Viruses

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Viruses
Four tons of wheat; eight tons of rye; four fat oxen; eight fat pigs; twelve fat sheep; two hogsheads of wine; four barrels of beer; two barrels of butter; one thousand pounds of cheese; one bed, with accessories; one full-dress suit; and one silver goblet.


Goods traded in 1625 for one bulb of the "broken" tulips that became an aesthetic and commercial craze: the "tulipomania" of the mid-1630s in Holland
The human mind has always been fascinated with the concepts of largest and smallest. For mathematicians, these are infinity and zero; for physicists, the ever-expanding universe and subatomic particles. Biologists have a special feeling for whales and redwood trees, each of which has a grandeur all its own. At the other pole are the smallest of all living things, the viruses. Viruses populate the world between the living and the nonliving, the molecules that can duplicate themselves and the ones that cannot. Inherent in the organization and properties of viruses are many of the secrets of life and life processes.
All life forms employ two categories of chemicals, one that stores information and a second that acts, based upon that information, to duplicate the organism. The information stored in a living entity provides a plan or blueprint to carry out the organism's life functions and to pass a nearly exact duplicate of the plan to the next generation. Just as on a computer tape, the information stored in living organisms can be read linearly; at each position on the "tape," a bit of information is given. ("Bit," an abbreviation of the compound "binary digit," refers to the smallest unit of information stored on a computer tape or disk; here it corresponds to the nucleotide, the smallest unit of information in a chromosome.)
Consider the smallest of viruses, the viroids, which duplicate themselves in plants--and, in so doing, cause serious plant diseases. The simplest viroid contains only 240 bits of information, about ten million times less than the human information base (three billion bits). These 240 bits are arranged on a circular chromosome (the equivalent of the computer tape) and contain a set of signals that permit the molecule to duplicate itself. The replication of this viroid must occur within a plant cell, because the host cell contributes all the components needed for the viroid chromosome to make copies of itself. Indeed, all viruses can duplicate themselves only inside cells, because they require significant contributions from their hosts. But because different viruses take different things from their hosts, there is a great diversity in both viruses and the toll they exact--that is, the diseases they cause.
The diseases caused by viruses have had an enormous impact upon human beings. The existence of these parasites has shaped the evolution of plant and animal hosts and played a significant role in who we are today. Viral diseases, moreover, have been important events in our history. For example, it is unlikely that a small band of Spanish soldiers in Mexico could have defeated the Amerindians in 1520 in the absence of a raging smallpox epidemic brought inadvertently to the New World by the soldiers themselves. On the other hand, there has not been a single case (barring lab accidents) of smallpox in the world since October 1977; there is every reason to believe that an intensive vaccination program has now eliminated a disease that had a two-thousand-year recorded history. At the opposite extreme of our understanding and control, the present-day epidemic of AIDS, which is caused by the human immunodeficiency virus (HIV), is an extraordinary puzzle that will be with us for many years to come. Clearly, the medical consequences of virus infections remain an important reason to study these organisms.
During the last half of the twentieth century, a revolution in the biological sciences has taken place. New approaches and technologies have permitted us to study life processes at the molecular level. We have learned both to count and to determine the sequence of the bits of information (nucleotides) that make up the blueprint of life. The genetic code required to translate those bits of information into the molecules that act has now been deciphered. The development of molecular biology was led in a significant way by the study of viruses and their hosts. We have learned that many life forms and processes display common features. Because viruses depend upon their hosts to supply the tools needed to replicate themselves, the virus must share the same rules and signals with the host. Lessons learned from the viruses are thus applicable to more complex organisms.
Some viruses cause cancer in their hosts, by distorting the functions and signals used by the host cell to maintain itself. Identifying these functions and signals has been critical to our present understanding of the molecular basis of cancer, and the study of viruses has pointed the way to new approaches for treatment and prevention. An experiment is under way in Taiwan, for example, in which 63,500 newborn infants were recently immunized to prevent hepatitis B virus infection; the prediction is that forty or fifty years from now there will be 8300 fewer cases of liver cancer in that population. What we have learned from viruses has contributed to our understanding of life at both the molecular level and the population level: from the smallest perspective to the largest.
The first viruses were recognized as special entities only about a hundred years ago, between 1886 and 1898. We have come a long way since then, proceeding by asking one question at a time and building upon the answers. What are viruses? Are they alive? What do they look like? Are there many different kinds of viruses? Does each virus cause one specific disease? How do viruses cause diseases? How do viruses duplicate themselves? What have we learned about viruses that can be applied to humans? Each of these questions was asked for the first time in the context of a set of observations and a scientific perspective. In this book, we will attempt to preserve that context so that we can better appreciate the often remarkable insights of the successive questions and the often unexpected nature of the answers. In that way, we can recapture the one-hundred-year-old science of virology.
Historical Perspectives and Definitions
The idea of a submicroscopic world of the viruses could not have been grasped before the discovery of the microscopic world. Living organisms too small to see with the unaided human eye were first observed by Antony van Leeuwenhoek (1632-1723). Leeuwenhoek lived in Delft, Holland, where he held a political sinecure as custodian of the town hall. He was also a cloth merchant who used simple one-lens microscopes to examine draperies and cloth textures. Despite comparative isolation and a lack of formal training, Leeuwenhoek devoted a great deal of time and effort to his hobby of lens grinding, producing the best lenses then available: they magnified an object about 300 times. Leeuwenhoek turned his attention and interest to common objects and began to examine "rain-well-sea-and-snow water." In a series of communications to the Royal Society of London, Leeuwenhoek described his microbial world of "wee animalcules," including what we know today as the bacteria (spheres, rods, spiral shapes), protozoa, algae, and yeasts, as well as sperm, eggs, red blood cells, and more. Leeuwenhoek's zest for discovery and his wonder at seeing the microbial world unfold are best conveyed by one of his letters to the Royal Society, about a decaying tooth he examined: "I took this stuff out of the hollows in the roots, and mixed it with clean rain water and set it before the magnifying-glass ... . I must confess that the whole stuff seemed to me to be alive. But notwithstanding, the number of these animalcules was so extraordinarily great that 'twould take a thousand million of some of 'em to make up the bulk of a coarse sand-grain."
The 300-fold magnification employed by Leeuwenhoek is theoretically below the limit required to visualize bacteria; it is likely that he had to rely upon indirect light (now called darkfield illumination) to visualize even the outlines of these organisms, although he never stated that in any of his letters. Indeed, Leeuwenhoek was quite secretive about his tools and methods; only the best lens grinders, working independently, could repeat his observations. Because of these limitations, Carolus Linnaeus was able to recognize only six species of microbes as distinct entities in the group of animals that he classified under the name Chaos in 1767. With the invention and improvement of the two-lens, or compound, microscope, which had the ability to magnify one to two thousand times, six hundred distinct microbes were recognized in Ehrenberg's Atlas by 1838. (Today, the list of bacteria in Bergey's Manual of Systematic Bacteriology literally fills several volumes.) Once the microbial world had gained a firm reality, the scientists of the nineteenth century began to debate two questions whose answers would bring us closer to recognizing the existence of the submicroscopic world of viruses.
The first question--whether microbes can appear spontaneously in decomposing matter (that is, by "spontaneous generation") or arise from the duplication of similar microorganisms--was hotly debated, with experiments apparently supporting both sides of the controversy. Although many good experimental proofs eliminating spontaneous generation as a viable theory predate Louis Pasteur (1822-1895), he is usually given credit for show-ing that boiled (sterilized) medium would indeed remain clear and free of bacterial growth in a specially designed "swan-neck" flask that allowed air access to the medium but did not permit airborne bacteria to enter. These vessels, with their original contents, remain free of bacteria to this day in the Pasteur Museum in Paris. Pasteur declared, in his usual confident manner, "Never will the doctrine of spontaneous generation recover from the mortal blow of this simple experiment"--and he was right. These new beliefs led directly to antiseptic surgery (through the efforts of Joseph Lister in the 1860s) and sterile techniques in medicine and in scientific research; isolating a single class of organism can be accomplished only by having a sterile field or culture within which to grow it.
The second great question tackled by nineteenth-century microbiologists was whether microbes cause specific diseases. From the time of Hippocrates (fifth century B.C.), miasmas, or poisonous vapors, had been invoked to explain epidemics of contagious disease. To distinguish a miasma from a microbe and to determine which one really causes disease were formidable tasks. The challenge was taken up by Robert Koch (1834-1910), who realized that what was needed was a good definition of "causative agent." Koch's postulates for distinguishing between a real pathogen (disease-causing agent) and a contaminant or adventitious microbe are employed to this day. These postulates state: (1) the organism must be regularly found in the lesions of the disease, (2) the organism must be isolated in pure culture (hence the need for sterile technique), (3) inoculation of such a culture of pure organisms into the host should initiate the disease, and (4) the organism must be recovered once again from the lesions of this host. In 1876 Koch proved by these means that anthrax in cattle was caused by the bacterium Bacillus anthracis. Koch went on to discover that these bacteria can form spores that are resistant even to boiling in water. This observation helped to explain why some experiments in spontaneous generation that had employed boiled water resulted in media filled with bacteria after the flasks had cooled; Pasteur's proof was thus reinforced. The existence of spores also explained why fields where anthrax-infected cattle had grazed could infect new herds even years later. The predictive value of these new sciences and the utility of their observations were becoming clear.
By the last decade of the nineteenth century, the microscopic world of bacteria was an important part of our understanding of disease, medicine, and agriculture, to say nothing of our newfound knowledge about bread, beer, and wine--the gifts of fermentation by yeast and other microbes. The microscope could resolve bacteria whose size was 1 to 2 microns (a micron is one millionth of a meter, or 0.0001 centimeter). The light microscope could, in the best cases, resolve a particle as small as 0.2 to 1.0 micron in diameter. The size of most common bacteria, 1 to 2 microns, was then the visual edge of the microscopic world, below which the human eye could not see. Armed with Koch's postulates, microbiologists began an intensive classification of bacteria and the diseases they cause. Their efforts went well until, in some cases, this already standard paradigm could not be repeated.
In 1886 Adolf Mayer, a German agricultural chemist working in Holland, was studying a disease of tobacco characterized by a pattern of light and dark areas on the infected leaves. He proposed the name "mosaic disease of tobacco" and proceeded to try to determine if this disease had an infectious origin. Mayer took the affected leaves and ground them up with water to produce a clear, soluble ex-tract of leaf components, which he then injected into healthy plants. In nine cases out of ten, these plants showed all the symptoms of the disease. Mayer then attempted to culture, in pure form, the organism that caused the disease, using the standard techniques of his day for growing bacteria. Surprisingly, this search failed to isolate a bacterium or fungus in pure form, even though the disease could be transmitted like any other infectious disease. Koch's postulates could not be satisfied, and Mayer concluded that this disease was caused by a bacterium whose special nature prevented its culture, but which certainly would be revealed in future studies.
In the meantime, Chamberland filter-candles were produced--special filters that had pore sizes too small (about 0.1 to 0.5 micron) to let bacteria through. In 1892 a young Russian scientist, Dimitrii Ivanovsky, who was studying tobacco mosaic disease, reported to the St. Petersburg Academy of Science: "I have found that the sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filter-candles." The tobacco mosaic agent, which failed to replicate (that is, to increase in strength) in cell-free culture but appeared to grow on leaves, was smaller than anything previously described. (Ivanovsky pointed out the possibility that a toxin secreted by a bacterium caused this disease; toxins do not replicate themselves.) Six years later in the Netherlands, Martinus W. Beijerinck repeated Ivanovsky's experiment and showed that an infectious agent, able to replicate in the leaves but not in the filtered solutions, could transmit the tobacco mosaic disease. The submicroscopic filterable agent could be diluted manyfold and, when placed upon tobacco leaves, would produce many copies of itself that could be diluted again and shown to transmit disease. This plant pathogen, now called tobacco mosaic virus, was the first living, replicating member of the submicroscopic world to be recognized.
These observations were rapidly followed by the isolation of a filterable animal virus, the foot-and-mouth disease virus of cattle, by F. A. J. Löffler and P. Frosch in 1898. The first human disease shown to be caused by a filterable agent smaller than any known bacteria was yellow fever, recognized by the U.S. Army Commission under the direction of Walter Reed in 1900. Transmissible and filterable agents were even found in clear fluid extracts of bacteria themselves by Frederick W. Twort in England (1916) and Felix d'Hérelle in France (1917). D'Hérelle named these viruses of bacteria bacteriophages.
The term virus, which comes from the Latin word for poison, had been used synonymously for infectious agents of all kinds throughout the nineteenth and into the twentieth centuries. Beijerinck, in his discovery of the tobacco agent, called it a "contagium vivum fluidum." By the 1930s, scientists routinely used the term filterable virus to refer to any agent that passed through a filter fine enough to retain bacteria, but today virus is applied only to submicroscopic agents (less than 0.3 micron) that can pass through filters that retain most bacteria. These agents, whether their hosts are plants, animals, or bacteria, are obligate intracellular parasites--that is, they are able to duplicate themselves only inside a host cell; as the first experiments had shown, viruses cannot replicate in a cell-free (fluid) environment. This parasitism may result in the death or alteration of the host cells, which is in large part the reason that viruses cause diseases. For the early virologists, unable to see viruses in their light microscopes, there was an element of faith in these studies. But many viruses have now been shown to satisfy Koch's postulates: like the bacteria that cause anthrax, they can replicate and cause disease.
Modern Definition of Viruses
By the 1930s viruses were classified according to the host that they infected. Three groups of viruses--plant, animal, and bacterial (the bacteriophages)--were recognized; they were identified and stored in liquid filtrates derived from the leaves, lesions, or juices of their hosts. As methods became available to purify viruses from the complex mixtures of chemicals found in these fluids, the chemical composition of a virus could be determined and used to classify it.
When, in 1935, Wendell Stanley succeeded in obtaining a crystalline form of the tobacco mosaic virus, this form was seen by some scientists as filling the gap between the living world of reproducing viruses and the chemical or nonliving world of molecules and crystals. In truth, the ability to form a crystal simply reflects the structural uniformity or symmetry of all the viruses in a population, together with a surface complementarity that permits an orderly aggregation of virus particles. German technology in the 1930s provided a new tool: the electron microscope. Instead of visible light, it employs beams of electrons focused by magnets--permitting the useful resolving power to go from one to two thousand diameters magnification to three hundred thousand diameters. Now the submicroscopic world of viruses could be visualized, and electron micrographs showed the beautiful symmetry of viruses predicted by the crystallization of the tobacco mosaic virus. Today, chemical composition, symmetry, and structure make up the basis of virus classification.
Almost all viruses are composed of two parts: a nucleic-acid core containing the genetic informationand, surrounding and protecting this core, a coat built from repeating protein subunits and, in some cases, further encased in a lipid (fatty) envelope. A virus's genetic information may be encoded in deoxyribonucleic acid (DNA), as it is for all nonviral living organisms, or in some viruses it may be encoded in ribonucleic acid (RNA). Both DNA and RNA are long, linear polymers composed of subunits (monomers) that are linked together by strong chemical bonds, as we will see in Chapter 2. The monomers, called nucleotides, are each composed of a base, a sugar, and a phosphate group. The type of sugar gives the nucleic acid its name: in DNA the sugar is deoxyribose, and in RNA it is ribose.
As we will examine in some detail in Chapter 2, DNA is usually found not as a single polymeric strand, but as a double strand with the two polymers wound about each other in a helix. The sequence of nucleotides in a DNA or RNA polymer determines the genetic information, and the chemical composition of this genetic information--whether it is encoded in DNA or RNA--is used to classify viruses. Viruses may contain double-stranded DNA (herpesviruses), single-stranded DNA (parvoviruses), double-stranded RNA (reoviruses), or single-stranded RNA (poliovirus). The RNA viruses are unique in that they are the only living organisms that use RNA to store their genetic information; all the other reproducing forms of life employ DNA. (The consequences of this will become clear when we discuss the RNA viruses in greater detail.)
In the simplest cases, the DNA or RNA of a virus is surrounded by proteins. Proteins are also polymers, composed of twenty different monomeric subunits: amino acids. The sequence of these amino acids determines the chemical properties of the protein by guiding the polymer to fold into ashape or structure that determines the protein's function. The sequence of nucleotides in DNA or RNA polymers specifies, in turn, the sequence of amino acids in a protein. In this way, genetic information determines a protein's sequence of amino acids, shape, and activity (function).
The function of a virus's coat protein is to form a cage or protective sphere around the genetic information (DNA or RNA) so that it is not attacked or altered by the environment. The protein coat also mediates the spread of a virus from host to host, ensuring that new viral generations will perpetuate themselves. Because a single protein is simply not large enough to encompass all the genetic information, hundreds or thousands of identical protein subunits are produced in the virus-infected cell. Several of these proteins come together into a symmetrical assembly unit called a capsomere. Capsomeres interact with each other through contacts on the protein surfaces to form a shell or coat surrounding the viral nucleic acid. This shell is often called a capsid, and the entire particle is called a nucleocapsid (DNA plus protein or RNA plus protein). The simplest viruses contain only this nucleic acid surrounded by a protein coat. More complex viruses surround the nucleocapsid with a lipid envelope and insert additional viral proteins into the envelope. A completed or mature viral particle is called a virion.
Different viruses encode the information in their DNA or RNA for distinct proteins that make up the capsomeres, capsids, and virions. Because of this, each virus has a unique set of capsomere proteins whose shape and composition determine how it will interact with other identical capsomeres and form a shell about the nucleic acid. Different viruses, therefore, produce virion particles with distinct shapes, sizes, and properties--each determined by the shape of the capsomere and its bonding patterns with neighboring capsomeres. Tobacco mosaic virus, for example, is composed of a single strand of RNA that produces a capsomere that assembles around the RNA to form a helical, rod-shaped structure. Other viruses, such as the rhinoviruses that cause the common cold, also contain a single strand of RNA, but the capsid is assembled into a spherical shape around the nucleic acid.
These closed-shell spherical virions have structures based on icosahedral symmetries. An icosahedron is an enclosed surface composed of twenty equilateral triangular faces. The symmetry of the icosahedron is characterized by rotational axes: each of the twelve vertices has a fivefold rotational axis (each of five rotations of 72 degrees produces an identical view), a threefold rotational symmetry around the center point of each of the twenty triangular surfaces, and a twofold rotational symmetry about each edge of the icosahedron, where two triangular surfaces meet. As recognized by the innovative architect Buckminster Fuller, who used the icosahedral shape to build his structurally sound geodesic domes, this class of polyhedron is the most efficient of all the closed shells that can be constructed. This is because the icosahedron uses the smallest subunits (capsomeres) to build an enclosed shell of a fixed size, which in turn conserves the genetic information needed to encode these subunits, resulting in a smaller DNA or RNA strand. In addition, small protein subunits conserve the energy it takes to synthesize proteins in a cell. There are sixty identical elements on the surface of any icosahedron, and these elements form a symmetrical structure related by the twofold, threefold, and fivefold rotational symmetries.
Today, viruses are classified by their hosts, chemical composition (nucleic acid, protein, presence or absence of lipid envelope), shape, size, and symmetry. Such a classification is presented in the table in the appendix, which emphasizes the viruses discussed in this book. Included in this table are the diseases or associated pathologies caused by some of these viruses.
Cellular Structure and Functions
To understand viruses, we must first review our knowledge of the indispensable environment for viral replication--the living cell. Just as viruses are built largely from repeating identical subunits (capsomeres) that form symmetrical structures, so too the body of a multicellular host is built essentially from repeating units of cells. There are several advantages to building a structure from identical repetitive units. First, it is economical to mass-produce the same part over and over again. Second, if a rare mistake is made, only one of a million units is different; if it doesn't fit into the symmetrical lattice (because it is different), it usually doesn't hurt the structure. If a systematic mistake is made, on the other hand, that may be lethal. In addition, self-assembly systems are guided by a small number of attractive forces between identical subunits, repeated over and over again, to provide strength in numbers. Such systems build the symmetric structures so often seen in living organisms.
We see these principles in action at almost every level of life. At the molecular level, proteins assume their shape from their chemical composition; oppositely charged amino acids in different portions of a protein, for example, may cause it to fold, bringing together the opposite charges by physical attractive forces. The shape of a protein provides surfaces for aggregation with other proteins to form multimeric subunits in cells or capsomeres in viruses. With viruses, identical capsomeres then assemble to form a particle; virus particles may come together to form a visible crystal, as in the case of the tobacco mosaic virus.
Similarly, parts of a cell are built from repeating units that are organized to produce subcellular organelles where specific functions are carried out. A cell collects its organelles into a package by surrounding these units in a lipid membrane, the plasma membrane. Thousands and millions of identical cells are brought together to form an organ that represents the sum of these cellular functions. Identical repetitive cellular units in an organ are often interrupted by unique cellular subunits that carry out specialized functions. Organs or systems of functions are combined to produce a body, which gains its unique qualities by integrating these organ systems and having them work in a coordinated way useful for the whole. It is clear that similar principles have evolved and been employed through the many levels of organization of life--from molecule to organelle to cell to organ to organism.
At the heart of any cell is the information needed to build the cell. All information is encoded in the DNA molecule housed in the cell nucleus. The first problem a cell or virus faces is that the DNA molecule is very long. In the case of a virus with 5200 nucleotides, the linear DNA polymer is several microns long, while the three billion nucleotides in human DNA are a meter long when stretched end to end. Written out as a linear series of one-letter abbreviations, the code or nucleotide sequence for a single virus would take a whole page of this text. The code for a human being would take five hundred thousand pages.
Packaging the genetic material, then, raises a problem. The diameter of a small virus may be0.045 micron, and a cell nucleus with its DNA is only about 7 to 10 microns across. How can such a long molecule of DNA be fitted into such a small volume of space, while still leaving it available to replicate? The solution, evolved in both viruses and cells, is to wrap the long DNA strand around a protein core. This is not too different from wrapping a computer tape about a spool. For all cells, the protein core is called a nucleosome; it is composed of eight protein subunits (histones) that assemble into a nucleosome core. These nucleosomes then condense to form a linear filament, which in turn forms a long series of loops, back and forth, packaging the DNA into tight protein-DNA (nucleoprotein) complexes.
This DNA-protein package is separated from the rest of the cell by an envelope, the nuclear membrane. This is really a double-membrane system that is interrupted at periodic intervals by large pores where the outer and inner membranes are continuous. Both the nuclear membrane and the pores are highly selective, letting out of the nucleus some--but not all--molecules made there and taking into the nucleus some--but not all--molecules made in the cytoplasm (the cell protoplasm outside the nuclear membrane but bounded in turn by the cell's plasma membrane). Molecules--for example, some proteins--contain signals, determined by their sequence of amino acids, that govern entry into the nucleus. These signals tell each protein where to localize in a cell; they are the ZIP codes of a molecule. By this mechanism, proteins like the histones reside only in the nucleus, while other proteins reside only in the cytoplasm. This is a key part of the self-assembly system of a cell. Molecules must "know" where to go in a cell in order to build a structure that has spatially distinct elements.
The nucleus of a cell, then, is separated by its membrane from the cytoplasm, which contains a number of structurally and functionally distinct organelles. At several places, however, the outer nuclear membrane appears to be continuous with an extensive network of membranes in the cytoplasm, the endoplasmic reticulum (ER). This system of membrane-enclosed spaces seems to be used for the transport of certain molecules around the cell, allowing the placement of a molecule in a particular cellular locale or compartment. Lining the inside portions of the ER in the cytoplasm are ribosomes--organelles made up of subunits of RNA and protein (nucleoprotein complexes) that are the sites upon which all protein synthesis occurs. Newly made proteins on a ribosome can be transported across the membrane of the ER into the fluid-filled spaces of the ER, where specialized vesicles transport proteins to specific organelles and locations in the cell. (A vesicle is a spherical particle made of lipid, surrounding a fluid-filled space--a kind of bubble.)
Places where ribosomes collect on the ER are called the rough ER; they synthesize proteins for export outside the cell or for transport to specific cellular locales. Some of the cellular ER elements do not have associated ribosomes (the smooth ER), and some ribosomes are free of ER elements and synthesize proteins that remain in the cytoplasm. Once again, a spatial feature--where a protein is destined to reside in a cell--is associated with the place it is synthesized; that is, the rough ER exports the protein or localizes it to membranous portions of the cell, while free ribosomes synthesize cytoplasmic proteins. Membranes thus serve to segregate the chemical reactions of a cell into compartments. The ER is also the site where new membranes are synthesized to increase the surface area on which chemical reactions may occur and to produce millions of vesicles to transport proteins about the cell for localization in the three-dimensional structure.
Some of these vesicles transport proteins to the Golgi apparatus, a system of membrane compartments organized into a stacked array. The different layers of membranes contain different sets of enzymes whose function is to add complex sugars to proteins or lipids, forming glycoproteins or glycolipids. This is often an important part of the preparation for either the secretion of a protein from a cell to the outside environment or the insertion of a protein into the plasma membrane of the cell. Modifying a protein by adding a sugar to it alters its chemical properties and may change its function or location in a cell. So-called exocytic vesicles (small, spherical lipid particles) bud from the Golgi and go to the cell surface. They fuse with the plasma membrane and secrete their contents to the environment outside the cell. In this way, cells communicate with each other, and the whole becomes greater than the sum of its parts.
Reciprocally, the plasma membrane is constantly budding new vesicles that contain elements of the outside environment into the cytoplasm; in this way, substances at the cell surface are transported into the cell and sampled to collect information about events occurring outside. After the information is processed, the so-called endocytic vesicle has fulfilled its task. But the cytoplasm of a cell contains a number of vesicles with other specialized functions: lysosomes, for example, are membrane-enclosed bodies that store enzymes that digest or degrade proteins, DNA, or RNA. The lysosomal membrane keeps these powerful enzymes away from the cytoplasmic cellular proteins essential to life. Once an endocytic vesicle's sampling task is complete, it fuses with a specialized lysosome (an endosome), and the contents of the vesicle are degraded. The monomers formed as a product of this digestion (amino acids, nucleotides, and so forth) are then reused in the synthesis of new molecules.
Although lysosomes are only a small part of a cell, when they do not function properly the entire organism is at risk. Tay-Sachs disease is an inherited disorder in which the lysosomes that digest lipids are missing a single enzyme needed to break down these fats. The resulting accumulation of lipids blocks nerve-cell impulses, and the consequences are fatal to the organism.
Viruses frequently take advantage of the target-specific or vesicle-specific signals (ZIP codes) that direct molecules to the lysosomes. Semliki Forest virus, a togavirus that replicates in both insects and humans, has a coat protein that permits it to attach to a specific receptor at the plasma membrane of a cell. This virus is taken into the cell in an endocytic vesicle that, by virtue of the ZIP code on the viral protein, targets the vesicle for fusion with an endosome. The viral-encoded coat protein has stolen the same chemical signal used by cellular proteins to fuse the endocytic vesicle to the endosome. The fusion of these two vesicles releases the virus into the cytoplasm, where the viral RNA uses the host's ribosomes to synthesize viral proteins. Then the virus uses the host's ER, Golgi apparatus, and exocytic vesicles to exit from the cell via the plasma membrane. The virus has ensured its own propagation by using the same ZIP codes as the host proteins to exploit cellular organelles.
Two more dimensions of cell function demand brief attention: energy supply and three-dimensionalstructure. The synthesis of proteins, the traffic of these vesicles and proteins about the cell, the duplication of the genetic information, and the constant synthesis of new vesicles are all processes that require enormous amounts of energy. The synthesis of polymers from their respective monomeric components is common to all four of the major chemicals in a cell: amino acids produce proteins, nucleotides produce DNA or RNA, sugars produce polysaccharides (complex sugars), and fatty acids produce lipids. In each case, synthesis consumes energy to link molecules together and generate polymers that store information or act biochemically. All cells, in all organisms, use the same currency of energy--a chemical that can channel its energy into heat (to keep body temperature constant), movement, synthesis of new polymers, and duplication of the cell and organism. (Even the processes of reading and thinking about this consume chemical energy.) The chemical in the cell that does all this is called adenosine triphosphate (ATP). This is a nucleotide that is also used as a precursor of RNA. ATP stores energy in its chemical bonds, especially the bonds that hold the three phosphate groups together. When ATP is degraded to adenosine diphosphate (ADP), energy is released that can be transferred to other molecules that effect movement or thought, assemble new polymers, or produce heat.
Cells have perfected systems to synthesize tremendous levels of ATP--a single healthy liver cell will synthesize ten million molecules of ATP every second. Given the billions of cells in the human body, we produce and consume staggering amounts of this chemical fuel all the time. ATP is generated during a series of reactions linked to the complete breakdown of glucose (a sugar) into carbon dioxide (we exhale it) and water (we excrete it). In the best cases (that is, with lots of oxygen around), we gain about thirty-six ATP molecules for every molecule of glucose converted to carbon dioxide and water. Most of our ATP is produced in a specialized organelle in the cytoplasm called a mitochondrion. Each mitochondrion has a convoluted membrane system upon which resides a set of enzymes that function as pumps, gates, and channels to transfer the energy gained from the breakdown of glucose into ATP. ATP is then translocated out of the mitochondria into the cytoplasm for use in hundreds of reactions that fuel the cell.
This picture of the compartmentalization of a cell--employing membranes to separate functional units and using ZIP codes, in the form of chemical sequences, to transport proteins from organelle to organelle--reflects the high degree of internal cellular organization. Most cells can change shape in response to external stimuli, repositioning their internal organelles and, in some cases, moving from place to place. The ability to accomplish these activities depends on a network of protein filaments and tubules in the cytoplasm. These elements are termed the cytoskeleton of the cell. Not surprisingly, both filaments and tubules are built fromprotein subunits that can assemble and disassemble rapidly in a cell. This process is controlled at several levels; the building of cytoskeletal elements at one position in a cell while disassembly proceeds at another location is the basis for shape changes, cell movement, or the redistribution of organelles. ATP-TO-ADP conversions release enough energy to slide one protein filament against another, generating a force for movement. While all cells have filaments and microtubule arrays, certain specialized examples of these subunit assembly systems in, for instance, muscle cells have been adapted for particular purposes.
The cell that we have been describing here is an average, nonspecialized cell from an animal or a human being. Plant cells have the same organelles, plus additional ones to collect sunlight and convert carbon dioxide and water into glucose. Bacteria, however, are quite different. One of the major distinctions between bacteria (and their relatives) and all other plants and animals is that the bacterial nucleus is not surrounded by a membrane--that is, the DNA lies in the cytoplasm without the separation provided by the nuclear membrane. In addition, bacteria do not have clearly identifiable sets of subcellular organelles like the ER, Golgi, or mitochondria. These differences are so fundamental that bacteria and their relatives are classified separately from the plant and animal kingdoms and are designated prokaryotes (having a primitive nucleus). Cells of animals or plants are designated eukaryotes (having a true nucleus). The advantages of specialization by the more highly evolved animals and plants do not result only from the differences between cells in a multicellular organism. As we have seen, the higher animal or plant cell is more specialized within itself, when compared to the cells of prokaryotic organisms.
Steps in the Replication of Viruses
Viruses are not cells, but they require cells. As obligate intracellular parasites, they must attach to and enter a host cell in order to reproduce. Although different groups of viruses have evolved diverse strategies to replicate themselves, it is possible to review the events in the life cycle (replication cycle) of a generalized virus to provide a background for understanding individual virus groups.
Virtually all viruses must accomplish eight key steps in their replicative cycle.
Step 1. The virus attaches itself to its host cell.
Step 2. The virus or its genetic information penetrates the cell.
Step 3. The nucleic acid is uncoated, which frees the DNA or RNA from its capsomeres or lipid envelope and permits the host cell to read out (express) the genetic functions of the virus.
Step 4. At this stage in the life cycle of many viruses, only a portion of the viral genetic information is expressed, resulting in the synthesis of only the subset of viral-encoded proteins collectively called the early viral gene functions (proteins). These proteins may function in one of several ways. In some cases, they contribute directly to the replication of the viral chromosome. In other cases, these viral proteins turn off many of the host-cell activities, maximizing the cell's available resources for virus production. Alternatively, some viruses that can duplicate themselves only in actively dividing host cells produce proteins that stimulate host-cell division.
Step 5. The viral nucleic acid is then synthesized to produce hundreds or thousands of copies of the viral chromosome.
Step 6. At this time, a second subset of the viral genetic information, commonly termed the late proteins, is expressed. These are the structural proteins, including the capsomeres of the virus.
Step 7. The capsomeres are assembled to form a shell around the nucleic acid of the virus.
Step 8. The mature virus, having duplicated its new copies, is released from the infected cell to attack a new cell and repeat this process.
Going from steps 1 through 8, a viral replicative cycle displays temporal organization: specific events occur in sequence, each dependent upon the successful completion of the previous step. This developmental process, which results in the synthesis of thousands of viral particles, is formally similar to the development of a multicellular organism from a single fertilized egg cell. Both processes require quantitative and qualitative changes over time. Early viral proteins, made from the single genome of the infecting virus, are expressed at low levels, while the late proteins are made from the newly replicated viral chromosomes produced in step 5. Quantitative changes are thus initiated and regulated chronologically.
Let us review each step in a little more detail.
Attachment or Adsorption
A virus uses specific proteins on its coat to recognize and attach to specific receptors on a cell surface (the plasma membrane). These receptors may be proteins or other components that are located only on certain cells; in effect, a virus may be able to attach only to a liver cell or to a lung cell and to no other cell of the host body. This may result in specific disease states, such as hepatitis or pneumonia caused by viruses that replicate only in liver or lung cells. Viruses are said to have a specific tissue preference (tropism), and in some cases this is due to tissue-specific receptors.
The human immunodeficiency virus (HIV), for example, attaches to a receptor called the CD4 protein. The CD4 protein is found on the surface of certain lymphocytes (white blood cells) that are critical for the vitality of the immune system in humans. By entering into and killing only cells that have the CD4 protein on their surfaces, HIV kills the cells that maintain our ability to protect ourselves from infection, thus causing AIDS (acquired immune deficiency syndrome; see Chapter 7). The tropism of HIV is determined by its adsorption to cells with the CD4 receptor. Few other animals contain the CD4 protein on their cell surfaces (it is specific for some primates), so the HIV agent grows only in these animals (chimpanzees and humans).Thus, the species limitations of some viruses may be due to restrictions in their ability to attach to specific cells. Other viruses replicate in many animals (influenza virus, for example), and this too can have profound consequences for the biology of the virus (see Chapter 8). There are also viruses that can replicate in many tissues or cell types of an animal, which means they use receptors that are found on most cells. Such viruses may cause widely disseminated disease throughout the body of the host.
Bacteriophages infect and kill bacteria, which are single-cell organisms. Like all viruses, bacteriophages attach to a specific receptor on the surface of their host cell. Occasionally, a mutation (a change in the genetic information) occurs in the bacterium so that the host cell can no longer synthesize the receptor on its surface. In many cases this is not detrimental to the bacterial host, which is then resistant to the virus that normally uses that receptor. As might be expected, resistant host cells arise in populations under attack by viruses--and these resistant cells, without receptors, survive, replicate, and eventually take over as a majority type in the population. In this case, resistance to virus infection is selected for by the presence of a killer virus.
But it is not in the best interest of the virus to kill all its host cells, leaving only resistant bacteria; if there are no hosts, there can be no viruses. Some viruses have developed alternative strategies for a live-and-let-live viral life cycle. Others have taken advantage of rare mutations in the virion coat protein, which may permit the virus to attach to a new receptor, even one found on the bacterial cell that was resistant to the original or parent virus. This rare virus is then selected for, by virtue of its newly acquired ability to attach itself to the otherwise resistant bacteria and duplicate itself in this new host. Host-range mutations, as they are called, that extend or restrict the ability of a virus to attach to susceptible host cells can thus occur in either the virus or the host cell. A virus evolves by altering its host range to be able to enter new environments. The attachment step provides a specificity and a selectivity that have profound consequences for the life cycle of a virus.
Penetration
For those animal viruses with lipid envelopes, penetration of the nucleocapsid core of a virion into a cell occurs when the virion envelope fuses with the plasma membrane to which the virus is attached. The fusion of viral and cellular lipid membranes is usually mediated by proteins encoded by the information (chromosome) found in the viral particle. Fusion leaves the nucleoprotein core of the virus on the inside of the cell; this penetration step is also part of the uncoating of such a virus.
Animal viruses without a lipid envelope (the so-called naked virions, composed only of protein plus DNA or RNA) are usually taken into cells to which the virus is attached by a process called phagocytosis, or endocytosis. As we saw earlier in this chapter, many cells constantly produce cytoplasmic vesicles by pinching off portions of their own membranes facing the outside of the cell to yield spherical vesicles that sample the extracellular environment. When these endocytic vesicles migrate to the cytoplasm and fuse with the endosomes, they transport a virus to a cellular location where the viral protein subunits are removed and the viral nucleic acid becomes accessible to the cellular environment. Here too, penetration and un-coating of the nucleic acid are coupled steps. In some cases, viral nucleocapsids are transported directly to the host-cell nucleus, where the viral chromosome resides during the entire life cycle.
Some bacteriophages are composed of a head, containing the nucleic acid, and a tail; they resemble sperm. At the base of the tail, the virus-specific attachment organs secure the virus to the cell wall of a sensitive bacterium with receptors. The tail then contracts, inserting a protein tube into the bacterium, just as a needle and a syringe act to inject a substance. The bacteriophage's nucleic acid then moves from the head through the tube into the bacterium--effecting attachment, penetration, and uncoating in a single step.
Strategies of Viral Multiplication
The early events during any virus infection use the cellular signals and machinery to deliver the viral chromosome into the cell. Some viruses have evolved elaborate mechanisms to accomplish this, but the end result is always the same: access to the intracellular environment so as to reproduce more viruses. When a viral chromosome composed of either DNA or RNA enters a cell, it must accomplish two things: (1) the expression of new viral proteins in some temporal order and (2) the replication or duplication of the viral nucleic acid. The various strategies for accomplishing this are different for different virus groups, so the detailed descriptions are best left for later chapters. The expression of the viral genetic information relies on a series of steps that translate a genetic code stored chemically in the sequence of DNA or RNA nucleotides. The translation takes the chemical form of the synthesis of a protein that is composed of a defined order of amino acids. DNA (or RNA) stores the information; proteins act by carrying out the functions required for replication of the DNA (or RNA).
Assembly, Maturation, and Egress
Animal viruses have evolved several different methods for their assembly in and release from infected cells. With many viruses, the viral nucleic acid is packaged with capsomeres in the nucleus or the cytoplasm, and the dying cell releases the virus particles as it disintegrates. In enveloped viruses, however, viral structural proteins or glycoproteins (proteins that have carbohydrates associated with them) are inserted through the plasma or other membranes of a cell. The viral chromosome-protein complex that has been assembled in the host cell then migrates to the position of the viral glycoprotein at the inner or cytoplasmic side of the cellular membranes and associates with it. This promotes the formation of buds where the cellular membrane containing the viral glycoproteins surrounds the nucleocapsid core, and the virus particle is extruded into the extracellular environment. Thus, the maturation and cellular release steps are coordinated, and virus production goes on for hours as a continuous release process proceeding at the cell surface.
Finally, some bacteriophages encode the information to synthesize a protein that actively promotes lysis (dissolution or disintegration) of a host cell. It is, of course, critical for the virus to withhold the synthesis of this protein, or to block its function, until very late in its replication cycle. Active lysis mechanisms, while efficient in spreading infection, can be lethal for the virus if the temporal events are not controlled properly (they are alwayslethal for the host cell). Premature cell lysis, before infectious virus particles are produced, would result in no progeny virus and loss of the species.
It is clear, then, that viruses have evolved to reproduce themselves in a remarkably diverse set of ways. As Chapters 4 through 9 will describe in detail, viruses have evolved life styles that are unique, in that some have even left the standard paradigm of information flow in life--DNA to RNA to protein. Yet viruses are dependent upon their host cells. They must obey the rules and laws of life in a cell.

Are Viruses Alive?
The problem with this question is the same one that faced Robert Koch in defining a causative agent. How do we define life? And, once we do, can viruses be included in this definition? Volumes have been written to elucidate the properties of a living thing, but consensus has been rare in these debates.
Viruses employ a common genetic code (the sequence of nucleotides in DNA and RNA that determines which amino acid is found at a specific position in a protein) to store information for their development and replication; indeed, the code is universal to all reproducing organisms. This could suggest a single origin event--or, alternatively, the evolution of the best-of-all-possible genetic codes from many origin events. Viruses must, of course, share the same code and signals as their host cells, in order to duplicate themselves within those cells.
Viruses, as we have seen, can program their own replication within the confines of a cell. They have a plan, satisfying Aristotle's definition of life, encoded in the format of all living things. And, like other living things, viruses evolve and respond to environmental changes. Whether this describes the simplest of living forms or an extraordinarily complex combination of nucleic acids and proteins that are merely chemicals depends upon one's view of life.
In the last chapter of this book, it will become clear that viruses, as we have now defined them, are part of a continuum of genetic elements--viruses, viroids-virusoids, plasmids, transposable elements, and insertion elements--that leads to ever-simpler forms of life: finally, to a naked DNA molecule that contains only the most primitive bits of information. We will see that it is possible that these simplest of elements could be part of a continuing creation of new life forms within a cell. (Is this a revival of the theory of spontaneous generation?) If they do play such a part, then viruses--with their ability to move between cells, between hosts, or even, in some cases, between species--will contribute to the evolutionary changes of the host by bringing it new genetic information. There is good evidence that the vestiges of viral DNA are present in the chromosomes of human beings, passed on from generation to generation as passengers in our genetic endowment. At present it is not clear whether this viral DNA results from evolutionary accident or is indeed a fellow traveler selected for a value we do not yet understand. Viruses, however, do have at least two clear roles in our evolution. First, the diseases they cause select for resistant or less sensitive hosts; the second role follows from the direct incorporation of viral genetic information into our own lineage. Clearly, viruses are intimately associated with our lives. If they are not themselves alive, they live with us.

Copyright © 1992 by Scientific American Library