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CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli . We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. [ABSTRACT FROM AUTHOR]

Abstract: A three-dimensional reconstruction of Sindbis virus at 7.0 Å resolution presented here provides a detailed view of the virion structure and includes structural evidence for key interactions that occur between the capsid protein (CP) and transmembrane (TM) glycoproteins E1 and E2. Based on crystal structures of component proteins and homology modeling, we constructed a nearly complete, pseudo-atomic model of the virus. Notably, this includes identification of the 33-residue cytoplasmic domain of E2 (cdE2), which follows a path from the E2 TM helix to the CP where it enters and exits the CP hydrophobic pocket and then folds back to contact the viral membrane. Modeling analysis identified three major contact regions between cdE2 and CP, and the roles of specific residues were probed by molecular genetics. This identified R393 and E395 of cdE2 and Y162 and K252 of CP as critical for virus assembly. The N-termini of the CPs form a contiguous network that interconnects 12 pentameric and 30 hexameric CP capsomers. A single glycoprotein spike cross-links three neighboring CP capsomers as might occur during initiation of virus budding. [Copyright &y& Elsevier]

Abstract: Filamentous fungus Fusarium poae is a worldwide cause of the economically important disease Fusarium head blight of cereal grains. The fungus is itself commonly infected with a bisegmented dsRNA virus from the family Partitiviridae. For this study, we determined the structure of partitivirus Fusarium poae virus 1 (FpV1) to a resolution of 5.6Å or better by electron cryomicroscopy and three-dimensional image reconstruction. The main structural features of FpV1 are consistent with those of two other fungal partitiviruses for which high-resolution structures have been recently reported. These shared features include a 120-subunit T =1 capsid comprising 60 quasisymmetrical capsid protein dimers with both shell and protruding domains. Distinguishing features are evident throughout the FpV1 capsid, however, consistent with its more massive subunits and its greater phylogenetic divergence relative to the other two structurally characterized partitiviruses. These results broaden our understanding of conserved and variable elements of fungal partitivirus structure, as well as that of vertebrate picobirnavirus, and support the suggestion that a phylogenetic subcluster of partitiviruses closely related to FpV1 should constitute a separate taxonomic genus. [ABSTRACT FROM AUTHOR]

Abstract: Nudaurelia capensis ω virus is a T=4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 Å diameter) and have T=4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP5.0) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 Å. VLP5.0 undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent γ peptide (residues 571–641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT7.6 ↔NT5.0). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT7.6, NT5.0, and VLP5.0 at about 8, 6, and 6 Å resolution, respectively. We employed the 2. 8-Å X-ray model of the mature virus, determined at pH 7.0 (XR7.0), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT5.0 is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T=4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic γ peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a γ peptide, while the other is bent with the γ peptide disordered) and dramatic stabilization of the particle. The γ peptide at the flat contact remains dynamic when cleavage cannot occur (NT5.0) but becomes totally immobilized by noncovalent interactions after cleavage (VLP5.0). [Copyright &y& Elsevier]

Abstract: The coat protein (CP) of cowpea chlorotic mottle virus assembles exclusively into a T =3 capsid in vivo and, under proper conditions, in vitro. The N-terminal domain of CP has been implicated in proper assembly and was viewed as a required switch for mediating hexamer and pentamer formation in T =3 assembly. We observed that a mutant CP lacking most of the N-terminal domain, NΔ34, assembles, in vitro, into statistically predictable numbers of: native-like T =3 capsids of 90 dimers; “T =2” capsids of 60 dimers; T =1 capsids of 30 dimers. We generated cryo-EM image reconstructions of each form and built pseudo-atomic models based on the subunits from the crystal structure of plant-derived T =3 virus allowing a detailed comparison of stabilizing interactions in the three assemblies. The statistical nature of the distribution of assembly products and the observed structures indicates that the N-terminus of CP is not a switch that is required to form the proper ratio of hexamers and pentamers for T =3 assembly; rather, it biases the direction of assembly to T =3 particles from the possibilities available to NΔ34 through flexible dimer hinges and variations in subunit contacts. Our results are consistent with a pentamer of dimers (PODs) nucleating assembly in all cases but subunit dimers can be added with different trajectories that favor specific T =3 or T =1 global particle geometries. Formation of the “T =2” particles appears to be fundamentally different in that they not only nucleate with PODs, but assembly propagates by the addition of mostly, if not exclusively PODs generating an entirely new subunit interface in the process. These results show that capsid geometry is flexible and may readily adapt to new requirements as the virus evolves. [Copyright &y& Elsevier]

Abstract: Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the “non-fusogenic” mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these “fusogenic” orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 Å, showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric λ-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric σ-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks “hub-and-spokes” complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T = 13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric μ-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features. [Copyright &y& Elsevier]

Abstract: We investigated the implicit learning of a linguistically relevant variable (animacy) in a natural language context (namely, the relation of forms of determiners to semantics). Trial by trial subjective measures indicated that exposure to a form–animacy regularity led to unconscious knowledge of that regularity. Under the same conditions, people did not learn about another form–meaning regularity when a linguistically arbitrary variable was used instead of animacy (size relative to a dog). Implicit learning is constrained to acquire unconscious knowledge about features with high prior probabilities of being relevant in that domain. [Copyright &y& Elsevier]

Abstract: Model-based, three-dimensional (3D) image reconstruction procedures require a starting model to initiate data analysis. We have designed an ab initio method, which we call the random model (RM) method, that automatically generates models to initiate structural analysis of icosahedral viruses imaged by cryo-electron microscopy. The robustness of the RM procedure was demonstrated on experimental sets of images for five representative viruses. The RM method also provides a straightforward way to generate unbiased starting models to derive independent 3D reconstructions and obtain a more reliable assessment of resolution. The fundamental scheme embodied in the RM method should be relatively easy to integrate into other icosahedral software packages. [Copyright &y& Elsevier]

Abstract: The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a β-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained. [Copyright &y& Elsevier]

Abstract: Single particle analysis is a valuable tool in cryo-electron microscopy for determining the structure of biological complexes. However, the conformational state and the preparation of the sample are factors that play a critical role in the ultimate attainable resolution. In some cases extensive analysis at the microscope of a sample under different conditions is required to derive the optimal acquisition conditions. Currently this analysis is limited to raw micrographs, thus conveying only limited information on the structure of the complex. We are developing a computing system that generates a three-dimensional reconstruction from a single micrograph acquired under cryogenic and low dose conditions, and containing particles with icosahedral symmetry. The system provides the microscopist with immediate structural information from a sample while it is in the microscope and during the preliminary acquisition stage. The system is designed to run without user intervention on a multi-processor computing resource and integrates all the processing steps required for the analysis. Tests performed on experimental data sets show that the probability of obtaining a reliable reconstruction from one micrograph is primarily determined by the quality of the sample, with success rates close to 100% when sample conditions are optimal, and decreasing to about 60% when conditions are sub-optimal. The time required to generate a reconstruction depends significantly on the diameter of the particles, and in most instances takes about 1min. The proposed approach can provide valuable three-dimensional information, albeit at low resolution, on conformational states, epitope binding, and stoichiometry of icosahedral multi-protein complexes. [Copyright &y& Elsevier]

Abstract: We demonstrated unconscious learning of task-irrelevant perceptual regularities in a Serial Reaction Time (SRT) task in both visual and auditory domains. Participants were required to respond to different letters (‘F’ or ‘J’, experiment 1) or syllables (‘can’ or ‘you’, experiment 2) which occurred in random order. Unbeknownst to participants, the color (red, green, blue or yellow) of the two letters or the tone (1–4) of the syllables varied according to certain rules. Reaction times indicated that people indeed learnt both the color and tonal regularities indicating that task-irrelevant sequence structure can be learned perceptually. In a subsequent prediction test of knowledge of the color or tonal cues using subjective measures, we showed that people could acquire task irrelevant knowledge unconsciously. [Copyright &y& Elsevier]