Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously

Plant-made polio type 3 stabilized VLPs—a candidate synthetic polio vaccine

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized viruslike particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single particle reconstruction reveals a structure almost indistinguishable from wild-type PV3

Insights into Minor Group Rhinovirus Uncoating: The X-ray Structure of the HRV2 Empty Capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process

Cryo-electron microscopy reconstruction of a poliovirus-receptor-membrane complex.

To study non-enveloped virus cell entry, a versatile in vitro model system was developed in which liposomes containing nickel-chelating lipids were decorated with His-tagged poliovirus receptors and bound to virus. This system provides an exciting opportunity for structural characterization of the early steps in cell entry in the context of a membrane. Here we report the three-dimensional structure of a poliovirus-receptor-membrane complex solved by cryo-electron microscopy (cryo-EM) at a resolution of 32 A. Methods were developed to establish the symmetry of the complex objectively. This reconstruction demonstrates that receptor binding brings a viral five-fold axis close to the membrane. Density is clearly defined for the icosahedral virus, for receptors (including known glycosylation sites) and for the membrane bilayer. Apparent perturbations of the bilayer close to the viral five-fold axis may function in subsequent steps of cell entry

Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry.

During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes

Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit - Differences in structure and function relative to unliganded UL44

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile(135) of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44

Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus-receptor-membrane complex at 30 A resolution.

As a long-term goal we want to use cryoelectron tomography to understand how non-enveloped viruses, such as picornaviruses, enter cells and translocate their genomes across membranes. To this end, we developed new image-processing tools using an in vitro system to model viral interactions with membranes. The complex of poliovirus with its membrane-bound receptors was reconstructed by averaging multiple sub-tomograms, thereby producing three-dimensional maps of surprisingly high-resolution (30 A). Recognizable images of the complex could be produced by averaging as few as 20 copies. Additionally, model-free reconstructions of free poliovirus particles, clearly showing the major surface features, could be calculated from 60 virions. All calculations were designed to avoid artifacts caused by missing information typical for tomographic data ("missing wedge"). To investigate structural and conformational variability we applied a principal component analysis classification to specific regions. We show that the missing wedge causes a bias in classification, and that this bias can be minimized by supplementation with data from the Fourier transform of the averaged structure. After classifying images of the receptor into groups with high similarity, we were able to see differences in receptor density consistent with the known variability in receptor glycosylation

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim.

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24