Principles of Virology, Volume 1. Jane Flint

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.

      Delbrück was a zealot for phage research and recruited talented scientists to pursue the fundamental issues of what is now known as the field of molecular biology. This cadre of scientists focused their attention on specific phages of the bacterium Escherichia coli. Progress was rapid, primarily because of the simplicity of the phage infectious cycle. By the mid-1950s it was evident that viruses from bacteria, animals, and plants share many fundamental properties. However, the phages provided a far more tractable experimental system. Consequently, their study had a profound impact on the field of virology.

One critical lesson came from a definitive experiment that established that viral nucleic acid carries genetic information. It was known from studies of the “transforming principle” of pneumococcus by Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) that nucleic acid was both necessary and sufficient for the transfer of genetic traits of bacteria. However, in the early 1950s, protein was still suspected to be an important component of viral heredity. In a brilliantly simple experiment that included the use of a common kitchen food blender, Alfred Hershey and Martha Chase showed that this hypothesis was incorrect; DNA, not protein, carries the information for virus reproduction (Box 1.5).

Bacteriophages were originally thought to be lethal agents, invariably killing their host cells after infection. In the early 1920s, a previously unknown interaction was discovered, in which the host cell not only survived the infection but also stably inherited the genetic information of the virus. It was also observed that certain bacterial strains could lyse spontaneously and produce bacteriophages after a period of growth in culture. Such strains were called lysogenic, and the phenomenon, lysogeny. Studies of lysogeny revealed many previously unrecognized features of virus-host cell interactions (Box 1.6). Recognition of this phenomenon came from the work of many scientists, but it began with the elegant experiments of André Lwoff and colleagues at the Institut Pasteur in Paris. Lwoff showed that a viral genome exists in lysogenic cells in the form of a silent genetic element called the prophage. This element determined the ability of lysogenic bacteria to produce infectious bacteriophages. Subsequent studies of the E. coli bacteriophage lambda established a paradigm for one mechanism of lysogeny, the integration of a phage genome into a specific site on the bacterial chromosome.

Bacteriophages became inextricably associated with the new field of molecular biology. Their study established many fundamental principles: for example, control of the decision to enter a lysogenic or a lytic pathway is encoded in the genome of the virus. The first mechanisms discovered for the control of gene expression, exemplified by the elegant operon theory of Nobel laureates François Jacob and Jacques Monod, were deduced in part from studies of lysogeny by phage lambda. The biology of phage lambda provided a fertile ground for work on gene regulation, but study of virulent T phages (T1 to T7, where T stands for “type”) of E. coli paved the way for many other important advances. As we shall see, these systems also provided an extensive preview of mechanisms of animal virus reproduction (Box 1.7).

       Animal Cells as Hosts

      The culture of animal cells in the laboratory was initially more of an art than a science, restricted to cells that grew out of organs or tissues maintained in nutrient solutions under sterile conditions. Cells so obtained from living tissues, called primary cells, have a finite life span. Their dependence for growth on natural components in their media such as lymph, plasma, or chicken embryo extracts, and the technical demands of sterile culture prior to the discovery of antibiotics, made reproducible experimentation very difficult. However, by 1955, the work of many investigators had led to a series of important methodological advances. These included the development of defined media optimal for growth of mammalian cells, incorporation of antibiotics into cell culture media, and development of immortal cell lines such as the mouse L and human HeLa cells that are still in widespread use. These advances allowed growth of animal cells in culture to become a routine, reproducible exercise.

BOX 1.5

      EXPERIMENTS

       The Hershey-Chase experiment

      By differentially labeling the nucleic acid and protein components of virus particles with radioactive phosphorus (³²P) and radioactive sulfur (³⁵S), respectively, Alfred Hershey and Martha Chase showed that the protein coat of the infecting virus could be removed soon after infection by agitating the bacteria for a few minutes in a blender. In contrast, ³²P-labeled phage DNA entered and remained associated with the bacterial cells under these conditions. Because such blended cells produced a normal burst of new virus particles, it was clear that the DNA contained all of the information necessary to produce progeny phages.