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F P Booy

Publications and source records attributed to F P Booy.

At least 19 recordsLinked to original sources

The capsid architecture of channel catfish virus, an evolutionarily distant herpesvirus, is largely conserved in the absence of discernible sequence homology with herpes simplex virus.

Although herpesviruses have a wide host range and their genomes vary substantially in size, the nucleocapsid appears to be a conservative element of viral design. The capsid shell is icosahedrally symmetric (T = 16), and 125 nm in diameter and 15nm thick in the case of herpes simplex virus 1 (HSV-1). Channel catfish virus (CCV) has the gross morphology of a herpesvirus, although no relationship to other herpesviruses is evident from the sequences of its proteins. To examine CCV capsid architecture more closely, we have determined its structure by cryoelectron microscopy and three-dimensional image reconstruction. The CCV capsid is smaller than that of HSV-1, but its 12% smaller genome is packed to essentially the same average density; its icosahedral facets are flatter, and its shell is about 20% thinner, consistent with the smaller size of its major capsid protein. Otherwise, their major features are remarkably similar: CCV has the same triangulation number; its hexons and pentons also have chimney-like protrusions with an axial channel through each capsomer; and there are "triplexes" on the outer surface at the sites of local threefold symmetry. The basic herpesvirus capsid architecture is, therefore, remarkably well conserved in CCV and implies a utilitarian basis to this design. The protein composition of CCV mirrors that of HSV-1, except for the absence of the 12-kDa protein, VP26, which is dispensable for assembly in the HSV-1 system and, apparently, wholly dispensable for CCV.

Animals

Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26.

Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of approximately 2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site.

Animals

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid

Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.

The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.

Capsid

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.

Carbon

Conformational changes of a viral capsid protein. Thermodynamic rationale for proteolytic regulation of bacteriophage T4 capsid expansion, co-operativity, and super-stabilization by soc binding.

We have used differential scanning calorimetry in conjunction with cryo-electron microscopy to investigate the conformational transitions undergone by the maturing capsid of phage T4. Its precursor shell is composed primarily of gp23 (521 residues): cleavage of gp23 to gp23* (residues 66 to 521) facilitates a concerted conformational change in which the particle expands substantially, and is greatly stabilized. We have now characterized the intermediate states of capsid maturation; namely, the cleaved/unexpanded, state, which denatures at tm = 60 degrees C, and the uncleaved/expanded state, for which tm = 70 degrees C. When compared with the precursor uncleaved/unexpanded state (tm = 65 degrees C), and the mature cleaved/expanded state (tm = 83 degrees C, if complete cleavage precedes expansion), it follows that expansion of the cleaved precursor (delta tm approximately +23 degrees C) is the major stabilizing event in capsid maturation. These observations also suggest an advantage conferred by capsid protein cleavage (some other phage capsids expand without cleavage): if the gp23-delta domains (residues 1 to 65) are not removed by proteolysis, they impede formation of the stablest possible bonding arrangement when expansion occurs, most likely by becoming trapped at the interface between neighboring subunits or capsomers. Icosahedral capsids denature at essentially the same temperatures as tubular polymorphic variants (polyheads) for the same state of the surface lattice. However, the thermal transitions of capsids are considerably sharper, i.e. more co-operative, than those of polyheads, which we attribute to capsids being closed, not open-ended. In both cases, binding of the accessory protein soc around the threefold sites on the outer surface of the expanded surface lattice results in a substantial further stabilization (delta tm = +5 degrees C). The interfaces between capsomers appear to be relatively weak points that are reinforced by clamp-like binding of soc. These results imply that the "triplex" proteins of other viruses (their structural counterparts of soc) are likely also to be involved in capsid stabilization. Cryo-electron microscopy was used to make conclusive interpretations of endotherms in terms of denaturation events. These data also revealed that the cleaved/unexpanded capsid has an angular polyhedral morphology and has a pronounced relief on its outer surface. Moreover, it is 14% smaller in linear dimensions than the cleaved/expanded capsid, and its shell is commensurately thicker.

Allosteric Regulation

Distinct monoclonal antibodies separately label the hexons or the pentons of herpes simplex virus capsid.

The surface shell of the capsid of herpes simplex virus type 1 (HSV-1) is 15 nm thick and 125 nm in outer diameter and has the form of an icosahedral (T = 16) surface lattice, composed of 150 hexons and 12 pentons. Hexons are traversed by axial channels and have six-fold symmetric external protrusions, separated by triangular nodules ("triplexes"). Pentons resemble hexons morphologically, apart from their different order of symmetry. To localize VP5, the major capsid protein, in the shell structure and to investigate whether pentons are composed of the same molecules as hexons, we have performed cryo-electron microscopy and three-dimensional image reconstructions of control HSV-1 B capsids and of B capsids immunoprecipitated with two monoclonal antibodies raised against purified VP5 and purified capsids. The results clearly map the epitope of the anti-VP5 monoclonal antibody to the distal tips of the hexon protrusions. In contrast, no detectable labeling of pentons was observed. We conclude that the hexon protrusions are domains of VP5 hexamers, other parts of these molecules forming the basic matrix of the capsid shell to which the other proteins are attached at specific sites. Conversely, the anti-capsid monoclonal antibody decorates the outer rim of pentons but does not bind to hexons. These observations imply that either pentons are composed of some other protein(s) or that they also contain VP5, but in a conformation sufficiently different from that assumed in hexons as to transform its antigenic character. Other evidence leads us to favor the latter alternative.

Antibodies, Monoclonal

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.

Algorithms

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.

Animals

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.

Gene Products, env

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.

Animals

Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment.

Marked differences were observed between the H2 and H3 strains of influenza virus in their sensitivity to pretreatment at low pH. Whereas viral fusion and hemolysis mediated by influenza virus X:31 (H3 subtype) were inactivated by pretreatment of the virus at low pH, influenza virus A/Japan/305/57 (H2 subtype) retained those activities even after a 15-min incubation at pH 5.0 and 37 degrees C. Fusion with erythrocytes was measured by using the octadecylrhodamine-dequenching assay with both intact virions and CV-1 monkey kidney cells expressing hemagglutinin (HA) on the plasma membrane. To study the nature of the differences between the two strains, we examined the effects of low-pH treatment on the conformational change of HA by its susceptibility to protease digestion, exposure of the fusion peptide, and electron microscopy of unstained, frozen, hydrated virus. We found that the respective HA molecules from the two strains assumed different conformational states after exposure to low pH. The relationship between the conformation of HA and its fusogenic activity is discussed in the context of these experiments.

Erythrocytes

Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron microscopic study.

The Brookhaven scanning transmission electron microscope was employed to measure the masses of two nucleocapsid species (of light and intermediate densities) of equine herpesvirus 1. These were found to be 196.7 +/- 9.2 and 229.0 +/- 9.5 megadaltons (MDa), respectively. Biochemical assays showed that neither nucleocapsid contained any significant amount of DNA (less than 0.2% [wt/wt]). Taking into account data on protein composition, we conclude that the difference between their masses is essentially contributed by viral protein 22 (46 kDa), which is an integral component of the maturable intermediate nucleocapsid but not of the abortive light nucleocapsid. In view of earlier ultrastructural information on capsomer symmetry, our mass determinations are consistent only with the 150 hexavalent capsomers being hexamers of the 148-kDa major capsid protein.

Animals

Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin.

The hemagglutinin of influenza virus undergoes a conformational change at low pH, which results in exposure of a hydrophobic segment of the molecule, crucial to expression of viral fusion activity. We have studied the effects of incubation of the virus at low pH either at 37 or 0 degrees C. Treatment of the virus alone at pH 5.0 induces the virus particles to become hydrophobic, as assessed by measuring the binding of zwitterionic liposomes to the virus. At 37 degrees C this hydrophobicity is transient, electron microscopic examination of the virus reveals a highly disorganized spike layer, and fusion activity toward ganglioside-containing zwitterionic liposomes, measured at 37 degrees C with a kinetic fluorescence assay, is irreversibly lost. By contrast, after preincubation of the virus alone at pH 5.0 and 0 degrees C fusion activity remains unaffected. Yet, the preincubation at 0 degrees C does result in exposure of the hydrophobic segment of hemagglutinin, but now hydrophobicity is sustained and viral spike morphology unaltered. Hydrophobicity also remains to a significant extent upon pH neutralization, but fusion activity is negligible under these conditions. It is concluded that for optimal expression of fusion activity the virus must be bound to the target membrane before exposure to low pH. Furthermore, even after exposure of the hydrophobic segment of hemagglutinin, fusion occurs only at low pH. Finally, fusion occurs only at elevated temperature, possibly reflecting the unfolding of hemagglutinin trimers or the cooperative action of several hemagglutinin trimers in the reaction.

Cell Membrane

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.

Animals

Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles.

An electron microscopical study was made of the influenza virus, type B/Hong Kong, in the unstained, frozen, hydrated state after quench-freezing in cooled liquid ethane. The results are compared with data from negatively stained specimens. It is shown that cryo-electron microscopy confirms and extends the data obtained by conventional methods. In particular, the virus is shown to be circular in projection with no indication of icosahedral symmetry, the lipid membrane is clearly resolved as a bi-layer and it is demonstrated that the distribution of material within the bi-layer is non-uniform, with a shell of more electron dense material surrounding a less dense central region. Neuraminidase spikes are not clearly distinguished from haemaglutinin spikes. The diameter of the complete B/Hong Kong virus was estimated from cryo-micrographs as 1270(+/- 70) A. Some preliminary data for influenza virus type A/X31 are presented.

Freezing

Electron microscopy of frozen biological suspensions.

The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench-frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze-dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen-hydrated, freeze-dried or sugar embedded crystals can withstand a three- to four-fold increase in electron exposure for the same damage when compared with similar sugar-embedded or freeze-dried samples at room temperature.

Catalase