Principles of Virology, Volume 1. Jane Flint

Principles of Virology, Volume 1 - Jane Flint


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to viral proteins (see Chapters 6, 7, and 9). The cell provides nucleotide substrates, energy, enzymes, and other proteins. Transport systems are required because the cell is compartmentalized: essential components might be found only in the nucleus, the cytoplasm, or within subcellular organelles. Study of the mechanisms of viral genome replication has established fundamental principles of cell biology and nucleic acid synthesis.

      EXPERIMENTS

      In vitro assembly of tobacco mosaic virus

      The ability of the primary sequence of viral structural proteins to specify assembly is exemplified by the coat protein of tobacco mosaic virus. Heinz Fraenkel-Conrat and Robley Williams showed in 1955 that purified tobacco mosaic virus RNA and capsid protein assemble into infectious particles when mixed and incubated for 24 h. When examined by electron microscopy, the particles produced in vitro were found to be identical to the rod-shaped particles produced from infected tobacco plants (Fig. 1.9B). Neither the purified viral RNA nor the capsid protein alone was infectious. The spontaneous formation of tobacco mosaic particles in vitro from protein and RNA components is the paradigm for self-assembly in biology.

       Fraenkel-Conrat H, Williams RC. 1955. Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc Natl Acad Sci U S A 41:690–698.

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      Organisms have many physical barriers to protect themselves from dangers in their environment, such as invading parasites. Vertebrates also possess an immune system to defend against anything recognized as foreign. Studies of the interactions between viruses and the immune system are particularly instructive, because of the many viral countermeasures that can frustrate this system. Elucidation of these measures continues to teach us about the basis of immunity (Volume II, Chapters 2 to 4).

      Cell Culture

       Types of Cell Culture

      Although human and other animal cells were first cultured in the early 1900s, contamination with bacteria, mycoplasmas, and fungi initially made routine work with such cultures extremely difficult. For this reason, most viruses were produced in laboratory animals. The use of antibiotics in the 1940s to control microbial infection was crucial to the establishment of the first cell lines, such as mouse L929 cells (1948) and HeLa cells (1951). John Enders, Thomas Weller, and Frederick Robbins discovered in 1949 that poliovirus could multiply in cultured cells. As noted in Chapter 1, this revolutionary finding, for which these three investigators were awarded a Nobel Prize in 1954, led the way to the propagation of many other viruses in cells in culture, the discovery of new viruses, and the development of vaccines such as those against the viruses that cause poliomyelitis, measles, and rubella. The ability to infect cultured cells synchronously permitted studies of the biochemistry and molecular biology of viral reproduction. Large-scale propagation and purification of virus particles allowed studies of the composition of virus particles, leading to the solution of high-resolution, three-dimensional structures (see Chapter 4).

      Cells in culture are still the most commonly utilized hosts for the propagation of animal viruses. To prepare a cell culture, tissues are dissociated into a single-cell suspension by mechanical disruption followed by treatment with proteolytic enzymes. The cells are then suspended in culture medium and placed in specialized plastic flasks or covered plates. As the cells divide, they cover the plastic surface. Epithelial and fibroblastic cells attach to the plastic and form a monolayer, whereas blood cells such as lymphocytes settle but do not adhere. The cells are grown in a chemically defined and buffered medium optimal for their growth. Commonly used cell lines double in number in 24 to 48 h in such media. Most cells retain viability after being frozen at low temperatures (−70 to −196°C).


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