Robley C. Williams

Electron microscopy of tobacco mosaic virus under conditions of minimal beam exposure

Abstract A new technique of electron microscopy has been developed which, when applied to the micrography of tobacco mosaic virus, results in a notably improved delineation of its fine structure. The technique stems from earlier observations that the amount of electron beam irradiation normally used in electron microscopy is destructive to biological specimens. A method is described to minimize such damage, wherein the specimen is subjected only to the irradiation necessary to obtain a photographic exposure. The contrast in the appearance of tobacco mosaic virus as micrographed by the new procedures, and by traditional ones, is demonstrated.

Infectivity of viral nucleic acid.

A method of preparation of infectious nucleic acid from TMV is described. The material is of similar infectivity at 10 γ/ml as is TMV at 0.02 to 0.5 γ/ml. No significant number of virus particles can be found in the ultracentrifuge sediment of such preparations, while TMV added to nucleic acid is quantitatively sedimented under the same conditions. The instability in 0.1 M salts, the remarkable sensitivity to ribonuclease, and the resistance to anti-TMV-γ-globulin clearly differentiate the nucleic acid infectivity from that of intact or detergent-damaged virus. From the behavior in a centrifungal field and other evidence it is concluded that the activity is associated with the bulk of the nucleic acid in the preparations (Mol. wt. = 200,000 to 300,000) and not with a small fraction of high molecular weight.

Mode of DNA packing within bacteriophage heads.

Abstract Electron micrographs of five different DNA bacteriophages, as prepared by drying in thin films of negative stain, frequently show their heads to be disrupted and flattened. In such cases DNA strands, no larger than 2.5 nm in diameter, become visible, either contained within partially ruptured capsids or completely ejected from severely ruptured ones. Seen in either aspect, the strands appear with circular outline; in some cases a set of concentric circles (or a tightly wound spiral) is evident. Two alternative models of DNA packing within phage heads are proposed. Both are consistent with the electron microscopic observations and, as applied specifically to T4 phage heads, they are also consistent with available data from birefringence studies. One model proposes that the DNA, in simple double-helix form, is wound into a ball. The other suggests that the DNA is wound like a spool, with a greater number of turns in the central region than at the two ends and with the spool axis perpendicular to the axis of the phage particle. The available evidence does not permit a choice to be made between the two models.

Electron Microscopy of Viruses

Publisher Summary This chapter examines the technical aspects of the science and practice of electron microscopy. The electron microscope yields its most detailed and unambiguous answers in delineating the three-dimensional surface contours of objects, such as viruses, after they have been dried on the supporting film and shadowed. The electron microscope is frequently used for the examination of virus preparations that are relatively impure and in which the morphological identity of the virus is uncertain or is completely unknown. The microscope cannot yield an “identification” of a particle such as a virus; however, it can be extremely useful in providing a correlation between the presence of a certain class of particle and an associated activity. The traditional procedure in attempting to establish a morphological identity of a virus is to take some partially purified material from both infected and noninfected cells, place drops of both suspensions on specimen screens and allow them to dry, shadow the dried specimens, and examine fields in an electron microscope. The electron microscope can be used to provide two types of information about purified virus suspensions: (1) the numbers of virus particles present in the suspension and (2) the size and shape of the particles.

Quantitative electron microscopy of microtubule assembly in vitro

The kinetics of microtubule assembly from purified porcine brain tubulin were examined by quantitative electron microscopy. Assembly at 37°C was initiated by the addition of GTP to 1 m m to the protein solution at ∼1 mg/ml in ammonium acetate buffer, pH 6·4. A solution of partially polymerized protein was mixed with a suspension of tomato bushy stunt virus at known particle concentration, and spray-deposited, in negative stain, upon specimen films. The resulting drop patterns were micrographed and the percent protein seen in distinguishable polymeric forms was calculated from the numbers, lengths, and known masses and mass/unit lengths of these entities. Clumping of protein by the uranyl acetate stain was avoided by use of a dual nebulizer which mixed protein and stain only upon spray droplet formation. The micrographs displayed ring-like forms, microtubules, and aggregates of parallel protofilaments—arrayed as flat, curved, and helical ribbons. Protein found as rings rapidly decreased from an initial 30% to a negligible amount at 2 minutes; the amount as microtubules increased asymptotically to an equilibrium value of 50% by 8 minutes; the quantity of ribbons was largest at 3 minutes (∼20%), but vanishingly small by 8 minutes. The results show that the fully formed microtubules consisted of protein which was initially both in the ring form and in the tubulin-dimer (6 S) form. They also quantitatively demonstrate that the twisted ribbons are assembly intermediates, those with a full protofilament complement presumably converting to microtubules by cylindrical folding.

The polyhedral form of the Tipula iridescent virus.

Abstract The particles of the cytoplasmic virus of Tipula paludosa , named Tipula iridescent virus (TIV), are remarkable for the association of large size (130 mμ) with a high degree of uniformity of size and shape. When air-dried they have an appearance that suggests a polyhedral morphology. Following freeze-drying the TIV particles are univormly hexagonal in contour. Owing to their large size it is possible to secure a knowledge of their polyhedral shape by analysis of the shadows cast by the particles. Both single and double shadowing have been employed, and a comparison made with the shapes of shadows cast by model polyhedra. It is concluded that the shape of the Tipula iridescent virus is that of an icosahedron.

Morphology of bovine fibrinogen monomers and fibrin oligomers

Abstract Molecules of bovine fibrinogen and small oligomers of thrombin-induced fibrin have been examined by electron microscopy after contrast enhancement by rotary shadowing and by negative staining. Rotary-shadowed molecules, in addition to exhibiting the well-known trinodular structure, showed a spine of fairly uniform width connecting the central nodule with the terminal protrusions. In specimens negatively stained with uranyl formate or acetate most of the molecules had a gently sigmoidal configuration, all of like handedness. The spines were visibly prominent and frequently had a slight central bulge. The terminal protrusions appeared with a distinctly oblong shape and were oriented parallel to each other, with the long dimension tilted about 45 ° to the axis of the contiguous part of the spine. After exposure of the fibrinogen molecules to millimolar Ca2+ the central bulge became more noticeable and each terminal protrusion took on the form of two contiguous, equal-sized spheres. Significant dimensions are: overall length of molecule, 45 nm; length and width of spine, 31 nm × 3 nm; length and width of terminal protrusions, 9 nm × 4 nm; diameter of Ca2+-induced spheres, ~ 4 nm. Specimens from fibrinogen solutions to which minute amounts of thrombin had been added (and the macroscopic clots removed) contained oligomeric assemblies that were small enough to be resolvable into their fibrin subunits. They consisted of two chains that were parallel and were similar to each other in that they never differed by more than one in their subunit number. The elements in one chain of an oligomer appeared to be bound to those of the other chain with a half-molecule staggered overlap. They retained the lengths they had as fibrinogen molecules.

Cyclic DNA of shope papilloma virus

Double-stranded DNA of Shope papilloma virus prepared for electron microscopy by the protein monolayer technique was found to exist in loop-like structures without ends. Mean contour lengths of such DNA ranged from 2·3 to 2·8 μ. The phenol-extracted material showed similar conformations whan the DNA was allowed to diffuse to a preformed monolayer of cytochrome c , and when a mixture of DNA and cytochrome c was spread on an aqueous surface. DNA diffusing from virus subjected to osmotic shock also appeared as rings, but the rings were less convoluted than those in the preparations extracted by phenol. The variation in length is tentatively attributed to varying degrees of local preparative denaturation in the DNA strands.

Structural and functional differentiation in T2 bacteriophage.

Abstract Particles of T2 bacteriophage and T2 ghosts have been disjoined into several component parts by treating them to cycles of alternate freezing and thawing. The distinguishable components are: intact heads, intact tails, head membranes, cross sections of tails, cores of tails, and fine fibers both attached to and removed from tails. These components have been examined for correlation of their structure and their host-adsorptive function. It is found that the intact heads, the membranes of ghosted heads, and probably the tail cores do not adsorb to sensitive bacteria. The tails and the tail fibers are found to adsorb. The possible functions of the tail cores and of the tail fibers in the mechanism of phage infection are discussed.

Structure of Shope papilloma virus particles.

Abstract Shope papilloma virus from warts of cottontail rabbits was partially purified by differential centrifugation and then subjected to rate zonal centrifugation in sucrose or glycerol density gradients where the particles were separated into three bands and a small pellet. The top component consisted of particles that were of characteristic size for papilloma virus but, upon examination in the electron microscope, after treatment with phosphotungstic acid, most of them appeared to be hollow. This fraction also gave a protein-like absorption curve in the ultraviolet, contained very little phosphorus, and was almost noninfectious. By contrast the particles in the middle band appeared completely formed, contained appreciable phosphorus, had a nucleoprotein-like absorption curve, and were highly infectious. It is not known whether the two types of particle are produced as such in the host or are a result of preparation. In all particles a pattern of spots was seen either over the surface or around the periphery. The spots were of uniform size and were arrayed in a definite pattern, suggesting that they represent a subunit structure.