Asimov's New Guide to Science

by Isaac Asimov

  A Dutch bacteriologist, Martinus Willem Beijerinck, repeated the experiment in 1897 and came to the decision that the agent of the disease was small enough to pass through the filter. Since he could see nothing in the clear, infective fluid under any microscope and was unable to grow anything from it in a test-tube culture, he thought the infective agent might be a small molecule, perhaps about the size of a sugar molecule. Beijerinck called the infective agent a filtrable virus (virus being a Latin word meaning “poison”).

  In the same year, a German bacteriologist, Friedrich August Johannes Löffler, found that the agent causing hoof-and-mouth disease in cattle could also pass through a filter. And, in 1901, Walter Reed, in the course of his yellow-fever researches, found that the infective agent of that disease also was a filtrable virus. In 1914, the German bacteriologist Walther Kruse demonstrated the common cold to be virus-produced.

  By 1931, some forty diseases (including measles, mumps, chicken pox, influenza, smallpox, poliomyelitis, and hydrophobia) were known to be caused by viruses, but the nature of viruses was still a mystery. Then an English bacteriologist, William Joseph Elford, finally began to trap some in filters and to prove that at least they were material particles of some kind. He used fine collodion membranes, graded to keep out smaller and smaller particles, and he worked his way down to membranes fine enough to remove the infectious agent from a liquid. From the fineness of the membrane that could filter out the agent of a given disease, he was able to judge the size of that virus. He found that Beijerinck had been wrong: even the smallest virus was larger than most molecules. The largest viruses approached the rickettsia in size.

  For some years afterward, biologists debated whether viruses were living or dead particles. Their ability to multiply and transmit disease certainly suggested that they were alive. But in 1935, the American biochemist Wendell Meredith Stanley produced a piece of evidence that seemed to speak forcefully in favor of “dead.” He mashed up tobacco leaves heavily infected with the tobacco-mosaic virus and set out to isolate the virus in as pure and concentrated a form as he could, using protein-separation techniques for the purpose. Stanley succeeded beyond his expectations, for he obtained the virus in crystalline form! His preparation was just as crystalline as a crystallized molecule, yet the virus evidently was still intact; when he redissolved it in liquid, it was just as infectious as before.

  For his crystallization of the virus, Stanley shared the 1946 Nobel Prize in chemistry with Summer and Northrop, the crystallizers of enzymes (see chapter 12).

  Still, for twenty years after Stanley’s feat, the only viruses that could be crystallized were the very simple plant viruses (those infesting plant cells). Not until 1955 was the first animal virus crystallized. In that year, Carlton E. Schwerdt and Frederick L. Schaffer crystallized the poliomyelitis virus.

  The fact that viruses could be crystallized seemed to many, including Stanley himself, to be proof that they were merely dead protein. Nothing living had ever been crystallized, and life and crystallinity seemed to be mutually contradictory. Life was flexible, changeable, dynamic; a crystal was rigid, fixed, strictly ordered.

  Yet the fact remained that viruses are infective, that they can grow and multiply even after having been crystallized. And growth and reproduction have always been considered the essence of life.

  The turning point came in 1936 when two British biochemists, Frederick Charles Bawden and Norman Wingate Pirie, showed that the tobacco mosaic virus contains ribonucleic acid! Not much, to be sure: the virus is 94 percent protein and only 6 percent RNA; but it is nonetheless definitely a nucleoprotein. Furthermore, all other viruses proved to be nucleoprotein, containing RNA or DNA or both.

  The difference between being nucleoprotein and being merely protein is practically the difference between being alive and dead. Viruses turned out to be composed of the same stuff as genes, and the genes are the very essence of life. The larger viruses give every appearance of being chromosomes on the loose, so to speak. Some contain as many as seventy-five genes, each of which controls the formation of some aspect of its structure—a fiber here, a folding there. By producing mutations in the nucleic acid, one gene or another may be made defective, and through this means, its function and even its location can be determined. The total gene analysis (both structural and functional) of a virus is within reach, though of course this represents but a small step toward a similar total analysis for cellular organisms, with their much more elaborate genic equipment.

  We can picture viruses in the cell as raiders that, pushing aside the supervising genes, take over the chemistry of the cell in their own interests, often causing the death of the cell or of the entire host organism in the process. Sometimes, a virus may even replace a gene, or series of genes, with its own, introducing new characteristics that can be passed along to daughter cells. A virus may also pick up DNA from a bacterial cell it has infected, and carry it to a new cell which it then infects. This phenomenon is called transduction—a name given it by Lederberg, who discovered the phenomenon in 1952.

  If the genes carry the “living” properties of a cell, then viruses are living things. Of course, a lot depends on how one defines life. I, myself, think it fair to consider any nucleoprotein molecule capable of replication to be living. By that definition, viruses are as alive as elephants and human beings.

  No amount of indirect evidence of the existence of viruses is as good as seeing one. Apparently the first man to lay eyes on a virus was a Scottish physician named John Brown Buist. In 1887, he reported that, in the fluid from a vaccination blister, he had managed to make out some tiny dots under the microscope. Presumably they were the cowpox virus, the largest known virus.

  To get a good look—or any look at all—at a typical virus, something better than an ordinary microscope was needed. The something better was finally invented in the late 1930s: the electron microscope, which can reach magnifications as high as 100,000 and resolve objects as small as 0.001 micrometers in diameter.

  The electron microscope has its drawbacks. The object has to be placed in a vacuum, and the inevitable dehydration may change its shape. An object such as a cell must be sliced extremely thin. The image is only two-dimensional; furthermore, the electrons tend to go right through a biological material, so that it does not stand out against the background.

  In 1944, the American astronomer and physicist Robley Cook Williams and the electron microscopist Ralph Walter Graystone Wyckoff jointly worked out an ingenious solution of these last difficulties. It occurred to Williams, as an astronomer, that just as the craters and mountains of the moon are brought into relief by shadows when the sun’s light falls on them obliquely, so viruses might be seen in three dimensions in the electron microscope if they could somehow be made to cast shadows. The solution the experimenters hit upon was to blow vaporized metal obliquely across the virus particles set up on the stage of the microscope. The metal stream left a clear space—a “shadow”—behind each virus particle. The length of the shadow indicated the height of the blocking particle. And the metal, condensing as a thin film, also defined the virus particles sharply against the background.

  The shadow pictures of various viruses then disclosed their shapes (figure 14.3). The cowpox virus was found to be shaped something like a barrel. It turned out to be about 0.25 micrometers thick-about the size of the smallest rickettsia. The tobacco-mosaic virus proved to be a thin rod 0.28 micrometers long by 0.015 micrometers thick. The smallest viruses, such as those of poliomyelitis, yellow fever, and hoof-and-mouth disease, were tiny spheres ranging in diameter from 0.025 down to 0.020 micrometers—considerably smaller than the estimated size of a single human gene. The weight of these viruses is only about 100 times that of an average protein molecule. The brome-grass mosaic virus has a molecular weight of 4.5 million. It is only one-tenth the size of the tobacco-mosaic virus.

  Figure 14.3. Relative sizes of simple substances and proteins and of various particles and bacteria. (An inch and a half
on this scale = 1/10,000 of a millimeter in life.)

  In 1959, the Finnish cytologist Alvar P. Wilska designed an electron microscope using comparatively low-speed electrons. Because they are less penetrating than high-speed electrons, they can define some of the internal detail in the structure of viruses. And in 1961, the French cytologist Gaston DuPouy devised a way of placing bacteria in air-filled capsules and taking electron microscope views of living cells in this way. In the absence of metal-shadowing, however, detail was lacking.

  The ordinary electron microscope is a transmission device because the electrons pass through the thin slice and are recorded on the other side. It is possible to use a low-energy electron beam that scans the object to be viewed, much as an electron beam scans a television tube. The electron beam causes material on the surface to emit electrons of their own. It is these emitted electrons that are studied. In such a scanning electron microscope, a great deal of surface detail can be made out. Such a device was suggested by the British scientist C. W. Oatley in 1948; and by 1958, such electron microscopes were in use.

  THE ROLE OF NUCLEIC ACID

  Virologists have actually begun to take viruses apart and put them together again. For instance, at the University of California, the German-American biochemist Heinz Fraenkel-Conrat, working with Robley Williams, found that gentle chemical treatment broke down the protein of the tobacco-mosaic virus into some 2,200 fragments, consisting of peptide chains made up of 158 amino acids apiece, and individual molecular weights of 18,000. The exact amino-acid constitution of these virus-protein units was completely worked out in 1960. When such units are dissolved, they tend to coalesce once more into the long, hollow rod (in which form they exist in the intact virus). The units are held together by calcium and magnesium atoms.

  In general, virus-protein units make up geometric patterns when they combine. Those of tobacco-mosaic virus, just discussed, form segments of a helix. The sixty subunits of the protein of the poliomyelitis virus are arranged in twelve pentagons. The twenty subunits of the Tipula iridescent virus are arranged in a regular twenty-sided solid, an icosahedron.

  The protein of the virus is hollow. The protein helix of tobacco-mosaic virus, for instance, is made up of 130 turns of the peptide chain, producing a long, straight cavity within. Inside the protein cavity is the nucleic-acid portion of the virus. This may be DNA or RNA, but, in either case, it is made up of about 6,000 nucleotides, although Sol Spiegelman has detected an RNA molecule with as few as 470 nucleotides that is capable of replication.

  Fraenkel-Conrat separated the nucleic acid and protein portions of tobaccomosaic viruses and tried to find out whether each portion alone could infect a cell. It developed that separately they could not, as far as he could tell. But when he mixed the protein and nucleic acid together again, as much as 50 percent of the original infectiousness of the virus sample could eventually be restored!

  What had happened? The separated virus protein and nucleic acid had seemed dead, to all intents and purposes; yet, mixed together again, some at least of the material seemed to come to life. The public press hailed Fraenkel-Conrat’s experiment as the creation of a living organism from nonliving matter. The stories were mistaken, as we shall see in a moment.

  Apparently, some recombination of protein and nucleic acid had taken place. Each, it seemed, had a role to play in infection. What were the respective roles of the protein and the nucleic acid, and which was more important?

  Fraenkel-Conrat performed a neat experiment that answered the question. He mixed the protein part of one strain of the virus with the nucleic-acid portion of another strain. The two parts combined to form an infectious virus with a mixture of properties! In virulence (that is, the degree of its power to infect tobacco plants), it was the same as the strain of virus that had contributed the protein; in the particular disease produced (that is, the nature of the mosaic pattern on the leaf), it was identical with the strain of virus that had supplied the nucleic acid.

  This finding fitted well with what virologists already suspected about the respective functions of the protein and the nucleic acid. It seems that when a virus attacks a cell, its protein shell, or coat, serves to attach itself to the cell and to break open an entrance into the cell. Its nucleic acid then invades the cell and engineers the production of virus particles.

  After Fraenkel-Conrat’s hybrid virus had infected a tobacco leaf, the new generation of virus that it bred in the leaf’s cells turned out to be not a hybrid but just a replica of the strain that had contributed the nucleic acid. It copied that strain in degree of infectiousness as well as in the pattern of disease produced. In other words, the nucleic acid had dictated the construction of the new virus’s protein coat. It had produced the protein of its own strain, not that of the strain with which it had been combined in the hybrid.

  This reinforced the evidence that the nucleic acid is the “live” part of a virus, or, for that matter, of any nucleoprotein. Actually, Fraenkel-Conrat found in further experiments that pure virus nucleic acid alone could produce a little infection in a tobacco leaf—about 0.1 percent as much as the intact virus. Apparently once in a while the nucleic acid somehow managed to breach an entrance into a cell all by itself.

  So putting virus nucleic acid and protein together to form a virus is not creating life from nonlife; the life is already there, in the shape of the nucleic acid. The protein merely serves to protect the nucleic acid against the action of hydrolyzing enzymes (nucleases) in the environment and to help it go about the business of infection and reproduction more efficiently. We might compare the nucleic-acid fraction to a man and the protein fraction to an automobile. The combination makes easy work of traveling from one place to another. The automobile by itself could never make the trip. The man could make it on foot (and occasionally does), but the automobile is a big help.

  The clearest and most detailed information about the mechanism by which viruses infect a cell has come from studies of the viruses called bacteriophages, first discovered by the English bacteriologist Frederick William Twort in 1915 and, independently, by the Canadian bacteriologist Felix Hubert d’ Herelle in 1917. Oddly enough, these viruses are germs that prey on germs—namely, bacteria. D’Herelle gave them the name bacteriophage, from Greek words meaning “bacteria eater.”

  The bacteriophages are beautifully convenient things to study, because they can be cultured with their hosts in a test tube. The process of infection and multiplication goes about as follows:

  A typical bacteriophage (usually called phage by those who work with it) is shaped like a tiny tadpole, with a blunt head and a tail. Under the electron microscope, investigators have been able to see that the phage first lays hold of the surface of a bacterium with its tail. The best guess about how it does this is that the pattern of electric charge on the tip of the tail (determined by charged amino acids) just fits the charge pattern on certain portions of the bacterium’s surface. The configurations of the opposite, and attracting, charges on the tail and on the bacterial surface match so neatly that they come together with something like the click of perfectly meshing gear teeth. Once the virus has attached itself to its victim by the tip of its tail, it cuts a tiny opening in the cell wall, perhaps by means of an enzyme that cleaves the molecules at that point. As far as the electron-microscope pictures show, nothing whatever is happening. The phage, or at least its visible shell, remains attached to the outside of the bacterium. Inside the bacterial cell, there is no visible activity. But, within half an hour, the cell bursts open, and hundreds of full-grown viruses pour out.

  Evidently only the protein shell of the attacking virus stays outside the cell. The nucleic acid within the virus’s shell must pour into the bacterium through the hole in its wall made by the protein. That the invading material is just nucleic acid, without any detectable admixture of protein, was proved by the American bacteriologist Alfred Day Hershey by means of radioactive tracers. He tagged phages with radioactive phosphorus and radioactive sulfur
atoms (by growing them in bacteria that had incorporated these radioisotopes from their nutritive medium). Now phosphorus occurs both in proteins and in nucleic acids, but sulfur will turn up only in proteins, because there is no sulfur in a nucleic acid. Therefore if a phage labeled with both tracers invaded a bacterium and its progeny turned up with radiophosphorus but no radiosulfur, the experiment would indicate that the parent virus’s nucleic acid had entered the cell but its protein had not. The absence of radiosulfur would suggest that all the protein in the virus progeny was supplied by the host bacterium. The experiment, in fact, turned out just this way: the new viruses contained radiophosphorus (contributed by the parent) but no radiosulfur.

  Once more, the dominant role of nucleic acid in the living process was demonstrated. Apparently, only the phage’s nucleic acid went into the bacterium, and there is superintended the construction of new viruses—protein and all—from the material in the cell.

  Indeed, the infectious agent that causes spindle-tuber disease in potatoes was found to be an unusually small virus. In 1967, in fact, the microbiologist T. O. Diener suggested the virus in question was a naked strand of RNA. Such infectious bits of nucleic acid (minus protein) he called viroids, and some half dozen plant diseases have now been attributed to viroid infection.

  The molecular weight of a viroid has been estimated at 130,000, only 1/300 that of a tobacco-mosaic virus. A viroid might consist of only 400 nucleotides in the string, yet that is enough for replication and, apparently, life. The viroids may be the smallest known living things.

  Such viroids may conceivably be involved with certain little-understood degenerative diseases in animals which, if virus-caused, are brought about by slow viruses that take a long time to produce the symptoms. This may be the result of the low infectivity rates of short strings of uncoated nucleic acid.