Frank P. Booy

Encapsidated Conformation of Bacteriophage T7 DNA

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.

Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

ABSTRACT Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution

The three-dimensional structure of bovine papillomavirus has been determined to 9 Å resolution by reconstruction of high resolution, low dose cryo-electron micrographs of quench-f rozen virions. Although hexavalent and pentavalent capsomeres form star-shaped pentamers of the major capsid protein L1, they have distinct high-resolution structures. Most prominently, a 25 Å hole in the centre of hexavalent capsomeres is occluded in the pentavalent capsomeres. This raises the possibility that the L2 minor capsid protein is located in the centre of the pentavalent capsomeres. Inter-capsomere connections ∼10 Å in diameter were clearly resolved. These link adjacent capsomeres and are reminiscent of the helical connections that stabilize polyomavirus.

L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

ABSTRACT BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy.

Virus capsids constitute a diverse and versatile family of protein‐bound containers and compartments ranging in diameter from ~200 Å (mass~1 MDa) to >1500 Å (mass> 250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 Å, is disclosing novel structural motifs, assembly mechanisms, and the precise locations of major epitopes. Capsids are essentially symmetric structures, and icosahedral surface lattices have proved to be widespread. However, many capsid proteins exhibit a remarkable propensity for symmetry breaking, whereby chemically identical subunits in distinct lattice sites have markedly different structures and packing relationships. Temporal differences in the conformation of a given subunit are also manifested in the large‐scale conformational changes that accompany capsid maturation. Larger and more complex capsids, such as DNA bacteriophages and herpes simplex virus, are formed not by simple self‐assembly, but under the control of tightly regulated programs that may include the involvement of viral scaffolding proteins and cellular chaperonins, maturational proteolysis, and conformational changes on an epic scale. In addition to its significance for virology, capsid‐related research has implications for biology in general, relating to the still largely obscure assembly processes of macromolecular complexes that perform many important cellular functions.—Steven, A. C., Trus, B. L., Booy, F. P., Cheng, N., Zlotnick, A., Caston, J. R., Conway, J. F. The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy. FASEB J. 11, 733–742 (1997)

Cryo-crinkling : what happens to carbon films on copper grids at low temperature

A study of the surface flatness of carbon films on copper grids used for cryo-electron microscopy has been carried out using a Hitachi S-900 low-voltage SEM. Dramatic changes in flatness were observed after cooling from room temperature to -170 degrees C. The changes were similar both for carbon films that had been floated from a mica surface and for those initially deposited on the surface of plastic films. Results demonstrate that films prepared on copper grids that appear flat at room temperature become extensively, but reversibly, puckered at -170 degrees C. The linear thermal expansion coefficient (alpha) for copper is 16.2 x 10(-6)/degrees C and the puckering can be explained by assuming that the coefficient for amorphous carbon is substantially less. Measurements on grids made of titanium, molybdenum and tungsten (coefficients 8.5, 5 and 4.5 x 10(-6)/degrees C, respectively) showed significantly less puckering.

Magnification mismatches between micrographs: corrective procedures and implications for structural analysis.

Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.