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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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Most dsRNA viruses have a genome-enclosing capsid that comprises 120 copies of a single coat protein (CP). These 120 CP subunits are arranged as asymmetrical dimers that surround the icosahedral fivefold axes, forming pentamers of dimers that are thought to be assembly intermediates. This scheme is violated, however, in recent structures of two dsRNA viruses, a fungal virus from family Partitiviridae and a rabbit virus from family Picobirnaviridae, both of which have 120 CP subunits organized as dimers of quasisymmetrical dimers. In this study, we report the CP backbone trace of a second fungal partitivirus, determined in this case by electron cryomicroscopy and homology modeling. This virus also exhibits quasisymmetrical CP dimers that are connected by prominent surface arches and stabilized by domain swapping between the two CP subunits. The CP fold is dominated by alpha-helices, although beta-strands mediate several important contacts. A dimer-of-dimers assembly intermediate is again implicated. The disordered N-terminal tail of each CP subunit protrudes into the particle interior and likely interacts with the genome during packaging and/or transcription. These results broaden our understanding of conserved and variable aspects of partitivirus structure and reflect the growing use of electron cryomicroscopy for atomic modeling of protein folds., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.

We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage phi29, at subnanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the phi29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, an approximately 60A diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host.

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, NDelta34, 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 NDelta34 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.

In multistep reactions, stability of intermediates is critical to the rate of product formation and a significant factor in generating kinetic traps. The capsid protein of cowpea chlorotic mottle virus (CCMV) can be induced to assemble into spherical particles of 30, 60, and 90 dimers. Based on examining assembly kinetics and reaction end points, we find that formation of uniform, ordered structures is not always a result of reactions that reach equilibrium. Equilibration or, alternatively, kinetic trapping can be identified by a straightforward analysis. Altering the assembly path of "spherical" particles is a means of controlling the distribution of products, which has broad applicability to self-assembly reactions.