Physiological, morphological, and physicochemical characterization of a novel Escherichia coli bacteriophage, phage MM

A double-stranded DNA containing, T even-like, Escherichia coli bacteriophage, called MM, has been isolated from the local sewage and purified by polyethylene glycol precipitation followed by banding on a cesium chloride three-step gradient. It yields a burst size of 75 particles per infected cell, and has an adsorption coefficient of 3.3 x 10(-10) cm3/min and a latent period of 45 min. Electron microscopy of phage MM reveals an isometric icosahedral head, 92 nm long and 81 nm wide, and a 112-nm-long contractile tail with six pairs of 40-nm-long fibers attached to its baseplate. Phage MM appears similar to E. coli phage T4 or Salmonella phage O1. The density of phage MM in cesium chloride is 1.515 g/ml, and its total mass is 144 MDa. Gel electrophoresis of purified MM capsids displays two major capsid proteins in approximately equimolar amounts and with apparent molecular masses of 38 and 15 kDa. Similarly, purified MM tails yield two major polypeptides with apparent molecular masses of 55 and 16 kDa, most likely representing the major tail sheath and tail tube polypeptides. Its double-stranded DNA has a G-C content of 50%, a length of 131 kilobases (kb), and a mass of 89 MDa.     

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins.

Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

Some properties of a new virus of Pieris rapae

A new insect virus of Pieris rapae was purified using a chloroform-butanol treatment followed by two differential and sucrose gradient centrifugations. The sedimentation coefficient of the purified virion was approximately 132 S, and it banded at a density of 1.39 g/cm³ in cesium chloride. The virion has a nonenveloped capsid with icosahedral symmetry. Several virions were shown to have a regular hexagonal contour about 25 nm in diameter and to be composed of many capsomeres. Full and empty viral particles, with 12 capsomeres around the periphery of the capsid, were noted. In some particles a small core has been observed which is spherical, about 15 nm in diameter. Both purified virus and partially purified virus preparations from dead, infected larvae gave only one precipitin band with a reaction of identity when tested against the antiserum to partially purified virus. When crude extracts of uninfected larvae and purified virus were tested against antiserum to partially purified virus, the pure virus produced a precipitin band. The band was formed independently and did not join to the band of the uninfected insect producing a typical reaction of nonidentity.     

A quasi-atomic model of human adenovirus type 5 capsid

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 A resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon-hexon and hexon-penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1.     

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells.

The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus.     

The role of capsid maturation on adenovirus priming for sequential uncoating

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.     

Structure and Development of Viruses as Observed in the Electron Microscope: XI. Entry and Uncoating of Herpes Simplex Virus

Two morphologically distinct types of capsids are described. The dense capsid appeared to be disrupted near the cellular membrane with release of core material. The light capsid was more stable and was frequently encountered close to the nucleus, where empty capsids were also found. Pretreatment of cells before infection with either puromycin or actinomycin D markedly decreased the percentage of empty capsids. It is suggested that the two types of capsids play different roles in the process of initiating infection. One (the dense capsid) releases deoxyribonucleic acid (DNA) shortly after entry. This DNA is transcribed into a virus-specific ribonucleic acid, which codes for an enzyme capable of altering the permeability of the second type of capsid (the light capsid). In proximity to the nucleus, the infectious DNA then escapes without gross disruption of the capsid.     

Detecting small changes and additional peptides in the canine parvovirus capsid structure

Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40 degrees C and 60 degrees C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50 degrees C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Comparison of Strategies for the Production of FMDV Empty Capsids Using the Baculovirus Vector System

Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.     

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.     

Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins.

The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.     

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.     

Characterization of post-translational products of herpes simplex virus gene 35 proteins binding to the surfaces of full capsids but not empty capsids.

We report on the properties of a genetically and immunologically related family of structural (gamma) polypeptides of herpes simplex virus 1 designated as infected cell polypeptides (ICP) 35. The members of this family were identified and studied with the aid of a panel of monoclonal antibodies exemplified by H745. This monoclonal antibody reacted with six bands (ICP35a to 35f) formed by ICPs contained in either HEp-2 or Vero cell lysates electrophoretically separated in denaturing gels and transferred to nitrocellulose sheets. The six bands had apparent molecular weights in the range 39,000 to 50,000. Traces of ICP35 with apparent molecular weights of 37,000 were also observed in some preparations. On two-dimensional separation ICP35 family members formed at least 20 spots reactive with H745. These differed in both isoelectric properties and electrophoretic mobility in denaturing gels. Pulse-chase experiments, together with results published earlier, indicate that ICP35a to 35d are cytoplasmic precursors to nuclear products. One of these corresponds to virion protein 22a, a component of capsids containing DNA accumulating in the nuclei of infected cells. ICP35 was labeled by 32Pi added to the medium, but the extent of phosphorylation varied and may be a determinant of isoelectric properties. Iodination studies indicate that ICP35e and 35f are the predominant forms of ICP35 present on the surface of full, nuclear capsids containing DNA. None of the members of the ICP35 family were detected in empty capsids. Surface iodination labeled the major capsid protein (ICP5) of empty capsids, but not of full capsids, indicating that ICP35e and 35f coat the surface of the viral capsid and block access to sites for iodination of ICP5, the major capsid protein.     

Bacteriophage P22 capsids with a subgenome length of packaged DNA

Bacteriophage P22 assembles a DNA-free procapsid that subsequently packages P22 DNA. To study the packaging of bacteriophage P22 DNA, attempts were made to isolate P22 capsids with a subgenome length of packaged DNA. With the use of cesium chloride buoyant density sedimentation and agarose gel electrophoresis, the following capsids with a subgenome length of packaged DNA were isolated and characterized: (i) a capsid with the solid-support-free electrophoretic mobility and radius of the DNA-free P22 procapsid; (ii) a capsid with the solid-support-free electrophoretic mobility and radius of the mature P22 bacteriophage; and (iii) a capsid with a solid-support-free electrophoretic mobility and possibly a radius intermediate to those of the procapsid and bacteriophage.     

Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.     

Alterations in nuclear matrix structure after adenovirus infection.

Infection of HeLa cells with adenovirus serotype 2 causes rearrangements in nuclear matrix morphology which can best be seen by gentle cell extraction and embedment-free section electron microscopy. We used these techniques to examine the nuclear matrices and cytoskeletons of cells at 6, 13, 28, and 44 h after infection. As infection progressed, chromatin condensed onto the nucleoli and the nuclear lamina. Virus-related inclusions appeared in the nucleus, where they partitioned with the nuclear matrix. These virus centers consisted of at least three distinguishable areas: amorphously dense regions, granular regions whose granulations appeared to be viral capsids, and filaments connecting these regions to each other and to the nuclear lamina. The filaments became decorated with viral capsids of two different densities, which may be empty capsid shells and capsids with DNA-protein cores. The interaction of some capsids with the filaments persisted even after lysis of the cell. We propose that granulated virus-related structures are sites of capsid assembly and storage and that the filaments may be involved in the transport of capsids and capsid intermediates. The nuclear lamina became increasingly crenated after infection, with some extensions appearing to bud off and form blebs of nuclear material in the cytoplasm. The perinuclear cytoskeleton became rearranged after infection, forming a corona of decreased filament number around the nucleus. In summary, we propose that adenovirus rearranges the nuclear matrix and cytoskeleton to support its own replication.     

Herpes simplex virus capsid structure: DNA packaging protein UL25 is located on the external surface of the capsid near the vertices

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.     

Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy.

Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, rising approximately 5 nm above a midlayer of density. The innermost layer, or "floor," is a 4-nm-thick sheet of virtually continuous density except for the orifices of the channels that traverse each capsomer. Hexon protrusions are distinctly hexagonal in shape, and penton protrusions are pentagonal. The structures of the three kinds of hexons (distinguished according to their positions on the surface lattice) are closely similar but differ somewhat in their respective orientations and in the shapes of their channels. The most prominent features of the midlayer are threefold nodules ("triplexes") at the trigonal lattice points. By analogy with other viral capsids, the triplexes may represent trimers of another capsid protein, possibly VP23 (36 kilodaltons [kDa]) or VP26 (12 kDa). Intermediate capsids differ from light capsids, which are empty, in having one or more internal components. In individual images from which the shell structure has been filtered away, these components are seen to have dimensions of 20 to 30 nm but to lack a visible substructure. This material--which is smeared out in the reconstruction, implying that its distribution is not icosahedrally symmetric or necessarily consistent from particle to particle--consists of aggregates of VP22 (46 kDa). From several lines of evidence, we conclude that this protein is located entirely within the capsid shell. These aggregates may be the remnants of morphogenetic cores retained in capsids interrupted in the process of DNA packaging.