investigation of bacteriophages established not only the foundations for the field of molecular biology but also fundamental insights into how viruses interact with their host cells.
The Defining Properties of Viruses
Throughout the early period of virology when many viruses of plants, animals, and bacteria were cataloged, ideas about the origin and nature of these distinctive infectious agents were quite controversial. Arguments centered on whether viruses originated from parts of a cell or were built from unique components. Little progress was made toward resolving these issues and establishing the definitive properties of viruses until the development of new techniques that allowed their visualization or propagation in cultured cells.
The Structural Simplicity of Virus Particles
Dramatic confirmation of the structural simplicity of virus particles came in 1935, when Wendell Stanley obtained crystals of tobacco mosaic virus. At that time, nothing was known of the structural organization of any biologically important macromolecules, such as proteins and DNA. Indeed, the crucial role of nucleic acids as genetic material had not even been recognized. The ability to obtain an infectious agent in crystalline form, a state that was more generally associated with inorganic material, created much wonder and speculation about whether a virus is truly a life form. In retrospect, it is obvious that the relative ease with which this particular virus could be crystallized was a direct result of its structural simplicity.
The 1930s saw the introduction of the instrument that rapidly revolutionized virology: the electron microscope. The great magnifying power of this instrument (eventually more than 100,000-fold) allowed direct visualization of virus particles for the first time. It has always been an exciting experience for investigators to obtain images of viruses, especially as they appear to be remarkably elegant (Fig. 1.9). Images of many different virus particles confirmed that these agents are very small (Fig. 1.10) and that most are far simpler in structure than any cellular organism. Many appeared as regular helical or spherical particles. The description of the morphology of virus particles made possible by electron microscopy also opened the way for the first rational classification of viruses.
The Intracellular Parasitism of Viruses
Organisms as Hosts
A defining characteristic of viruses is their absolute dependence on a living host for reproduction: they are obligate parasites. Transmission of plant viruses such as tobacco mosaic virus can be achieved readily, for example, by applying extracts of an infected plant to a scratch made on the leaf of a healthy plant. Furthermore, as a single infectious particle of many plant viruses is sufficient to induce a characteristic lesion (Fig. 1.11), the concentration of the infectious agent could be measured. Plant viruses were therefore the first to be studied in detail. Some viruses of humans and other species could also be propagated in laboratory animals, and methods were developed to quantify them by determining the lethal dose. The transmission of yellow fever virus to mice by Max Theiler in 1930 was an achievement that led to the isolation of an attenuated strain, still considered one of the safest and most effective ever produced for the vaccination of humans.
After specific viruses and appropriate host organisms were identified, it became possible to produce sufficient quantities of virus particles for study of their physical and chemical properties and the consequences of infection for the host. Features such as the incubation period, symptoms of infection, and effects on specific tissues and organs were investigated. Laboratory animals remain an essential tool in investigations of the pathogenesis of viruses that cause disease. However, real progress toward understanding the mechanisms of virus reproduction was made only with the development of cell culture systems. The first and the simplest, but crucial to both virology and molecular biology, were cultures of bacterial cells.
Lessons from Bacteriophages
In the late 1930s and early 1940s, the bacteriophages, or “phages,” received increased attention as a result of controversy centering on how they might have arisen. John Northrup, a biochemist at the Rockefeller Institute in Princeton, NJ, championed the theory that a phage was a metabolic product of a bacterium. On the other hand, Max Delbrück, in his work with Emory Ellis and later with Salvador Luria, regarded phages as autonomous, stable, self-replicating entities characterized by heritable traits. According to this paradigm, phages were seen as ideal tools with which to investigate the nature of genes and heredity. Probably the most critical early contribution of Delbrück and Ellis was the perfection of the “one-step growth” method for synchronization of the reproduction of phages, an achievement that allowed analysis of a single cycle of phage reproduction in a population of bacteria. This approach introduced highly quantitative methods to virology, as well as an unprecedented rigor of analysis. The first experiments showed that phages indeed multiplied in the bacterial host and were liberated in a “burst” following disruption of the cell.
Figure 1.9 Electron micrographs of virus particles following negative staining. (A) The complex, nonenveloped virus bacteriophage T4. Note the intricate tail and tail fibers. Reproduced with permission from Dr. Robert L. Duda, University of Pittsburgh, Pittsburgh, PA. (B) The helical, nonenveloped particle of tobacco mosaic virus. Courtesy of Plant Resistance Gene Wiki (http://prgdb.crg.eu/wiki/Species:Tobacco_mosaic_virus), licensed under CC BY-SA 3.0. (C) Enveloped particles of the rhabdovirus vesicular stomatitis virus. Courtesy of CDC/Dr. Fred. A. Murphy (CDC PHIL ID#5611). (D) Nonenveloped, icosahedral human rotavirus particles. Courtesy of F. P. Williams, U.S. Environmental Protection Agency, Washington, DC.
Figure 1.10 Size matters. (A) Sizes of animal and plant cells, bacteria, viruses, proteins, molecules, and atoms are indicated. The resolving powers of various techniques used in virology, including light microscopy, electron microscopy, cryo-electron microscopy (Cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy, are indicated. Viruses span a broad range from that equal to some small bacteria to just above ribosome size. The units commonly used in descriptions of virus particles or their components are the nanometer (nm [10-9 m]) and the angstrom (Å [10-10 m]). (B) Illustration of the size differences among two animal viruses and a typical eukaryotic host cell.
Figure 1.11 Lesions induced by tobacco mosaic virus on an infected tobacco leaf. In 1886, Adolph Mayer first described the characteristic patterns of light and dark green areas on the leaves of tobacco plants infected with tobacco mosaic virus. He demonstrated that the mosaic lesions could be transmitted from an infected plant to a healthy plant by aqueous extracts derived from infected plants. Following application of the preparation to healthy plant leaves, the number of characteristic lesions containing dead cells is directly proportional to the number of infectious particles in the test sample. Courtesy of USDA Forest Service, under license CC BY 3.0.
