Viral Genome Replication
Replication of viral genomes requires the cell’s synthetic machinery in addition 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.
Assembly of Progeny Virus Particles
The various components of a virus particle, the nucleic acid genome, capsid protein(s), and in some cases envelope proteins, are often synthesized in different cellular compartments. Their trafficking through and among the cell’s compartments and organelles requires that they be equipped with the proper homing signals (see Chapter 12). Components of virus particles must be assembled at some central location, and the information for assembly must be preprogrammed in these molecules (see Chapter 13). The primary sequences of viral structural proteins contain sufficient information to specify assembly; this property is exemplified by the remarkable in vitro assembly of tobacco mosaic virus from coat protein and RNA (Box 2.1). Successful virus reproduction depends on redirection of the host cell’s metabolic and biosynthetic capabilities, signal transduction pathways, and trafficking systems (see Chapter 14).
Viral Pathogenesis
Viruses command our attention because of their association with animal and plant diseases. Viral pathogenesis is the process by which viruses cause disease. The study of viral pathogenesis requires investigating not only the relationships of viruses with the specific cells that they infect but also the consequences of infection for the host organism. The nature of viral disease depends on the effects of viral reproduction on host cells, the responses of the host’s defense systems, and the ability of the virus to spread in and among hosts (Volume II, Chapters 1 to 5).
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.
Overcoming Host Defenses
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).
Cultivation of Viruses
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).
There are three main kinds of monolayer cell cultures (Fig. 2.2), each with advantages and disadvantages for virus research. Primary cell cultures are prepared from animal tissues as described above. They have a limited life span, usually no more than 5 to 20 cell divisions. Commonly used primary cell cultures are derived from chicken or mouse embryos, monkey kidneys, or human tissues that are otherwise typically disposed of, such as embryonic amnion, kidney, foreskin, and respiratory epithelium. Such cells are used for experimental virology when the state of cell differentiation is important or when appropriate cell lines are not available. They are also used in vaccine production: for example, infectious attenuated poliovirus vaccine strains may be propagated in primary monkey kidney cells. Primary cell cultures are used for the propagation of viruses to be used as human vaccines to avoid contamination of the product with potentially oncogenic DNA from continuous cell lines (see below). Some viral vaccines are now prepared in diploid cell
