The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins ("ejection ...proteins") have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100-110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.
The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in ...its icosahedral capsid, and capsids as compartments for transcription and replication. ϕ6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an α-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
•Cryo-EM structure at 4.5 Å resolution•Visualize maturation-related conformational switches•Capsid protein fold
Nemecek et al. report a structure of a pentameric capsid protein from a dsRNA virus and uncover an unusual a-helical subunit fold. This structure adapts in the two conformations found in the procapsid. The capsid maturation process thus involves massive conformational changes.
UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose ...architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.
1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
2 Imaging Sciences Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA
...3 Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
4 Laboratoire de Génétique et Pathologie, IFREMER, 17390 La Tremblade, France
Correspondence Andrew J. Davison a.davison{at}vir.gla.ac.uk
Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster ( Crassostrea gigas ) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T =16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (U L , 167·8 kbp; U S , 3·4 kbp) each flanked by inverted repeats (TR L /IR L , 7·6 kbp; TR S /IR S , 9·8 kbp), with an additional unique sequence (X, 1·5 kbp) between IR L and IR S . Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses.
The GenBank/EMBL/DDBJ accession number of the OsHV-1 DNA sequence reported in this paper is AY509253 .
SDS-PAGE analysis of the OsHV-1 capsid proteins is available as supplementary material in JGV Online.
Present address: 9 rue Traversière, 17200 St Sulpice de Royan, France.
"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, ...as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the
giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them "mottled capsids". Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a "twisted hook" conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.
In yeast cells infected with the PSI+ prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled ...in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal ‘prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter (∼8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield ∼1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.
The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending ...inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three "ejection proteins" (E-proteins gp7, gp16, and gp20) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins' locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection.
While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins E-proteins), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.
The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 ...different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes' departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation.
This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit "protomers" are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.
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