block this step. Because the pathway of antibody-mediated infection has not yet been well characterized it is not clear whether this pathway parallels that of receptor-mediated infection.
Is the 135S particle an intermediate in cell entry? While there is some consensus that receptor binding induces structural changes in the virion, the identities of the intermediates and the details of the entry pathway remain uncertain. Characterization of the cell entry pathway for poliovirus (and many other viruses) is complicated by the very high ratio of particles to PFU (typically 100 to 1,000 particles/PFU). Since most optical and biochemical experiments sample the bulk population of particles, one is never certain whether the measurements are relevant to productive infection. For example, biochemical studies that show significant levels of native virions in clathrin-coated vesicles early in infection have been interpreted as evidence that the virus enters the cell via classical clathrin-mediated endocytosis (61). However, in a more recent experiment in which infection was assayed, cells expressing a dominant-negative dyamin mutant showed that entry via clathrin-mediated endocytosis is not obligatory for poliovirus infection (21).
Similarly, the failure to detect a possible intermediate under certain conditions does not prove that it is not an intermediate. In a steady-state process such as cell entry, an intermediate will accumulate only if it is upstream of a rate-limiting step in the pathway. If the rate-limiting step is upstream, the intermediate will be consumed as fast as it is produced and may not be detected. Dove and Racaniello recently characterized the early stages of infection by cold-adapted mutants of poliovirus. They showed that the cold-adapted virus did not accumulate 135S when grown at 25°C and concluded that the 135S particle was therefore not a true intermediate (22; see also references 48 and 49). However, the receptor-mediated production of the 135S particle is known to be very slow at low temperatures (27) and may have become rate limiting at 25°C. In contrast, some step immediately downstream is rate limiting at physiological temperatures. Indeed, studies with neutral red to probe the kinetics of RNA release (with infection being assayed) suggest that RNA release is rate limiting at physiological temperatures (35). Since the receptor alone appears to be unable to convert 135S to 80S (2, 27), this second step may require an additional, as yet uncharacterized trigger. Several lines of evidence support designation of the 135S particle as an intermediate in cell entry. (i) The 135S particle (but not the 80S particle) binds to liposomes (24). (ii) 135S particles can form ion channels in synthetic bilayers without acidification (57). (iii) The cell-associated fraction of 135S is the dominant form of the virus early in infection and subsequently chases into the 80S form (24). (iv) Ligands (including antiviral drugs) that bind in a pocket in the capsid protein VP1 prevent the accumulation of 135S particles and result in the loss of infectivity (28, 44, 51). (v) Viral mutants that reversed the effects of a mutant poliovirus receptor were more labile to 135S conversion (60). (vi) The 135S particle is infectious in a receptor-independent manner (20). Although the per particle infectivity of the 135S particle is several orders of magnitude lower than that of virus, it should be noted that the receptor-independent infection by 135S particles did not benefit from a locally high concentration of virions at the cell membrane which is induced by receptor binding. Indeed, recent experiments show that the infectivity of the 135S particle can be raised to levels that are comparable to virions when the 135S particles are first bound to appropriate antibodies and the complexes are used to infect cells expressing the Fc receptor (M. Chow, personal communication). We argue that the evidence (especially the infectivity of the 135S particle) suggests that the 135S particle is either an intermediate or is sufficiently similar to an intermediate to serve as a template for developing (ultimately testable) models for viral entry and uncoating.A revised pore model for translocation of RNA into the
infected cell.
Taken together with prior information,
the present observations lead us to propose (Fig.
7) a modification of previous models for
poliovirus cell entry (29, 38, 53). The virion initially attaches by binding to the receptor, triggering the conformational change to the 135S state. In this transition, VP4, together with the N
termini of VP1, is extruded from the bottom of the canyon and arranged
around the outside of the mesa (near the fivefold axis), where five
copies of the predicted amphipathic helix at the N terminus of VP1
(24, 29) would be ideally positioned for membrane insertion.
The N-terminal myristate of VP4and perhaps other regions of VP4also
may be imbedded in the membrane and may facilitate the insertion of the
VP1 N termini into the membrane. To open a channel at the fivefold axis
by local or symmetric expansion, a transmembrane pore is formed and the
VP3 plug is moved out of the way (perhaps like a float valve). RNA then
exits the particle and enters the cytoplasm. During this process, the
80S particle is formed by shifts of the VP1, VP2, and VP3 subunits and
the fivefold plug is restored.
|
| |
ACKNOWLEDGMENTS |
|---|
We thank R. Grant for a 135S preparation and A. Zlotnick and M. Chow for helpful discussions.
This work was supported in part by NIH grant AI20566 (to J.M.H.) and by an NSF grant for High Performance Computing and Communication (NSF grant MCB 9527181 to G. Wagner). S.C. was supported by a postdoctoral fellowship from the Wellcome Trust. The Harvard Center for Structural Biology is supported by the Giovanni Armenise-Harvard Foundation for Advanced Scientific Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115. Phone: (617) 432-3918. Fax: (617) 432-4360. E-mail: hogle{at}hogles.med.harvard.edu.
Present address: Department of Biochemistry, Wolfson Laboratory,
Imperial College, London SW7 1AY, United Kingdom.
Present address: Biophysics Group, Blackett Laboratory, Imperial
College, London SW7 2BZ, United Kingdom.
§ Present address: Fresenius Medical Care North America, Lexington, MA 02420.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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