Aids Research Spurs New Interest in Some Ancient Enemies

By Claudia Wallis

The invader is tiny, about one sixteen-thousandth the size of the head of a pin. It consists basically of a double-layered shell or envelope full of proteins, surrounding a bit of ribonucleic acid (RNA), the single-stranded genetic molecule, and often enters the bloodstream of its victim after sexual contact. It is an AIDS virus, and its intrusion does not go unnoticed. Scouts of the body's immune system, large cells called macrophages, sense the presence of the diminutive foreigner and promptly alert the immune system. It begins to mobilize an array of cells that, among other things, produce antibodies to deal with the threat.

Single-mindedly, the AIDS virus ignores many of the blood cells in its path, evades the rapidly advancing defenders and homes in on the master coordinator of the immune system, a helper T cell. On the surface of that cell, it finds a receptor into which one of its envelope proteins fits perfectly, like a key into a lock. Docking with the cell, the virus penetrates the cell membrane and is stripped of its protective shell in the process. Within half an hour, the strand of RNA and an enzyme the virus carries with it are floating in the cytoplasm, the fluid interior of the cell.

Now a remarkable transformation takes place. With the help of the enzyme, the naked AIDS virus converts its RNA into double-stranded deoxyribonucleic acid (DNA), the master molecule of life. The molecule then penetrates the cell nucleus, inserts itself into a chromosome and takes over part of the cellular machinery, directing it to produce more AIDS viruses. Eventually, overcome by its alien product, the cell swells and dies, releasing a flood of new viruses to attack other cells, including more helper T cells and macrophages. The immune system, deprived of a crucial number of those vital T cells, is unable to direct the fight against infection. A host of opportunistic diseases, normally warded off by a healthy immune system, attacks the body. Gradually weakened by the onslaught, the AIDS victim dies, sometimes in months, but almost always within a few years of the first symptoms. By last week this appalling scenario had been played out to its fatal conclusion in some 15,000 Americans. Another 11,500 were under assault, showing the telltale symptoms of the disease, and from 1 million to 2 million others harbored the virus, vulnerable at any time to a final, all-out attack.

Viruses.

The word comes from the Latin for slimy liquid, stench, poison -- and the connotation is appropriate, not only for the AIDS virus but for the untold number of other varieties that have been preying on animals and plants since long before Homo sapiens appeared on earth. Indeed, the current AIDS epidemic is a grim reminder that these infinitesimal, bizarre creatures may be mankind's deadliest enemy. And scientists are warning that a perennial viral threat, the upcoming flu season, could be far more dangerous than usual -- more evidence that these tiny foes are responsible for a large share of human suffering.

In addition to causing AIDS and flu, viruses have brought the scourges of smallpox, yellow fever and polio. They bear responsibility for many of the familiar rashes of youth -- chicken pox, measles, rubella -- as well as such disparate disorders as the common cold, gastroenteritis, herpes, shingles, warts and mononucleosis. Viruses are known to cause at least one form of human cancer and are prime suspects in several other kinds of malignancies. Just last week Dr. Robert Gallo of the National Cancer Institute in Bethesda, Md., announced that he and his team had isolated a new virus that may cause certain kinds of lymphoma and may even play a role in a chronic fatigue illness that seems to strike adults. One prominent virologist, Dr. William Haseltine of Harvard's Dana-Farber Cancer Institute, ventures that "at least 25% of human cancers are caused by viruses." Viruses may even initiate so- called autoimmune diseases by tricking the immune system into attacking its own body tissue.

For all their malevolence and mischief, viruses may have played an important, perhaps crucial, role in evolution. And now, as recombinant DNA technology advances, molecular biologists are engineering viruses that may ! soon benefit rather than devastate humans.

Stripped of their harmful genes, viruses are becoming the stuff of safe and potent vaccines. And within the next few years, scientists hope to gain federal approval to conduct gene therapy. Their goal: the use of viruses as "vectors" to carry healthy genes to the chromosomes of people with genetic diseases, genes that may permanently cure them.

Mankind has long been familiar with the ravages of viruses, if not the creatures themselves. Dried pustules on the mummified face of Ramses V testify to the fact that smallpox killed even the mighty in Egypt 3,000 years ago. In the 16th century, Spanish conquistadors may have unknowingly used viruses in a primitive form of germ warfare; they apparently supplied their intended victims, the Aztecs and the Incas, with blankets taken from houses with smallpox in Europe. Viruses helped cause a fiscal crisis in 17th century Holland, where infections of tulip bulbs produced a new variety of the flower with spectacular, rippling patterns of color. The government was unable to control the resulting speculation, which threatened the economy before tulipomania, as it became known, died down.

Although the agents of all these infections remained a mystery, the first safe vaccine against a viral disease was developed in the 18th century by Edward Jenner, a doctor in rural England. Jenner noticed that farmhands who contracted cowpox, a mild disease related to smallpox, did not develop the more deadly disease. In 1798 he inoculated a boy with material from a milkmaid's cowpox sore, then demonstrated that the lad had developed immunity to smallpox.

It was not until the late 19th century, the "golden age of bacteriology," that scientists began to suspect the existence of some kind of infectious agents even smaller than the bacteria that were clearly visible through their microscopes.

Contaminated liquids that had been passed through porcelain filters designed to purify laboratory solutions and capable of blocking the passage of the smallest known bacteria were still able to infect both plants and test animals. However, careful microscopic scrutiny of the filtered liquids failed to reveal the "filterable agents" that caused the diseases. Also, unlike bacteria, these agents could apparently not be grown in culture dishes, where scientists hoped they might form colonies large enough to be seen with the naked eye. The source of such diseases as mumps, smallpox, yellow fever, rabies and dengue remained a mystery. And yet, wrote frustrated Bacteriologist William Henry Welch in 1894, "these are the most typically contagious diseases, which it might have been supposed would be the first to unlock their secrets."

One clue to the activity of viruses emerged during World War I, when a British and a French scientist independently noticed the appearance of clear circular spots in laboratory cultures grown over with bacteria. When material from a clear spot was applied to a different location in the bacteria culture, another circular area devoid of bacteria soon appeared. Felix d'Herelle, the French bacteriologist, thought he knew why. "What caused my clear spots," he wrote, "was in fact an invisible microbe, a filterable virus, but a virus parasitic on bacteria." D'Herelle named the unseen bug a bacteriophage (from the Greek phage, to devour).

While some of the properties of viruses were becoming evident in the 1920s, no one had yet seen one; on the average, scientists now know, viruses are ten to 100 times as small as the typical bacterium, and in fact far smaller than the wavelength of visible light. That makes them too diminutive to be seen with the most powerful optical microscopes. But in 1931 the invention of the electron microscope -- for which German Physicist Ernst Ruska finally won the Nobel Prize this year -- broke the light barrier. The new instrument -- along with a technique called X-ray crystallography (in which X rays are diffracted through crystallized virus particles to reveal their molecular structure) -- at last provided a view of the bizarre and startling world of the tiny creatures.

Some viruses, like the ones that cause the common cold, look vaguely like soccer balls: round with a surface of bumpy triangular facets. Others, particularly the larger bacteriophages, resemble lunar landing modules. The flu virus looks like the head of a Roman mace, with spikes protruding in all directions; herpes viruses are spherical, as is the AIDS variety. Whatever their shape, all viruses have something in common. They are models of biological minimalism, consisting simply of a core of genetic material -- either a DNA or RNA molecule -- and a protective envelope made of proteins (most varieties have a double coat, the outer one consisting either of another protein shell, or of proteins and lipids, fatty substances similar to those in a cell membrane). "There's no waste in a virus," says Dr. Stephen Straus of the National Institutes of Health (NIH). "Every piece is there for a reason. It's a magnificent little structure."

Unlike any other known creature, the virus occupies a strange netherworld somewhere between living and inanimate objects. While made up of protein and genetic material, it lacks the cell structures common to all life. And unlike true lifeforms, it does not need and cannot metabolize nutrients, does not grow, and cannot replicate without the help of its host. Says David Baltimore, head of the Whitehead Institute in Cambridge, Mass.: "Viruses are the most extreme form of parasite." Anthony Faras, director of the Institute of Human Genetics at the University of Minnesota, emphasizes the virus' utmost dependence on its host. "Put a virus in a test tube," he says, "and it can't do anything. It can't even make copies of itself."

To circumvent its shortcomings the virus must commandeer the protein-making facilities and cellular power plants of its host. It does so by staging a genetic coup, either inserting its genes into the DNA of the host cell -- as the AIDS virus does -- or establishing a floating command center either in the nucleus or the cytoplasm. Once activated, the viral genes order the cell to begin producing more viruses, carbon copies of the original invader (see chart).

In general, viruses are particular in choosing their hosts. Though some, including the rabies and flu viruses, are capable of infecting both animals and man, most favor not just a single species but a limited number of cell types within that species. "If it's AIDS, it commonly goes to the T cells," says Dr. Bernard Fields, chairman of the department of microbiology and molecular genetics at Harvard. "If it's polio, it goes to certain subsets of nerve cells in the spinal cord. If it's hepatitis, it goes to the liver." Until recently the virus' ability to discriminate mystified researchers. How, after all, does a rabies virus, entering the muscle tissue through a dog bite in the leg, know how to find its way along the nerves to particular cells in the brain? The answer lies in the configurations of both the invader and the cell it targets. The rabies virus carries a protein on its envelope shaped in such a way that it meshes precisely with another protein, or receptor, found on the surface of certain brain cells, just as AIDS virus surface proteins fit helper T cell receptors.

Nature did not provide these docking sites for the convenience of viruses; they serve as receptors for hormones and other substances vital to the workings of the cell. It is the peculiar genius of viruses that they have mutated and evolved protein shapes that enable them to use these receptors as ports of call. Once the virus has docked, it is taken in as if it were a valued visitor. "This is a lovely event," says Harvard Biochemist Stephen Harrison admiringly. "The virus gets taken into (the cell), and pow! -- the genes of the virus are dumped into the cytoplasm." In the typical acute infection, like the common cold or the flu, those genes go straight to work, producing proteins that eventually take over and retool the cell machinery to make viruses.

The response to acute infection is also immediate. Like a fire, the incipient infection sets off alarms that alert the immune system to bring out its defensive weapons. It is an awesome arsenal. First, natural killer cells and the Pac-man-like macrophages rush to the scene to gobble up infected cells. After about a week, if this first-tier defense fails to control the threat, says Fields, "you bring out the guided missiles." These are antibodies -- produced by B cells upon the order of helper T cells -- that are custom-designed to home in on certain antigens, distinctively shaped proteins that characterize a particular type of virus, and destroy the enemy or render it harmless.

Although acute infections like influenza kill thousands each year, most people defeat their tiny attackers. Still, they may suffer while the battle is being waged. Indeed, many of the typical symptoms of infection -- fever, chills, itchy rashes, localized swelling -- are due less to the virus than to the vigorous activity of the immune system. However, once the body has created a population of antibody-producing B cells designed to combat a specific virus, immunity to that virus often lasts for decades, or even a lifetime. Then why does the common cold return again and again? One reason, scientists explain, is that colds can be caused by any one of hundreds of strains of bugs, most of them belonging to a group called the rhinovirus. A new cold can be brought on by a strain the immune system has not previously encountered.

Other viruses are responsible for longer-lasting effects. In so-called latent infections, the viral genes lie low, becoming active only intermittently, but throughout a lifetime. Herpes simplex (HSV), for example, makes its presence felt either in the form of genital lesions (usually caused by HSV-2) or as cold sores around the mouth (usually HSV-1), and comes under immediate attack by the immune system, which most of the time wins the battle.

But not the war. For between attacks, the latent herpes viruses hide out in the nerve centers, or ganglia. There they are so quiescent, expressing only five to ten of their 70 genes, that the immune system fails to detect them. Occasionally, for reasons that are poorly understood but that usually involve stress, fatigue, sexual activity and even sunburn, the immune system can no longer keep the hibernating viruses in check; they awaken, reproduce and head for the skin. "As long as the virus remains latent in the ganglia, it remains shielded," says Bernard Roizman, a leading herpes researcher at the University of Chicago. As a result, no permanent cure for herpes exists, and none is in sight.

Latency is a characteristic common to all members of the troublesome herpes family. Herpes zoster, which causes chicken pox, sometimes hides in nerve cells, where no drug or antibody can reach it. Years after the pox attack, usually in middle or old age, zoster can sneak out and cause excruciating attacks of shingles. The Epstein-Barr virus, a herpes family member that causes infectious mononucleosis, follows a similar strategy, though its hiding place is not in the nerves but in the B cells, the very cells that make antibodies to viruses. In contrast to the dormant staying power of herpes viruses, the persistent hepatitis B virus can linger in the liver for decades while continuing to multiply. Those who are infected as infants, as many newborns in China, Southeast Asia and Africa are, almost always become lifelong carriers. "The virus doesn't do much damage for a long time," says Jesse Summers of the Fox Chase Cancer Center in Philadelphia, "but then, after 20 or 30 years, chronic liver problems develop." Papillomaviruses, which cause warts, may also take up permanent residence in the body, biding their time in skin cells.

Some DNA viruses become inactive and escape detection by the host's immune system by insinuating their genetic material into the DNA of the host cell. A retrovirus, however, must first use its enzyme called reverse transcriptase to convert its RNA into a DNA molecule, which can then insert itself into the cell's DNA and order the cellular machinery to begin producing more retroviruses. Or it can remain dormant and invisible to the immune system, / awaiting some signal to begin causing trouble. Hidden in the cell's DNA, says David Baltimore, who shared a Nobel Prize for the discovery of reverse transcriptase, the viruses "have found the perfect niche."

Many of these chronic viruses are now being linked to cancer. A landmark study conducted in Taiwan between 1975 and 1978 by Dr. R. Palmer Beasley, now at the University of California, San Francisco, found a striking connection between chronic hepatitis B infection and liver cancer, a leading killer in the Third World. "Someone infected with hepatitis B has 100 times the normal risk of developing liver cancer," says Beasley, "and that's being conservative." The Epstein-Barr virus has been associated with a couple of types of cancer. In Central Africa and New Guinea, it has been linked to Burkitt's lymphoma, an immune-cell cancer that primarily strikes children. In southern China, the virus plays a role in nasopharyngeal carcinoma, a malignancy of the nose and throat that afflicts more than 50,000 people a year. Retroviruses are known to cause cancer in a wide range of animals, from mice to chickens. In the early 1980s, Dr. Robert Gallo and his co-workers, as well as a group of Japanese researchers, showed for the first time that a retrovirus is responsible for cancer in humans. The culprit is now called human T-cell lymphotropic virus, type 1 (HTLV-1), which causes a rare and extremely aggressive adult leukemia that occurs mainly in the southern islands of Japan and parts of Central Africa and the Caribbean.

Recent research also shows a link between papillomavirus and cervical cancer, which annually strikes 15,000 women in the U.S. The human papillomavirus (HPV) family is a large one, including 46 types capable of causing everything from common plantar warts (HPV-1 and 4) to choking growths in the throat (HPV-11) to a bizarre warty rash found almost exclusively on the hands of butchers and meat handlers (HPV-7). So far, six types of HPV, all responsible for benign genital lesions, have also been associated with malignant cervical growths. Says Dr. Harald zur Hausen of the German Cancer Research Center in Heidelberg: "At least 80% of all cervical cancers are linked to papilloma."

But infection with these viruses does not always lead to malignant growth. In fact, that happens infrequently. Explains M.I.T. Biologist Nancy Hopkins: "Cancer arises from a number of insults to the DNA. Viruses are one insult. They start the process rolling." Years usually elapse between infection and the development of a related cancer. When liver cancer strikes a hepatitis carrier, for example, it generally does so 30 to 50 years after the victim was first infected. These long delays, Zur Hausen observes, "suggest the need for other events besides infection to occur in order to progress to cancer."

Several such events or "co-factors" have been suggested in the papillomavirus-cervical cancer connection. They include smoking (which appears to increase risk of this cancer fourfold), poor hygiene and concurrent infection with the herpes simplex virus type 2. Says Dr. Carlos Lopez of the Centers for Disease Control: "Maybe one virus is the instigator and the other is the promoter."

A growing amount of evidence suggests that whenever viral infection leads to cancer or chronic disease, some sort of breakdown or weakness of the immune system plays a contributing role. For instance, organ-transplant patients whose immune systems have been suppressed by antirejection drugs have a greatly increased risk of developing virus-related malignancies. "There is a very intimate relationship between viruses and immunity," says Dr. Thomas Merigan of Stanford's school of medicine. "If our immunity is a little deficient for one reason or another, then we are more likely to have progressive disease."

This may be true of AIDS. One of the great mysteries surrounding the disease is why only some of those infected get sick while others have carried the virus in their cells for several years and have so far remained healthy. Dr. Jay Levy, an AIDS researcher at the University of California, San Francisco, cites the possible role of other infections, use of drugs, poor nutrition, stress and lack of sleep, any of which may weaken the immune system. "If the person's immune system is not compromised by such events," he says, "I believe they will be able to fight off the virus and not develop the disease."

Other researchers have their doubts. They point out that although the immune systems of most AIDS victims make antibodies to the virus, the antibodies do not seem to halt the progression of the disease. There are several apparent explanations:

1) The virus can avoid detection by hiding inside a cell's DNA. It also can spread from cell to cell without ever entering the bloodstream; it does so, in part, by causing cells to fuse together. Thus, says Microbiologist Ashley Haase of the University of Minnesota, "even if you have antibodies to the ; virus circulating in the blood, they won't be able to destroy the infection."

2) The AIDS virus is present in very small quantities in the blood, even in the case of a full-blown infection, and thus makes a difficult target. (The small quantities may also help explain why AIDS is not especially contagious and spreads only by intimate contact.)

3) The AIDS virus reproduces and mutates at a much faster rate than most other viruses, frequently changing the structure of its surface antigens, the protein markers on its envelope. By the time the immune system has produced an antibody that recognizes and goes after a particular antigen, the antigen may have changed beyond recognition.

4) As its "ultimate weapon," notes Immunologist Elaine DeFreitas of Philadelphia's Wistar Institute, the virus thrives in helper T cells and macrophages, "the very cells that are sent to destroy it."

Viruses have even been implicated in such autoimmune diseases as insulin- dependent diabetes, rheumatoid arthritis and multiple sclerosis. In these disorders, the immune system appears to be confused and attacks body tissue as well as foreign invaders. How might a virus provoke the immune system to attack its own body? Immunovirologist Robert Fujinami of the University of California, San Diego, may have discovered one way.

Fujinami has been investigating an autoimmune disease called acute postinfectious encephalomyelitis, which occurs as a complication in about one out of 1,000 children who get measles. Like MS, it is a degenerative disease of the myelin sheath, the insulating layer that surrounds nerve fibers in the central nervous system and helps speed the passage of the nerve signals. Strangely enough, the disease strikes some ten to 14 days after the measles virus has completely disappeared. Fujinami has found that portions of the protein component of the measles virus, in what is called "molecular mimicry," closely resemble myelin's basic protein. Thus, he suggests, "if one makes an immune response against the virus, then conceivably the same response could attack the central nervous system."

Some scientists believe that still undiscovered "slow viruses" are responsible for neurological disorders like kuru and Creutzfeldt-Jakob disease -- which may develop decades after the victim is infected -- and perhaps for Alzheimer's disease too. But because such viruses have never been isolated and because of the nature of these diseases, some researchers suspect that a mysterious, entirely different infectious agent is involved.

Viruses are not all bad news. Despite the woes that they have brought, they probably have contributed more to humanity than just exotic varieties of Dutch tulips. Some scientists think the ubiquitous creatures may have an important and perhaps even beneficial impact on evolution. They suggest that by rearranging the DNA in chromosomes, and by transferring genes from one species to another, viruses can impart characteristics to a plant or animal that help it to not only survive but dominate and edge out competitors.

And medical researchers hope soon to have a powerful ally in their campaign against viruses: vaccines made from genetically engineered viruses. At the NIH, Dr. Bernard Moss is using recombinant DNA techniques to convert vaccinia, a large virus that causes cowpox, into a one-shot, multidisease vaccine. He plans to insert only the antigen-coding genes of eight to ten kinds of dangerous viruses into the DNA of live but weakened vaccinia viruses. The re- engineered vaccinia would then sport the antigens of the harmful viruses, but not their ability to cause disease. Once inoculated, it would stimulate the immune system into producing cells that could later act against infection by any of the harmful viruses.

In an even more dramatic development, a dozen teams of scientists are working on techniques to use viruses for "gene therapy" on humans with genetic disorders. Using recombinant DNA techniques, they plan to make retroviruses harmless by removing key genes, and to endow the viruses with other genes -- the ones lacking or inoperative in people with genetic diseases. These re-engineered retroviruses would be employed as vectors that would invade the appropriate human cells and insert the healthy genes correctly into the cells' DNA.

The first candidates for this therapy would be people with life-threatening hereditary disorders that are caused by a single, known defective gene. Among the illnesses being considered for gene therapy: beta-thalassemia, a severe form of anemia, and three rare disorders caused in each case by a defect in a gene that orders the production of a single, vital protein.

Although experimental work on mammals is proceeding slowly, Richard Mulligan, who has been practicing gene therapy on mice at the Whitehead Institute, is optimistic. "We are pretty damn close," he says. "We have retrovirus vectors that transfer efficiently. It looks like we can infect the ( appropriate types of cells reasonably safely." But, he concedes, he has not yet been able to induce the genes to "turn on" and order the cells to produce the missing proteins.

The solution, Mulligan hopes, is to attach the protein's gene to a section of DNA that acts as an on-switch, or promoter, before implanting it in the retrovirus. In his experiments to date, he says, the strategy is "working fantastically," and he expects encouraging results within two or three months.

Still, despite the viruses' apparent potential for good, their much greater capacity for evil has been amply demonstrated. Smallpox. Yellow fever. Rabies. Polio. And now the cruel AIDS epidemic. Concludes David Baltimore: "You could get rid of all the viruses from the world and the world would not be the worse for it."

With reporting by Christine Gorman/Park City, J. Madeleine Nash/Atlanta and Dick Thompson/Washington