Quenching of CdSe quantum dot emission, a new approach for biosensing.

The emission of CdSe quantum dots linked to the 5'-end of a DNA sequence is efficiently quenched by hybridisation with a complementary DNA strand with a gold nanoparticle attached at the 3'-end; contact of the quantum dot and gold nanoparticle occurs.

Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.

Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.

The filamentous type III secretion translocon of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

L1 interaction domains of papillomavirus l2 necessary for viral genome encapsidation.

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.

Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

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 approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 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 A 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.

Conformational intermediates and fusion activity of influenza virus hemagglutinin.

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).

Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins.

An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37 degrees C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to approximately 30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

Two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution.

Complexes between bovine papillomavirus type 1 (BPV1) and examples of two sets of neutralizing, monoclonal antibodies (mAb) to the major capsid protein (L1) were analyzed by low-dose cryo-electron microscopy and three-dimensional (3D) image reconstruction to 13 A resolution. mAb #9 is representative of a set of neutralizing antibodies that can inhibit viral binding to the cell surface, while mAb 5B6 is representative of a second set that efficiently neutralizes papillomaviruses without significantly inhibiting viral binding to the cell surface. The 3D reconstructions reveal that mAb #9 binds to L1 molecules of both pentavalent and hexavalent capsomeres. In contrast, 5B6 binds only to hexavalent capsomeres, reflecting the significant structural or environmental differences for the 5B6 epitope in the 12 pentavalent capsomeres. Epitope localization shows that mAb #9 binds monovalently to the tips of capsomeres whereas 5B6 binds both monovalently and bivalently to the sides of hexavalent capsomeres approximately two-thirds of the way down from the outer tips, very close to the putative stabilizing intercapsomere connections. The absence of mAb 5B6 from the pentavalent capsomeres and its inability to prevent viral binding to the cell surface suggest that receptor binding may occur at one or more of the 12 virion vertices.

At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease.

ClpAP, a typical energy-dependent protease, consists of a proteolytic component (ClpP) and a chaperone-like ATPase (ClpA). ClpP is composed of two apposed heptameric rings, whereas in the presence of ATP or ATPgammaS, ClpA is a single hexameric ring. Formation of ClpAP complexes involves a symmetry mismatch as sixfold ClpA stacks axially on one or both faces of sevenfold ClpP. We have analyzed these structures by cryo-electron microscopy. Our three-dimensional reconstruction of ClpA at 29-A resolution shows the monomer to be composed of two domains of similar size that, in the hexamer, form two tiers enclosing a large cavity. Cylindrical reconstruction of ClpAP reveals three compartments: the digestion chamber inside ClpP; a compartment between ClpP and ClpA; and the cavity inside ClpA. They are connected axially via narrow apertures, implying that substrate proteins should be unfolded to allow translocation into the digestion chamber. The cavity inside ClpA is structurally comparable to the "Anfinsen cage" of other chaperones and may play a role in the unfolding of substrates. A geometrical description of the symmetry mismatch was obtained by using our model of ClpA and the crystal structure of ClpP (Wang et al., 1997, Cell 91, 447-456) to identify the particular side views presented by both molecules in individual complexes. The interaction is characterized by a key pair of subunits, one of each protein. A small turn (8.6(o) = 2pi/42; equivalent to a 4-A shift) would transfer the key interaction to another pair of subunits. We propose that nucleotide hydrolysis results in rotation, facilitating the processive digestion of substrate proteins.

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.

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.

Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

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 approximately 200 A (mass approximately 1 MDa) to >1500 A (mass>250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 A, 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.

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

The three-dimensional structure of bovine papillomavirus has been determined to 9 A resolution by reconstruction of high resolution, low dose cryo-electron micrographs of quench-frozen 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 A 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 approximately 10 A in diameter were clearly resolved. These link adjacent capsomeres and are reminiscent of the helical connections that stabilize polyomavirus.

Structure of the herpes simplex virus capsid: peptide A862-H880 of the major capsid protein is displayed on the rim of the capsomer protrusions.

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T = 16 icosahedral lattice. The major capsid protein, VP5 MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid promoters, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.

Intermediate filament structure: hard alpha-keratin.

Structurally there are four classes of intermediate filaments (IF) with distinct but closely related axial organisations. One of these, hard alpha-keratin IF, has been studied to clarify several apparently exceptional features which include the number of molecules in the IF cross-section and the mode by which the axial organisation of its constituent molecules is stabilised. Using the dark-field mode of the STEM at the Brookhaven National Laboratory (USA) mass measurements were obtained from unstained IF isolated from hair keratin. The data thus obtained show that the number of chains in cross-section is about 30 (+/-3: standard deviation) and is very similar to the numbers determined in previous STEM experiments for the dominant filament type in other classes of IF (about 32). Furthermore, re-analysis of the low-angle equatorial X-ray diffraction pattern reveals, in contrast to earlier work, solutions that are compatible with the number of chains in cross-section indicated by the STEM data. The absence of the head-to-tail overlap between parallel molecules characteristic of most of IF may be compensated in hard alpha-keratin by a network of intermolecular disulfide bonds. It is concluded that native IF of hard alpha-keratin and desmin/vimentin--and probably many other kinds of IF as well--contain about 32 chains in cross-section, and that the axial structures of these various kinds of IF differ in small but significant ways, while generally observing the same basic modes of aggregation.

Assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation.

The herpes simplex virus-1 (HSV-1) capsid is an icosahedral shell approximately 15 nm thick and 125 nm in diameter. Three of its primary structural components are a major capsid protein (VP5; coded by the UL19 gene) and two minor proteins, VP19C (UL38 gene) and VP23 (UL18 gene). Assembly of the capsid involves the participation of two additional proteins, the scaffolding protein (UL26.5 gene) and the maturational protease (UL26 gene). With the goal of identifying morphological intermediates in the assembly process, we have examined capsid formation in a cell-free system containing the five HSV-1 proteins mentioned above. Capsids and capsid-related structures formed during progressively longer periods of incubation were examined by electron microscopy of thin-sectioned specimens. After one minute, 90 minutes and eight hours of incubation the structures observed, respectively, were partial capsids, closed spherical capsids and polyhedral capsids. Partial capsids were two-layered structures consisting of a segment of external shell partially surrounding a region of scaffold. They appeared as wedges or angular segments of closed spherical capsids, the angle ranging from less than 30 degrees to greater than 270 degrees. Partial capsids are suggested to be precursors of closed spherical capsids because, whereas partial capsids were the predominant assembly product observed after one minute of incubation, they were rare in reactions incubated for 45 minutes or longer. Closed spherical capsids were highly uniform in morphology, consisting of a closed external shell surrounding a thick scaffold similar in morphology to the same layers seen in partial capsids. In negatively stained specimens, closed spherical capsids appeared round in profile, suggesting that they are spherical rather than polyhedral in shape. A three-dimensional reconstruction computed from cryoelectron micrographs confirmed that closed spherical capsids are spherical with T = 16 icosahedral symmetry. The reconstruction showed further that, compared to mature HSV-1 capsids, closed spherical capsids are more open structures in which the capsid floor layer is less pronounced. In contrast to closed spherical capsids, polyhedral capsids exhibited distinct facets and vertices, indicating that they are icosahedral like the capsids in mature virions. Upon incubation in vitro, purified closed spherical capsids matured into polyhedral capsids, indicating that the latter arise by angularization of the former. Partial capsids, closed spherical capsids and polyhedral capsids were all found to contain VP5, VP19C, VP23, VP21 and the scaffolding protein; the scaffolding protein being predominantly in the immature, uncleaved form in all cases. Polyhedral capsids and closed spherical capsids were found to differ in their sensitivity to disruption at 2 degrees C. Closed spherical capsids were disassembled while polyhedral capsids were unaffected. Our results suggest that HSV-1 capsid assembly begins with the partial capsid and proceeds through a closed, spherical, unstable capsid intermediate to a closed, icosahedral form similar to that found in the mature virion. Structures resembling HSV-1 partial capsids have been described as capsid assembly intermediates in Salmonella typhimurium bacteriophage P22. HSV-1 capsid maturation from a fragile, spherical state to a robust polyhedral form resembles the prohead maturation events undergone by dsDNA bacteriophages including lambda, T4 and P22. Because of this similarity, we propose the name procapsid for the closed spherical capsid intermediate in HSV-1 capsid assembly.

The herpes simplex virus procapsid: structure, conformational changes upon maturation, and roles of the triplex proteins VP19c and VP23 in assembly.

The proteins coded by the five major capsid genes of herpes simplex virus 1, VP5 (gene UL19), VP19c (UL38), VP23 (UL18), pre-VP22a (UL26.5), and pre-VP21 (UL26), assemble into fragile roundish "procapsids", which mature into robust polyhedral capsids in a transition similar to that undergone by bacteriophage proheads. Here we describe the HSV-1 procapsid structure to a resolution of approximately 2.7 nm from three-dimensional reconstructions of cryo-electron micrographs. Comparison with the mature capsid provides insight into the large-scale conformational changes that take place upon maturation. In the procapsid, the elongated protomers (VP5 subunits) make little contact with each other except around the bases of the hexons and pentons, whereas they are tightly clustered into capsomers in the mature state; the axial channels, which are constricted or blocked in the mature capsid, are fully open; and unlike the well observed 6-fold symmetry of mature hexons, procapsid hexons are distorted into oval and triangular shapes. These deformations reveal a VP5 domain in the inner part of the protrusion wall which participates in inter-protomer bonding in the procapsid and is close to the site where the channel closes upon maturation. Remarkably, there are no direct contacts between neighboring capsomers; instead, interactions between them are mediated by the "triplexes" at the sites of local 3-fold symmetry. This observation discloses the mechanism whereby the triplex proteins, VP19c and VP23, play their essential roles in capsid morphogenesis. In the mature capsid, density extends continuously between neighboring capsomers in the inner "floor" layer. In contrast, there are large gaps in the corresponding region of the procapsid, implying that formation of the floor involves extensive remodeling. Inside the procapsid shell is the hollow spherical scaffold, whose radial density profile indicates that the major scaffold protein, pre-VP22a, is a long molecule (> 24 nm) composed of three domains. Since no evidence of icosahedral symmetry is detected in the scaffold, we infer that (unless higher resolution is required) the scaffold may not be an icosahedral shell but may instead be a protein micelle with a preferred radius of curvature.