Improvements to our modeling procedure.
Rigid-body pseudoatomic modeling involves many subjective choices, particularly about which parts of the atomic model to include, which parts to group together, what positions to start a refinement with, and what residuals to optimize. At the current resolution limit, we have no objective test for the correctness of the result, but we believe that the latest models are more plausible than the previous ones, and we present them as testable hypotheses, providing landmarks that make it easier to navigate through the maps.
Over the past 9 years, our efforts to make pseudoatomic models by rigid-body fitting have gradually improved, partly due to the increased resolution of the reconstructions. In particular, the current reconstructions at ∼9.5 Å resolution include low-density areas within the capsid that correspond to the centers of the β-barrels and to gaps between β-barrels and which help us to identify unique positions for the rigid bodies. Additionally, we now have a better understanding of how to handle the polypeptides whose conformations (in the 160S crystal structures) depend entirely on the binding contacts that they are seen to make with neighboring proteins. These are polypeptides that extend across the inner and outer surfaces of the capsid to make interactions with the β-barrel cores of neighboring proteins. Such contacts are largely responsible for holding the proteins of the capsid together, and the polypeptides in question would have “no visible means of support” if their protein neighbors were removed. In rigid-body refinement, grouping these segments together with their own β-barrel cores would obviously not have been a sensible choice. Instead, whenever possible, we include these segments and group them with the neighboring barrel to which they are bound. Partly owing to the clarity of the protein-solvent boundary in 80S reconstructions, we can incorporate a greater number of the loops and amino- and carboxyl-terminal extensions into our pseudoatomic models. Overall, the result is more similar in shape to the isocontour surfaces of the density maps than our previous cryo-EM models were. These more-inclusive models fill the available density more completely, thus reducing the number of alternative models that give similar refinement scores. Most importantly, they permit a better understanding of those portions of the structure that affect the structural transitions of the virus and change during the transitions.
Thus, when the density map for 135S particles was originally calculated to 9.5 Å resolution, it became obvious that the amino terminus of VP3 (residues 3014 to 3050) travels with the main VP1 rigid body, as that chain wraps around the lower surface of the VP1 barrel in mature virus. Similarly, it became clear that the β-hairpin at the amino terminus of VP2 (residues 2010 to 2018) continued to extend the back β-sheet of a VP3 β-barrel that belongs to a neighboring pentamer (10
), as it does in the crystal structures of 160S and 73S particles (Fig. 1E
). However, in the Bubeck et al. (10
) model of 135S, the presence of RNA on the interior of the capsid made it impossible to tell exactly where the protein ended. Despite this limitation, we were able to build and refine models for VP1 and VP3 that fit the inner and outer density contours well. In contrast, building a satisfactory model for VP2 based on rigid-body fitting and refinement of the VP2 coordinates from mature 160S virus particles was problematic. We could be sure that the modeling was wrong from the fact that the large EF loop of VP2, located at the tip of a 3-fold propeller, protruded through the outer contour of the reconstruction while, at that same contour, the loop was flanked by two empty density peaks that the model failed to account for. We speculated that VP2 might deform in some way or that the two empty peaks might correspond to VP2 in two or more partially occupied conformational arrangements. As we pointed out at the time, conservative rigid-body modeling had not provided a good description of the VP2 density, but the limited resolution restricted the number of parameters that we were entitled to use.
The recent reconstructions of 80S particles have allowed us to solve several of the earlier problems, partly because the absence of strong RNA density near the capsid shell has made the limits of the protein more obvious than in 135S. Moreover, when we docked the VP2 model (from 160S) to fit the current contour of the outer solvent boundary, we could see clearly that residues 2044 to 2057 were sticking outside the density contour on the inner surface. This was very reminiscent of the crystal structure of myrVP0, the precursor protein that is cleaved into myrVP4 and VP2 upon capsid maturation, in the atomic model of 73S empty capsids (4
). As we showed in Basavappa et al. (4
), this loop in myrVP0 is disordered, presumably correlated (both in 73S and in the present 80S density maps) with the disorder or absence of the amino terminus of VP1 and with the absence of RNA (Fig. 4C
). We therefore initiated modeling and refinement with the VP2 portion of myrVP0 from the 73S structures, and we found that it yielded a significantly improved fit to both the inner and outer solvent boundaries in both the 80S and 135S reconstructions.
Once the myrVP0 β-barrel was reliably placed, it also became apparent that additional flexible peptides previously omitted from the model should be assigned to specific rigid bodies and included. These parts were the GH loop of VP1, which remains associated with the outer surface of VP2 and accounts for one of the “empty” density peaks that previously caused a problem in modeling 135S, and the extreme carboxyl terminus of VP2, which makes extensive contacts with the GH loop of VP1. Following this approach, we have been able to go back to the Bubeck et al. reconstruction of the 135S infectious intermediate, as well as the two current 80S reconstructions, and obtain more satisfactory models to describe the cryo-EM density maps for all three uncoating intermediates. Furthermore, now that the obvious problems with VP2 placement have been solved, we feel that we are on much firmer ground in describing which aminoacyl residues are located in the interfaces that change during the conformational transitions of the virus and allow a much more accurate and detailed analysis of the mutational data.
The current model indicates that much of the GH loop of VP1 remains attached noncovalently to the large EF loop of VP2 and to the VP2 β-barrel during the 160S-to-135S transition, even though VP1 and VP2 are shifted and tilted differently from one another during the expansion of the virus. It is hard to imagine how the GH loop could remain associated with VP2, unless some of the GH-loop polypeptide functions as a slack linker to provide an expansion joint. This is a biologically important result because a number of residues of the VP1 GH loop (including 1226, 1228, and 1234) lie within the footprint of the poliovirus receptor (Pvr/CD155) and several residues (1225, 1226, 1228, 1231, 1234, and 1236) (6
) have been shown to affect receptor binding. Mutants containing single-amino-acid substitution at residues of the VP1 GH loop (including 1226, 1228, 1231, and 1236) result in a virus particle with a reduced propensity to undergo the 160S-to-135S transition. Interestingly, residue 1236 is flanked by residues 1235 and 1237, which both contact the pocket factor directly in the native 160S crystal structure (33
). In addition, both the amino and carboxyl ends of the VP1 GH loop (residues 1199 and 1237, respectively) flank the opening of the pocket (the so-called “pore”) and are also in direct contact with the “pocket factor” in the native 160S crystal structure. Notably, the “pocket factor” appears to be either reduced or missing in 135S, 80S.e, and 80S.l (Fig. 3C
). The relative shifts of VP2 and VP1, together with the loss of pocket factor and conformational rearrangement of the GH loop of VP1 during the 160S-to-135S transition, are collectively likely to be responsible for the fact that poliovirus loses its affinity for the receptor once the 135S transition has taken place. Taken together, these data suggest that the GH loop of VP1, along with peptides that interact with it, is involved in transmitting the receptor-binding signal to regions of the capsid that are responsible for the structural transitions.
The only modeled peptide that now remains outside our map contours when viewed at a generous contour level (0.5σ) is the extreme carboxyl terminus of VP1 (residues 1293 to 1302) in the 135S model. Regions from the carboxyl-terminal extension of VP1 have previously been shown to influence structural transitions. Thus, single mutations at residue 1290 lower the stability of 135S particles (17
), and monoclonal antibodies raised against a peptide that corresponds to residues 1280 to 1286 are able to immunoprecipitate 80S but not 135S particles (36
). In mature 160S virions, the loop of VP1 that includes residues 1280 to 1286 makes extensive contacts with a portion of the EF loop of VP2 that includes residue 2142 from the receptor-binding footprint (6
). Viruses with single mutations at 2142 have been shown to influence receptor-binding affinity, to expand host range and receptor specificity, and to become more labile to heat conversion from 160S to 135S (13
). Our conservative rigid-body modeling procedure and the resolution of our density maps do not permit us to make a more specific suggestion for how the carboxyl-terminal extension of VP1 may have changed. However, taken together with the mutagenesis and antibody-binding data, our maps and models both suggest that this polypeptide is unlikely to maintain its native (160S) conformation in the cell entry intermediates.
As we noted previously, VP2 has historically been the most problematic of the major capsid proteins to fit into our 135S and 80S maps; it appears to undergo the most dramatic changes in the normally well-ordered portions of the protein, and its interfaces with neighboring proteins are changed most extensively. In our 135S and 80S.l models, and to a lesser extent 80S.e, VP2 is the only one of the capsid proteins that tilts appreciably, relative to its orientation in 160S and 73S particles (Fig. 6
). This tilting would move the broader end of the VP2 wedge outward and the narrow end (the end closer to the 3-fold axes) inward. This rearrangement might disrupt the interaction (seen in mature 160S virions) between the carboxyl-terminal extension of VP1 and the EF loop of VP2. The difference in amount of tilting of VP2 seen between the two 80S structures may also account for differences in the density maps surrounding the 3-fold axes (VP3 tilting also influences changes in this region, although to a lesser extent) and at the 3-fold propeller tips, as illustrated by the variance map (Fig. 5
; see Movies S1 and S2 in the supplemental material).
Common structural changes associated with both RNA packaging and release.
The major structural differences that we observe between 135S and the 80S reconstructions occur on the inner surface of the capsid. Some of these structural changes, which may be associated with the RNA-release transition, are located in areas that also are seen to change during assembly (i.e., during the packaging of RNA and the cleavage of myrVP0), and are evident when the structures of 73S and 160S particles are compared (Fig. 4
). For example, we observe a 73S-like trefoil depression in our 80S structures (Fig. 3B
), while the resemblance of the 135S trefoil to that of 73S particles is less close. In contrast, on the inner surface of 135S particles, along the 3-fold axis, there is instead a 160S-like arrangement of density, which we attribute to the amino-terminal residues 2006 to 2013 and which caps off a low-density bubble in the capsid (Fig. 3C
). This structural difference suggests that the extreme amino terminus of VP2 becomes rearranged during the initiation of RNA release (in the 135S-to-80S.e transition). Note that this polypeptide terminus is the same one that is liberated by myrVP0 cleavage and that it moves by more than 20 Å during the 73S-to-160S transition. Indications of where the largest changes are taking place in the 80S.e-to-80S.l transition can be obtained by noting which regions have the highest variance between 80S particle forms (Fig. 5
). One region of high variability occurs in the floor of the trefoil and corresponds to the DE loop from the narrow end of the VP2 β-barrel, which exhibits a high degree of sequence conservation among picornaviruses. A second region of high variability occurs at the 5-fold plug, corresponding to five intertwined copies of the amino terminus of VP3. A third area occurs around the 2-fold axis, at the bridge of density (Fig. 3B
), which we suspect is likely to have been formed by a rearrangement of the amino-terminal extension of VP2. Interestingly, this suggests that peptides at all three symmetry axes become altered during the 80S.e-to-80S.l transition.
The most notable difference between the 135S and 80S structures occurs on the inner surface of the capsid, in the area that surrounds the 2-fold symmetry axis. This region is 73S-like in the 135S structure (Fig. 3B
), having a 2-fold depression flanked by protrusions, but becomes bridged across the 2-fold axis, with the addition of an oblong density feature in 80S particles. In 73S the flanking protrusions occur near the transition point where the amino-terminal polypeptide of VP1 becomes disordered (around residue 1067) and near the disordered loop in the amino-terminal extension of myrVP0 (corresponding to the neighborhood of residue 2043). Both of these dynamic peptides become well ordered in 160S, making numerous electrostatic interactions that stabilize them, including contacts between the 1063-to-1069 and 2045-to-2051 polypeptides in adjacent protomers. We are limited by the resolutions of our reconstructions of the cell entry intermediates, and by our rigid-body modeling, and therefore cannot yet model the specifics of the structural changes that we see near the 2-fold axis and which we are attributing to the rearrangement of these two polypeptides.
One interesting additional candidate for a participant in forming the bridging structure is the two-stranded β-hairpin from the amino-terminal extension of VP2, which forms the fifth and sixth strands of the interpentameric seven-stranded β-sheet (Fig. 1E
), since the hairpin is located close to the 2-fold bridge and because the density for it in the reconstruction, as noted above, is weaker than expected. As an aside, it is probably relevant that the corresponding residues of uncoated equine rhinitis A virus (ERAV) were shown to rearrange during RNA release (47
), though the specifics of the rearrangement are different. Thus, changes in atomic coordinates for ERAV before and after RNA release (47
) did not correspond to the density changes that we see in the poliovirus intermediates.
Holes in the capsid at the interface between 5-fold-symmetry-related protomers may serve as routes for RNA egress.
The 80S.e and 80S.l reconstructions were each made from a common, CTF-corrected data set of 2D images, classified into two distinct groups, using a structure-based method. Additionally, the current relatively high resolution of the reconstructions was impossible to achieve without carrying out the classification. For those reasons, the differences in density that we observe between these maps at a single contour level can be believed. Our radial-density plots illustrate that between the inner and outer capsid surfaces, there is less total protein density in the 80S.l than the 80S.e density map (Fig. 2F
). Additionally, when viewed at a stringent contour level (3σ) both maps have holes that span the capsid shell (Fig. 7
). These twisting channels start on the inner surface at openings close to the 5-fold plug, continue through the large low-density bubble on the 5-fold axis, run out through the VP1 lipid-binding pocket, and terminate where the pocket opens to the outer surface of the capsid, at the bottom of the “canyon,” near the quasi-3-fold axes (Fig. 7A and B
). The 80S.l map has additional, more direct holes, running through the capsid shell, which are located at points on the surface that lie between the 2-fold and quasi-3-fold axes of symmetry (Fig. 7B, D, and F
The 80S and 135S density maps are generally displayed at a contour level between 1σ and 2σ (Fig. 2
, and 5
), but when viewed at a slightly higher, stringent contour level (3.5σ, not shown), the 80S.l map displays large oblong holes along the 2-fold axis that provide straight pathways from inside to outside. In their longer dimension, these holes run from one quasi-3-fold axis to the other; in their shorter dimension, the holes measure about 15 Å in width, roughly corresponding to the width of the hole in the pseudoatomic model (Fig. 8
). In the 80S.e density map at 3.5σ, a similar direct hole can be seen, but instead of one large hole across the 2-fold axis there are two smaller holes separated by the 2-fold-bridge of density. Differences between 80S.e and 80S.l in this region suggest that rearrangement of the peptides that make up the 2-fold bridges facilitates the 80S.e-to-80S.l transition during RNA release. The bridging region seen in 80S.e at 3.5σ is part of the more extensive bridge seen in both 80S reconstructions at lower contour levels (Fig. 3B
), which is broader and extends further inward, to a lower radius. 80S.e and 80S.l thus differ from one another in the contour levels required to see specific features, and the majority of the bridge occurs at a distinctly lower contour level than most of the rest of the capsid, which suggests partial occupancy, whose extent differs between the two 80S populations and most likely increases over time.
The holes through the 80S density maps are located between 5-fold-symmetry-related protomers and extend between the 2-fold and quasi-3-fold axes of symmetry (Fig. 8B
). The holes in the density maps are blocked in the immediate vicinity of the 2-fold axes by a bridge of density that we attribute to residues of the amino-terminal extension of VP2. The holes in the 80S pseudoatomic models (Fig. 8C and D
) appear to extend all the way across the 2-fold axes because the residues that contribute to the bridge density have not been included in the atomic models. Note that the holes in the 80S pseudoatomic models are easily large enough for single-stranded RNA to pass through them and that the low density levels for the bridges that block these holes in the density maps suggests that the bridges are only partially occupied. The partial occupancy raises the possibility that at least some number of the large holes at the 2-fold axes may be open during RNA release. In the native 160S structure, there is continuous density in these interface regions. Thus, in 160S, portions of the VP1 GH loop, the VP3 EF loop and GH loop, the VP2 and VP3 carboxyl-terminal polypeptides, and portions of the N-terminal peptide of VP2 all interact with one another across interfaces between symmetry-related protomers, in places where holes exist in 80S. Mutations to residues in this particular interface have been shown to influence the formation of pentamers (38
), receptor binding (13
), and the ability of viruses to undergo both the 73S-to-160S transition (1
) and the 160S-to-135S transition (13
) (Fig. 9
). Based on these data and the symmetrized reconstructions, the locus of RNA exit could be anywhere along the intersubunit interface that extends from the pseudo-3-fold to the 2-fold.
In order to elucidate the exact egress point from the capsid shell of the single copy of the RNA genome and to provide details of the interaction with RNA, we would need asymmetric information. In the present study, we have used icosahedral averaging across multiple particles to determine these structures, which provides greater resolution but limits what we can learn about the unique site. In a related study of this same heat-treated virus sample, we have used cryo-tomography and asymmetric single-particle reconstruction procedures to map the footprint of the exiting RNA in a subset of the particles that have density for RNA inside and outside the capsid, and confirmed that the RNA does indeed exit at the interface between 5-fold-symmetry-related protomers, in an area between the 2-fold symmetry axis and the quasi-3-fold axis (Bostina et al., submitted).
Why are the structural changes so small?
We had anticipated that the structural changes associated with the 160S-to-135S and 135S-to-80S transitions would be much larger than the changes that we have observed. Those expectations had been based on the large changes in sedimentation in the 135S particle and on the need to externalize large peptides and RNA. In our previous reports of the 80S (5
) and 135S (5
) structures, we rationalized the small magnitude of the changes by suggesting that there might be additional transient intermediates in both structural transitions that are significantly expanded, creating openings for the externalization of peptides or RNA. This argument was based in part on an analogy with the expansion of T=3 plant viruses, which is induced by exposing the virus to slightly alkaline pH in the presence of chelators of divalent cations and which (as in poliovirus) results in the externalization of the amino-terminal extensions of the capsid proteins. Furthermore, when the expansion of tomato bushy stunt virus (TBSV) (26
) or turnip crinkle virus (TCV) (34
) is quenched by lowering the pH rapidly, the particles are trapped in a partially expanded state with their amino-terminal extensions caught on the outside of the particle. According to this analogy, the more fully expanded state of TBSV or TCV would correspond to the imagined transient fully expanded intermediate of poliovirus, and their quenched state would correspond to the 135S particle.
However, our current cryo-EM studies of poliovirus 80S particles provide no direct evidence for the existence of a form that is more expanded than the ones we have observed. In contrast to 135S particles (which each contain a full complement of RNA), there is good experimental support for the idea that much of the population of 80S particles has been “caught in the act,” with RNA trapped partway through the capsid. Supporting evidence includes the existence of at least two states of the 80S particle, with a variable amount of density on the inside that corresponds to residual RNA, and the finding that a small but significant percentage of the 80S.e particles have putative RNA density features both inside and outside the particle at the same time. Nevertheless, 135S particles and both forms of 80S particles all appear to be very similar in overall size (Fig. 2F
). Plausible explanations for this include the possibility that the holes observed at the particle 2-fold axes (perhaps in symmetry copies where the bridging density is absent) are large enough to allow passage of single-stranded regions of the RNA. Alternatively, if the observed holes are not large enough, and a still greater icosahedrally symmetric expansion of the capsid is required (as in TBSV and TCV), then it remains possible that the RNA externalization steps are tightly coupled with a transient expansion, with recontraction to the observed structure occurring during pauses. As a final alternative, we cannot rule out the possibilities that the icosahedral symmetry of the capsid is broken only locally at the site of RNA externalization and that the capsid expansion (or hole creation) at that site is too limited in its extent to have a detectable impact on the icosahedrally symmetrized cryo-EM reconstructions that we report here. In an effort to address those possibilities, we are currently attempting to extend the resolution of our asymmetric reconstruction of “caught in the act” particles (work in progress), though at present, its resolution is not yet high enough to answer the question.
Why are there two different forms of the 80S particle, and what does it mean for there to be residual RNA density inside the shell?
The presence of two structural classes of 80S particles, each containing a variable amount of residual RNA, was first observed in cryo-EM studies of 80S particles of rhinovirus (27
). However, the significance of the residual RNA and of the existence of two classes was not clear. Our observations that similar classes of 80S particles are seen in poliovirus argue strongly that the existence of these two forms is an inherent property of the RNA-release process, at least in the enterovirus and rhinovirus genera of the picornavirus family. In thinking about the significance of these observations, it is important to note that the RNA genome, which is extensively folded into complex double-stranded structures in the virion, must be locally unfolded into single-stranded regions during RNA release. Thus, our experimental protocols for producing the 80S particles in vitro
call for the virus to be heated to temperatures which are close to the bulk melting temperature of the RNA (49
), and we have previously demonstrated that the dyes that bind double-stranded RNA are released during the release of RNA from virions, implying that RNA unfolding must be taking place in natural infections as well (8
To account for the observation that 80S particles contain variable levels of RNA on the inside, we postulate that unfolding of secondary structure is more difficult in some regions of the genome than at others. Thus, RNA release could take place in stages, with pauses occurring at points where the secondary structure is more stable. As an interesting aside, note that unfolding, followed by externalization, and then refolding of the externalized segments of the RNA provide a ratchet that could help to drive the externalization of the RNA to completion once initiated. We can further postulate that the failure to find externalized RNA in the majority of particles that have residual RNA density on the inside is due to hydrolysis by RNases in the sample during pauses. The existence of two classes of particles might then be explained by whether or not RNA continued to be engaged by the exit machinery. Thus, the 80S.l class might include both the particles that had finished releasing their genomes and those whose RNA had disengaged after hydrolysis, whereas the 80S.e class might represent those particles that have partially released RNA but in which the RNA remains engaged with the exit site even after hydrolysis.