Cryo-Electron Tomography of Bacterial Viruses

Virology 435 (2013) 179–186

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Virology

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Review Cryo-electron tomography of bacterial viruses

Ricardo C. Guerrero-Ferreira, Elizabeth R. Wright n

Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA article info abstract

Bacteriophage particles contain both simple and complex macromolecular assemblages and machines Keywords: that enable them to regulate the infection process under diverse environmental conditions with a broad Bacteriophage range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible Cryo-electron microscopy to observe the interactions of bacteriophages with their host cells under native-state conditions at Cryo-EM unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to Cryo-electron tomography studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of Cryo-ET investigation and future directions in the ﬁeld are also discussed. Sub-tomogram averaging & 2012 Elsevier Inc. All rights reserved.

Contents

Introduction...... 179 Biological electron microscopy and the development of cryo-electron microscopy ...... 180 Imaging whole virus (isolated particles) ...... 182 Imaging attachment and DNA injection processes...... 183 Imaging the interactions of bacteriophages with bacterial appendages ...... 183 Imaging bacteriophage structural modiﬁcations during DNA ejection ...... 184 Ultrastructure applied to study bacteriophage evolution ...... 185 Conclusions ...... 185 Acknowledgments ...... 185 References ...... 185

Introduction icosahedral reconstruction methods has been used to solve the high-resolution three-dimensional (3D) structures of numerous Electron microscopy (EM) is one of the best direct imaging bacteriophage capsids. This information has proven essential for methods for studying the interactions between bacteriophages determining common pathways of capsid maturation. However, for and bacterial cells. However, conventional EM carries the intrinsic bacteriophage structures that either do not maintain a relationship disadvantage of requiring the use of heavy metal stains, ﬁxatives, with the icosahedral symmetry of the capsid or are present at one dehydration, and sectioning, all of which introduce preservation vertex of the capsid, reconstruction techniques that impose sym- artifacts to the specimen and may limit the amount of useful metry and, therefore limits to the structural information that may structural information (Lucic et al., 2005,2008). Plunge-freezing be resolved from the resultant 3D map. To this end, a number of and high-pressure freezing methods were introduced in the 1980s groups developed approaches for reconstructing bacteriophages and rapidly became the best methods to employ for the preserva- using algorithms that do not apply icosahedral averaging. Using tion of ﬁne ultrastructure in biological specimens (Dubochet et al., phi-29 as a model system, Morais et al. (2001),introducedamethod 1983; Lepaultetal.,1983; McDowall et al., 1983). Cryo-EM imaging for the reconstruction of symmetry mismatches in tailed bacter- of bacteriophages plunge frozenonEMgrids,combinedwith iophages. This algorithm emerged as the basis implemented in most of the current asymmetric reconstruction techniques. These methods have been used to determine key bacteriophage structures, n Corresponding author. Fax: þ1 404 727 9223. including the complete architecture of T7 (Agirrezabala et al., 2005), E-mail address: [email protected] (E.R. Wright). P22 (Chang et al., 2006), phi-29 (Tang et al., 2008), and Epsilon15

Table 1 Overview of structural studies of bacteriophages that use cryo-electron tomography.

Study Phage Host Equipment References

Genome ejection T5 Escherichia coli Philips CM 120 Bioﬁlter (120 kV) Philips CM 300 FEG (Bohm et al., 2001) (300 kV) Genome ejection P-SSP7 Prochlorococcus JEOL JEM3200FSC (300 kV) (Liu et al., 2010) Initial interaction P1 Escherichia coli FEI Tecnai G2 Polara (300 kV) (Liu et al., 2011) Initial interaction FCbK/ Caulobacter crescentus FEI Tecnai G2 Polara (300 kV) FEI Tecnai G2 F30 (300 kV) (Guerrero-Ferreira et al., FCb13 2011) Whole cell/virus assembly STIV Sulfolobus solfataricus FEI Tecnai G2 Polara (300 kV) (Fu et al., 2011) Whole virus F12 Pseudomonas syringae pv. Tecnai F20 (200 kV) (Hu et al., 2008) phaseolicola Whole virus Epsilon 15 Salmonella anatum JEOL JEM2200FS (200 kV) (Murata et al., 2010) Whole virus BPP-1 Bordetella spp. FEI Tecnai G2 Polara (300 kV) (Dai et al., 2010) Whole virus F6 Pseudomonas syringae FEI Tecnai-12 (120 kV) (Nemecek et al., 2010) Whole virus F6 Pseudomonas syringae FEI Tecnai-12 (120 kV) (Nemecek et al., 2011) Whole virus/DNA ejection F8a Bacillus anthracis FEI Tecnai G2 Polara (300 kV) (Fu et al., 2011) Whole virus/Initial F12 Pseudomonas syringae pv. Tecnai F20 (200 kV) JEOL JEM3200FSC (300 kV) (Leo-Macias et al., 2011) interaction phaseolicola

(Jiang et al., 2006). All together, these studies provided substantial very wide spread because of its speed and simplicity and that it information regarding the native structures of bacteriophage tail allows one to determine the shape, size, and external structure of complexes and the DNA packaging and infection apparatus. The an isolated or purified specimen. Since then, many other heavy success of obtaining complete structures of bacteriophages in metal stains have been tested and are available for staining a wide isolation provided greater impetus for determining the 3D struc- range of specimen types. Single particle 3D reconstruction methods tures of bacteriophage and the host cell during the infection process. were originally adapted for and applied to particles that were In order to visualize the complex 3D ultrastructure of entire negatively stained. However, even with its many benefits, the major eukaryotic and prokaryotic cells and pleiomorphic viruses, cryo- disadvantages to using negative stain techniques are the loss of electron tomography (cryo-ET) has matured as an unparalleled sample hydration, limitation of specimen size, and the lack of imaging technology (Baumeister, 2004,2005; Lucic et al., 2005; internal structure information. Murphy and Jensen, 2007; Yahav et al., 2011). Over the past few In order to visualize the entire structure of thicker cells and years, researchers have used cryo-ET to study the bacteriophage tissues, it is necessary to preserve the specimen in such a way infection cycle and a number of the specific events that take place that it may be cut into thinner sections for viewing in the TEM. have been visualized at unmatched resolution (Table 1). This review A number of methods were developed for the preservation of highlights the history of biological EM, the development of cryo-EM specimens prior to their embedment in polymer resins (Claude and cryo-ET, and the recently published contributions to the field of and Fullam, 1945; Glauert et al., 1956; Luft, 1961; Porter et al., bacteriophage biology that utilize cryo-ET as the main structural 1945; Watson, 1958a,b); the basic steps are presented below. technique. With the goal of offering an overview of the broad range Prior to being embedded in resin, the macromolecules of the of applications of cryo-ET to the study of bacterial viruses, the specimen need to be fixed and the water removed through structural studies discussed here answer questions regarding bac- dehydration. The most commonly used primary fixation scheme teriophage attachment, genome ejection, assembly and egress. includes a combination of glutaraldehyde and paraformaldehyde at concentrations ranging from 0.5% to 4% for crosslinking the proteins followed by secondary fixation with 2%–4% osmium Biological electron microscopy and the development tetroxide that reacts with proteins and unsaturated lipids. After of cryo-electron microscopy fixation, the specimen is dehydrated through a graded series of acetone or ethanol and is then suspended in a liquid polymer Biological EM has a long history, which began with the devel- resin that is subsequently cured in an oven. Once thin sections are opment of the transmission electron microscope (TEM) in the 1930s cut from the polymerized resin-specimen block, the sections are and early 1940s (Bozzola and Russell, 1998). Hydrated specimens, further stained with a combination of two heavy metal stains, such as cells, tissues, and purified macromolecules, require fixation, uranyl acetate and lead citrate. This methodology can be adapted dehydration, and staining prior to their introduction into the high and modified for many specimens and procedures; however, vacuum environment of the TEM. Two approaches were established the major limitations are that the specimen is chemically and to address the TEM imaging needs of the biological research physically altered through the introduction of fixatives, resins, community. First, negative-stain techniques were developed for and heavy metal stains. the rapid preservation and staining of biological materials in Cryo-EM was born out of the desire to maintain biological suspension; and second, procedures were pioneered for embedding specimen hydration for both direct imaging and electron diffrac- thicker specimens in resin for sectioning. Both methods are still tion in the TEM. The first frozen hydrated specimen imaged by widely used for processing samples prior to imaging, a brief electron diffraction in the TEM was catalase crystals by Taylor and summary of each is provided below (Bozzola and Russell, 1998). Glaeser (1974). Here, they froze catalase crystals in a thin film of Negative staining of small particles for TEM investigation began ice using liquid nitrogen as the cryogen. Several years later, in the 1950s. Three independent research groups, Hall (1955); cryogens other than liquid nitrogen were used to freeze samples Huxley (1956),andBrenner and Horne (1959) used solutions of and it was determined that the freezing rates were faster and phosphotungstic acid to fix and stain virus particles. Some of limited the introduction of crystalline ice. Now, most aqueous the viruses first imaged by negative stain TEM include tomato samples are prepared for cryo-EM or cryo-ET by first applying a bushy stunt virus (BSV), tobacco mosaic virus (TMV), and T2 small aliquot of a suspension to an EM grid, blotting the grid to bacteriophage. The technique of negative stain TEM has become near dryness, and then rapidly plunge freezing it in a cryogen R.C. Guerrero-Ferreira, E.R. Wright / Virology 435 (2013) 179–186 181

(typically liquid ethane or liquid propane) cooled to liquid tilt range of the specimen holder and microscope goniometer, nitrogen temperatures (196 1C). This method for plunge which creates what is known as a ’’missing wedge’’ of data in the freezing grids preserves the biological sample in a thin layer of 3D reconstruction. The missing wedge corresponds to the incom- vitreous, non-crystalline ice in a near native state and eliminates plete sampling of Fourier space, and may directly affect subsequent artifacts that result from sample fixation, dehydration or staining 3D alignment and averaging procedures. Primary data collection (Dubochet et al., 1988; Lepault et al., 1983). Once prepared, a that incorporates dual axis tomography (Iancu et al., 2005; cryo-EM grid is stored under liquid nitrogen until it is transferred Mastronarde, 1997; Penczek et al., 1995) allows the investigator to a ‘cryo-stage.’ The cryo-stage is designed to maintain the to acquire tilt series of the specimen along two orthogonal axes specimen at close to liquid nitrogen temperatures while the (i.e. sample rotated by 901). By sampling Fourier space along two specimen is imaged in the electron microscope. axes at a tilt range of 7601, one reduces the amount of unmeasured There are a few key features to the electron microscopes used space, or the ‘‘missing wedge,’’ from 33% (one axis) to 16% (two for cryo-ET studies of bacteriophages, whole bacterial cells, or the axes) (Iancu et al., 2005).Thisresultsinanincreaseintheisotropic interactions between the two (Table 1). First, when thicker samples resolution of the reconstructed data; consequently, improving data are imaged in the electron microscope, multiple scattering events quality for downstream processing such as sub-tomogram aver- result due to the limitation of the mean free path of the electrons aging (see below), template-directed macromolecule identification (200 nm for 120 kV and 350 nm for 300 kV) (Grimm et al., (Bohm et al., 2000; Frangakis et al., 2002), and the selection or 1998). In order to overcome these limits, groups generally use a identification of a feature that was not observed along only one axis microscope with an intermediate accelerating voltage (200–300 kV) (Iancu et al., 2005; Mastronarde, 1997). However, there are two for improved sample penetration by the electron beam and/or an major limitations of dual axis cryo-ET. First, the low total electron energy filter to remove noise-generating inelastically scattered dose used during image acquisition is further fractionated over a electrons from the final image that is acquired on the CCD greater number of images, which may negatively impact image (Grimm et al., 1996). Specimens that are substantially thicker than alignment during the reconstruction process. Second, additional 1 mm may be thinned by a number of methods prior to introduction improvements in the software for merging the two axes may be into the microscope (Al-Amoudi et al., 2004; Marko et al., 2007). necessary to increase the quality of the final reconstruction. Electron tomography involves the specimen of interest being semi- In addition to better 3D reconstructions through dual axis automatically or automatically tilted to angles that range between tomography, algorithms have been developed for averaging sub- 7601 and 7701 (Baumeister et al., 1999; Mastronarde, 2005; volumes of tomograms (Briggs et al., 2009; Leo-Macias et al., Suloway et al., 2009; Zheng et al., 2004). A series of individual 2D 2011; Schmid and Booth, 2008). Sub-tomogram averaging proce- images are acquired of the area of interest over the tilt range at tilt dures generate isotropic, higher signal-to-noise structures of a intervals of 1-21. The ‘tilt series’ is then computationally processed complex of interest. It has proved to be especially powerful in to generate a 3D reconstruction of the specimen (Gilbert, 1972a,b; studies of viral ultrastructure, specifically those of viral glycopro- Kremer et al., 1996; Lee et al., 2008; Marabini et al., 1997). teins (Fontana et al., 2012; Liu et al., 2008; Zanetti et al., 2006; However, there is a physical limit of 1401 associated with the Zhu et al., 2008), lattice-forming structural proteins (Briggs et al.,

Fig. 1. 3D rendering of F12 bacteriophage structure. (A) Surface rendering of an individual virion depicting two types of protruding densities, labeled with red (donut- shaped) and blue (elongated). (B) 7 nm thick section through a tomographic reconstruction of bacteriophage F12. The membrane and the two classes of protruding densities are clear. Red and blue lines represent the segmentation of donut-shaped and elongated protrusions, respectively. (C) 3D rendering resulting from the segmentation represented in panel ‘B’ with yellow label representing the membrane surface and red and blue the surface spikes. (D) Cross-section of the bacteriophage particle from panel ‘A’ illustrating two discrete shells. (E) Section through a F12 bacteriophage displaying the connection of the inner face of the envelope with the nucleocapsid (arrow). (F) Aligned average of 180 F12 bacteriophage particles clearly depicting two distinct shells. Reprinted from Virology, 372, Hu et al., Electron cryo- tomographic structure of cystovirus phi 12, 1–9, Copyright (2008), with permission from Elsevier. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 182 R.C. Guerrero-Ferreira, E.R. Wright / Virology 435 (2013) 179–186

2009; de Marco et al., 2010; Wright et al., 2007), and bacterioph- description of a new kind of receptor binding protein, with a age as presented below. toroidal or donut-like shape (Fig. 1). To complement these findings, Leo-Macias et al. (2011) carried out sub-tomogram averaging of the surface complex of F12, averaging 744 copies of this surface density from 158 bacterioph- Imaging whole virus (isolated particles) age particles. Approximately five, randomly distributed, toroidal, surface complexes were found to be 13 nm from the membrane of Cryo-EM followed by icosahedral image reconstruction was the cystovirus. With a resolution of 2.6 nm, obtained by sub- used to determine the 3D organization of the nucleocapsid of the tomogram averaging, the toroid was found to be 19 nm in cystovirus F6aswellasEscherichia coli-expressed and assembled diameter and to have six-fold symmetry. A 5 nm connecting procapsid-related particles (Butcher et al., 1997). In a more recent density was also observed, that attaches the surface protein to study of another cystovirus, F12, cryo-ET was employed to study the virus envelope. The authors proposed that the toroidal the overall substructure of the phage and the shape and distribu- structure evolved as an alternative strategy for the cystovirus to tion of the surface proteins (Hu et al., 2008). Cryo-ET was the infect additional cells. method of choice to determine the structure of the surface proteins Cryo-ET and sub-tomogram averaging were used by Nemecek because their positions do not relate to the icosahedral symmetry et al. (2011) to establish the occupancy of the packaging NTPase of the nucleocapsid. The particles exhibit two discrete shells (P4) and the RNA-dependent RNA polymerase (RdRP, P2) of resulting from the nucleocapsid and the membranous envelope, bacteriophage F6ofP. syringae. 560 individual procapsids were respectively. Connections between the nucleocapsid and the inner extracted from three tomograms and aligned to an existing single surface of the envelope appear to center the nucleocapsid within particle reconstruction. With this approach, it was feasible to the viral envelope. This study resolved the distribution of both identify both P2 and P4 on individual procapsids, count individual elongated densities that maintained close associations with the molecules, and map their locations. Their results suggest that membrane surface and donut-shaped densities set further away both subunits are randomly incorporated and that copy numbers from the membrane surface (Fig. 1). It was previously determined may be determined by their individual rates of synthesis. Their that the cystovirus F6 does not have donut shaped complexes on evaluation of P2 and P4 locations also showed that these proteins its surface. The viral surface is instead exclusively decorated with do not tend to cluster or co-localize, suggesting that their horizontal spikes that are necessary for attachment to the host activities are not physically coupled. In another study, cryo-EM bacterium Pseudomonas syringae (Bamford et al., 1987). Therefore, and cryo-ET were applied to demonstrate the process of procapsid cryo-ET was pivotal for elucidating the structural diversity of expansion in F6(Nemecek et al., 2011). By using sub-tomogram cystoviruses by establishing the presence of two asymmetrical averaging, this study classified individual vertices of compact and surface densities. Moreover, this technique allows the initial expanded F6. The aim was to visualize specific features on

Fig. 2. Binding of a T5 bacteriophage particle to a proteoliposome before DNA release. (a) Segmented 3D rendering of a T5 bacteriophage (depicted in blue) bound to a vesicle (displayed in gold) before DNA release. (b) Four XY slices (1.4 nm thick) through the same reconstruction depicted in (a). Arrows point to the tip of the bacteriophage tail inside the vesicle (Reprinted from Current Biology, 11/15, Bohm¨ et al., FhuA-mediated phage genome transfer into liposomes: A cryo-electron tomography study, 1168-1175, Copyright (2001), with permission from Elsevier.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) R.C. Guerrero-Ferreira, E.R. Wright / Virology 435 (2013) 179–186 183 individual procapsids rather than averaging these features tube closer to the cell, which causes the tail tube to puncture the through single particle methods. The process of classifying all cell envelope. vertices extracted from compact and expanded forms from the Other studies have also shown that structural alterations occur same tomograms revealed four distinct classes of vertices: two of in bacteriophages upon binding to the host cell and those changes them were similar to the compact procapsid vertex and the other are associated with genome release. Liu et al. (2010) carried two to expansion intermediates. out structural analysis of bacteriophage P-SSP7 of Prochlorococcus marinus (a cyanobacterium) using cryo-ET. Their results indicate that during the process of infection, the tail fiber of P-SSP7 has Imaging attachment and DNA injection processes the same conformation as that of empty bacteriophage particles. Furthermore, their results suggest that the tail-fibers induce In a structural study of T5 bound to receptor-containing a series of structural alterations in the portal vertex complex. proteoliposomes, Bohm et al. (2001) imaged the process of The accumulation of such conformational changes results in genome transfer. It is well established that during bacteriophage the successful ejection, or release, of bacteriophage genomic infection the viral genome is efficiently transferred into the material. bacterial host. The structural details of this process are, however, poorly understood. Cryo-ET revealed that after binding to the receptor, the tip of the bacteriophage tail passes through the lipid Imaging the interactions of bacteriophages with bacterial bilayer of the proteoliposome (Fig. 2) and undergoes a conforma- appendages tional change that increases its diameter from 2 to 4 nm and decreases its length from 50 to 23 nm. There are numerous examples in nature of how bacteriophage Cryo-ET and sub-tomogram averaging methods were also used accessory filaments promote bacteriophage interactions with by Liu et al. (2011) to study P1 bacteriophage particles attached to bacterial appendages and facilitate the infection of their hosts. E. coli mini-cells to determine the mechanism by which the Reports have shown that several bacteriophages utilize their tail bacteriophage interacts with the cell membrane of its host. filaments to adsorb to the bacterial flagellum. This interaction is A significant separation between the bacteriophage’s baseplate very specific, as in the cases of coliphage chi (Schade et al., 1967) and the cell’s outer membrane was identified during the transition or Bacillus subtilis bacteriophage PBS1 (Raimondo et al., 1968). In from an extended tail conformation to a contracted tail conforma- both cases, the bacteriophage benefits from rotation of the tion. They determined that long tail fibers attach to the cell flagellum, which allows the bacteriophage to access the sites of membrane in order to anchor the bacteriophage to the cell. Upon irreversible attachment and genome injection on the cell surface. tail sheath contraction, the long tail fibers become extended, Recently, Guerrero-Ferreira et al. (2011) used cryo-ET and causing the baseplate to move farther away from the cell. microbiology approaches to determine the role of a head filament This results in the movement of the bacteriophage’s head and tail during bacteriophage adsorption and the attachment of the FCbK/

Fig. 3. Segmented 3D volume from a cryo-electron tomogram of a FCb13-infected, C. crescentus cell illustrating head ﬁlament interacting with the ﬂagellum (Reprinted from PNAS, 108/24, Guerrero-Ferreira et al., Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus, 9963-9968, Copyright (2011). 184 R.C. Guerrero-Ferreira, E.R. Wright / Virology 435 (2013) 179–186

Fig. 4. The top panel depicts cryo-ET of representative bacteriophage 8a particles at each of the four states of DNA ejection. Differences in genome content and tail sheath length are evident. Notice the difference in baseplate morphology indicated by the arrows. The bottom panel depicts the corresponding segmented volumes of the four states, indicating viral components and size differences between the extended and contracted tail. Modiﬁed from Fu et al. (2011). Reprinted from Virology, 410/2, Fu et al. The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography, 141-148, Copyright (2011), with permission from Elsevier.

FCb13 tail to their host, Caulobacter crescentus. FCbK (and related on the multiple structural conformations that this bacteriophage bacteriophage FCb13) have an elongated head (60 nm in diameter goes through in order to release its genome from the capsid and 200 nm in length), a non-contractile tail (290 nm in length) during infection. The group describes four distinct structural with tail filaments (Leonard et al., 1972,1973) and an additional conformations that mainly vary in their genome content and tail filament protruding from the head of the bacteriophage (Leonard length (Fig. 4). Structural changes are also observed in the base- et al., 1972). The study by Guerrero-Ferreira et al. (2011) concludes plate in addition to changes in tail length during contraction. that the head filament is necessary for the initial interaction of the The authors concluded that these distinct states, which range from bacteriophage with the bacterial flagellum (Fig. 3). Flagellar rotation a genome-filled capsid and extended tail to an empty capsid and then promotes the translation of the bacteriophage towards the cell contracted tail, result in the translocation of the viral DNA from the pole, where irreversible attachment occurs. In addition, 2D cryo-EM bacteriophage 8a capsid into the cytoplasm of B. anthracis. and microbiology techniques confirmed the site of attachment to be No apparent difference was observed in the head structure the pili portals located at the C. crescentus pole. when comparing states 1 (Fig. 4A: DNA-filled capsid, extended tail) and 2 (Fig. 4B: DNA-filled capsid, contracted tail). The heads of bacteriophages in state 3 (Fig. 4C: partially DNA-filled capsid, Imaging bacteriophage structural modifications during contracted tail) contain densities that either corresponds to DNA ejection genome or protein. These densities were completely absent in state 4 (Fig. 4D: empty capsid, contracted tail) virions. The portal A recent study by Fu et al. (2011) analyzed more than 2000 protein is clearly visible in each of the four states, although its particles of bacteriophage 8a targeting spores of Bacillus anthracis. morphology is more easily depicted in bacteriophage particles in The results of this high throughput cryo-ET approach shed light state 4 (empty, contracted-tailed bacteriophages). The portal has R.C. Guerrero-Ferreira, E.R. Wright / Virology 435 (2013) 179–186 185 a typical wide-end inside the bacteriophage head, a central region of processes in the context of whole cells and at the same time the and a central channel sized for double-stranded DNA. A unique investigation of the macromolecular complexes involved in these feature of the bacteriophage 8a portal is an extra goblet-like processes (Muller-Reichert et al., 2007; Plitzko et al., 2009; van structure that extends from the 8a ’’portal crown domain’’ region Driel et al., 2009). toward the center of the capsid suggesting that it may play a role Bacteriophage biology developed as a means to the search for in DNA translocation. antibacterial therapies, the development of modern molecular biology and the generation of tools for recombinant DNA technology and genetic engineering. However, a great deal of information Ultrastructure applied to study bacteriophage evolution has been missing in regard to the complex structural relationships that exist between bacteria and bacteriophages. Now, bacterioph- In addition to elucidating viral functions by biochemical age biology has at its disposal a powerful new tool for studying methods and microbiological assays, the evaluation of virus cellular ultrastructure and infrastructure: cryo-ET. This technol- architecture by high-resolution structural methods has allowed ogy is rapidly evolving and has already been used to examine the clarification of existing long-range phylogenic relationships in several of the basic stages of the bacteriophage infection cycle. viral lineages (Ravantti et al., 2003). An example is bacteriophage Combining the strengths of bacteriophage biology with cryo-ET PRD1, which has an icosahedral protein coat. The major coat will open new frontiers in bacteriophage biology. protein, P3, has three interlocking subunits, each with two eight- stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. The P3 structure closely resembles Acknowledgments the equivalent protein in human adenovirus. Both viruses have similar overall architecture, with identical capsid lattices and The authors thank Dr. Ian Molineux for the invitation to attachment proteins at their vertices. Although the two dsDNA submit a minireview article to Virology and for critical reading viruses infect hosts from very different domains of life, their striking of the manuscript. We are grateful to Drs. Jens Holl and Gabriella similarities from major coat protein through capsid architecture Kiss for valuable help with manuscript preparation and helpful strongly suggest an evolutionary relationship (Benson et al., 1999). discussions. This work was supported in part by the Emory A common ancestor has also been proposed for these two viruses University, the Children’s Healthcare of Atlanta, the Georgia (Martin et al., 2001). High-throughput cryo-ET, combined with sub- Research Alliance, and the HFSP Grant RGP0051 to E.R.W. tomogram averaging, are ideal complementary approaches to genome-based methods to address questions regarding the evolution of viral ultrastructure in bacteriophage lineages. References

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