If biologists succeed, the common cold will be a kinder, gentler disease.
A cold virus enters your body through the twin portals of the nose. Swept along by hairlike cilia in the nasal passage, it's borne on a carpet of mucus to the back of the throat, a journey that takes at most 10 or 12 minutes. Almost always it's the end of the line. The virus either flows into the adenoids, the throat's garbagedisposal unit, or is washed down the esophagus to the gut, where it's obliterated by digestive acids.
But once in a while the virus remains, lodging against the lining of your nasal passage and, through a precise docking mechanism, binding tightly to a cell's receptors. Almost immediately an astonishing violation takes place. The virus enters the cell, disassembles, and unleashes its genes, taking ova the cell's reproductive machinery. The cell stops making new copies of itself and starts making new viruses instead. At this point you're still unaware of the invasion. But in a few days it'll be all too clear. Sore throat, a stuffed and runny nose, headache, perhaps a touch of fever-you've got a cold again.
The common cold is more annoying than debilitating, but it probably causes more acute illness than any other disease. Chances are that one in 20 of you reading this article is sniffling and sneezing right now. Chances are that you've suffered between one and six colds this past year. And chances are that you don't have the foggiest idea of how you caught it or what to do about it, except wait it out.
Until recently, physicians and virologists didn't know much more. The chief difficulty is that the common cold can actually be caused by some 200 different viruses. The culprits go by such names as parainfluenza virus, coronavirus, respiratory syncytial virus, and adenovirus. Yet most colds, perhaps up to half of them, are caused by members of the rhinovirus family (from the Greek word rhino, for "nose"). Exactly 100 rhinoviruses have been identified, each of which can give you a snootful.
Viral infections are notoriously difficult to cure. The best way to combat them is to stop them before they take hold, and for this purpose a vaccine is generally the weapon of choice. But it is unlikely that a vaccine will ever be developed against the common cold: it would have to stimulate the immune system to make antibodies against not just one particular cold virus but all 200. Other weapons are going to be needed to fight this ubiquitous disease, and to forge them molecular biologists will need to understand the fine structure of these viruses.
Rhinoviruses consist of fragile genes packaged in a shell of proteins. Ten years ago our knowledge of what they looked like was largely limited to what could be seen through the electron microscope: rhinoviruses were shaped somewhat like soccer balls, their shells made up of regular subunits arranged according to icosahedral symmetry. But no one could visualize how the tangled proteins in these shells were organized, how they twisted and folded up and interacted with one another. In other words, no one had any idea of what the surface landscape of these viruses looked like. That's what molecular biologist Michael Rossmann of Purdue University set out to discover in 1981. For four years he pieced together the structure of one particular rhinovirus, number 14 in the family of 100. Then he accomplished something that had been considered impossible: he produced an accurate three-dimensional model of the virus.
Imagine the scale involved. All virusff are tiny, and rhinoviruses are especially minute. It would take 10,000 rhinoviruses lined up side by side to span the space between words in this article. To model such an elusive creature, Rossmann took advantage of the virus's propensity to form crystals.
High school students make crystals by saturating water with salt or sugar and then letting the water evaporate. Rossmann did essentially the same thing to produce hundreds of rhinovirus crystals. Each tiny crystal contained on the order of a million million viruses, inert; stable, and frozen in time. The viruses were lined up beside one another, as well as stacked one atop another in geometric array. Because the millions of viruses in each crystal were virtually identical and all oriented in precisely the same direction, information gleaned from the entire crystal could be used to delineate the form of a single virus.
To produce an atomic map of the virus, Rossmann used the Comell High Energy Synchrotron Source, one of three such high-powered x-ray instruments in North America. He and his colleagues bombarded hundreds of crystals with an extremely bright thin pencil of x-rays. When the x-rays encountered the atoms within the virus crystal, they were reflected onto sheets of film, forming a complex pattern of millions of dots that contained a record of the virus's structure.
A computer then deciphered the patterns and translated them into images that the eye could begin to make sense of. These images represented slices of the virus-imagine looking at slices of a cut tomato-each one containing swirling shapes representing cross sections of the viral proteins. To get a three-dimensional view of how these shapes come together, Rossmann converted the computer images into large transparencies. When he stacked these images-much like stacking the slices of the cut tomato to reconstitute its shape and viewed them over a light box, the shapes fitted together to reveal the tiny virus's structure.
Four years ago Rossmann stood at the light box in his laboratory at Purdue and, in what Harvard biologist Don Wiley has called "a tour de force of modern x-ray crystallography," became the first person to view the topography of a rhinovirus. Indeed, he was the first to see the topography of any virus that infects humans. "It was April sixth," recalls Rossmann. "It was terribly exciting, maybe the most exciting day of my scientific life."
Poring over their maps of the virus, Rossmann and his colleagues discovered that its shell was made up of four recurring proteins that formed protrusions and depressions. The virus resembled a miniature planet, replete with mountains that rimmed deep, circular canyons. The mountains are used as landing sites by antibodies produced by the human immune system; it is there that the antibodies bind to the virus in an attempt to prevent infection. The shape of these mountaintops, however, varies from one generation of virus to the next, so that our antibodies can't consistently recognize them. Even if we are able to fight off the virus today, we may well succumb to its offspring tomorrow.
Rossmann realized that although the constantly changing mountaintops were a highly effective way of outmaneuvering our immune system, some part of the virus had to remain constant so that it could, time and again, attach to a particular receptor on the human cells it invaded. Moreover, this viral attachment site, wherever it was, would have to elude the body's protective antibodies. Quickly the thought came to him: What if the attachment site was buried in the canyon? The canyons were certainly deep enough; they are moatlike gashes fully one-twelfth the virus's diameter, a scale far beyond anything on Earth. Even the mile-deep Grand Canyon, for which Rossmann named these depressions ("We even talk about the north wall and the south wall"), doesn't come close to their relative size. "It was the obvious explanation of how to solve this paradox that on the one hand the virus must maintain at least a part of its surface as constant, and yet not allow antibodies to bind to it," Rossmann says. "It simply hides this region in a canyon where antibodies cannot reach it."
What Rossmann knew of antibodies gave support to his theory. Antibodies are relatively fat Y-shaped molecules. While a thin, protruding receptor on a cell might readily slip inside a canyon, antibodies are much too large to fit.
Pretty slick-if you're a virus, that is. You disguise your surface while keeping your attachment site out of harm's way. This bit of viral legerdemain means that any successful preventive treatment must either block the stable attachment sites in the canyon, shield the target cell itself, or interfere with the virus once it gets inside the human cell.
Rossmann and his colleague Tom Smith have recently experimented with the last approach, testing a series of compounds that may be able to prevent colds by paralyzing the virus in the cell. These drugs sink into the canyon and then pass through a "pore" into a cavity below the canyon floor. The researchers suspect that this cavity, like the empty core of a rubber ball, adds to the flexibility of the virus's structure; by filling in the cavity, the drugs stiffen the virus, locking its surface so that it can't disassemble and let loose its genetic material in order to reproduce. Sterling Research Group, manufacturer of the compounds, expects to begin human testing this year.
If it works, the ramifications could go beyond rhinoviruses. "Since many viruses seem to have basically the same structure," Rossmann says, "maybe we can use the same target in other viruses. HIV, the AIDS virus, for example, may have a pocket like this. If we find it, then we can try to design drugs that will fit into it. There are so many properties of viruses that we can explain just by looking at the structure. It's just fantastic."
Elliot Dick of the University of Wisconsin, one of the country's senior cold researchers, can only shake his head in admiration at Rossmann's work. "Now, that's the future of antivirals," he says."That's nice."
While Rossmann has focused on the virus, Richard Colonno of Merck Sharp & Dohme Research Laboratories has been looking at the cells in the nose that the virus targets. Colonno's goal is to block the receptors, thereby denying the virus access to the cells and preventing the disease. It is a logical approach and one that is theoretically promising, but when Colonno began his work in 1982 there were many difficulties. "If you have a hundred different viruses," says Colonno, "how many different cellular receptors do they bind to in a person's nose?" If there were as many varieties of receptors as there were rhinoviruses, it would be impossible to effectively block them. But if the number was manageable, his approach might work.
Colonno began by testing 24 rhinoviruses. He found that 20 of them bound to the identical receptor. This was good news. From there he could project that almost 90 percent of all rhinoviruses bind to the same receptor.
Now he needed to block that receptor. Colonno wanted to target it with custom-tailored antibodies, but first he had to find a way of producing the antibodies in large quantities. He injected laboratory mice with fragments of human cells bearing the receptor. Since the immune system of the mice would recognize the human cells as foreign, it would promptly make antibodies to them.
The mice did their part, producing thousands of kinds of antibodies. Colonno set out to determine by process of elimination which kind was directed against the receptor. Thus ensued what became famous around Merck as Colonno's Brute Force Method. In 8,000 different tests he covered a thin layer of human cells with mouse antibodies, then infected them with rhinovirus and left the whole business in an incubator overnight. If any of the antibodies bound to the cell receptor used by the virus, they would protect the cells, blocking the virus's point of entry. If not, the virus would advance unimpeded, leaving behind a collection of dead cells.
For 11 months Colonno's team repeated the test, checking the cultures each morning and finding nothing but dead cells. In May 1984, he was ready to quit, but he decided to do one last test. "We looked at one another," he recalls, "and said, 'Look, we haven't even seen a hint of protection. If it doesn't work this time, there's something wrong in the whole design.' It came to that. Sometimes, you have to threaten the experiment."
The next morning Colonno opened the incubator to find that of the final test plates, one displayed a batch of healthy cells. He couldn't believe his eyes. "The first thing I thought was, it's a nuke. Somebody didn't add virus to the cells. So we tested the same sample of antibodies again the next day, and it repeated positive. That got everybody excited, and we started jumping up and down. We clearly had something."
Indeed, subsequent experiments showed that the antibody Colonno had isolated was a powerhouse; it didn't just bind to the cell's receptor but actually knocked rhinovirus off the molecule in its rush to grasp hold of it. Colonno also discovered that almost every cell in the body (with the notable exception of red blood cells) contains the identical receptor. If rhinoviruses were hardy enough to survive outside the nasal passage, we might face the fantastic prospect of suffering a cold through every inch of our body.
In November 1985 Colormo enlisted the help of Urİiversity of Virginia cold researchers Jack Gwaltney and Fred Hayden to test the antibody on humans by applying it directly into the nose. Expectations ran high. Here was a chance to do what had never been done before: prevent the common cold. But the experiment was a huge disappointment. The antibody seemed to offer no protection. Almost everyone in the study came down with a cold.
"We outsmarted ourselves," Colorulo says now. "We knew how much antibody it took to protect cells on a plate, but how that related to your nose was completely unknown." He suspected that the dose was much too low and administered in too many separate applications. So in a second test four months later, the researchers gave volunteers eight times as much antibody. Although most of the people again came down with colds, this time the results were more heartening.
"There were two things that were very promising," says Colonno. "The first was that the people treated with the antibodies developed symptoms one to two days later than those in the untreated group. The second was that their symptoms were only half as severe." All of which suggested that the antibody was doing what it had done in the lab, blocking receptors and knocking the virus off cells.
Gwaltney agrees with Colonno. "The effect in volunteers was not as good as in cell culture, but in humans you've got so many variables. There are all the nooks and crannies in the nose-did we get all the spots, did we get good distribution? And there's the dose problem. I think the studies were positive."
Colonno then went on to the next step. The mouse antibody had served to prove that a receptor blockade was possible, and it was time to retire it. Because a mouse antibody was used, a protein foreign to humans, there was a slight possibility that after repeated exposures a person's immune system might attack it-in other words, produce an allergic reaction. Colonno faced the arduous task of finding molecules that mimic the antibody's blocking ability while presenting absolutely no risk to humans.
To do so, he distilled the antibody down to its essential functioning structure. "Using enzymes," he explains, "you can clip off the tail end of the Yshaped antibody-the part that identifies it as mouse rather than human. The two arms will still work as well as the whole antibody. But the odds of having an immune response against these arms is very remote." The initial results look promising.
Colonno and his colleagues are also exploring other intriguing territory: the canyons. They have determined, as Rossmann suspected, that the cell's protruding receptor does indeed penetrate to the bottom of the canyon. And by examining Rossmann's rhinovirus model, they've found a cluster of four amino acids on the canyon floor that seem likely candidates for the structure of the key attachment site.
To test their hunch, Colonno's lab used genetic engineering to tinker with these amino acids, then checked whether or not the altered viruses still bound to the cell's receptor. The mutant viruses bound poorly, suggesting that the four amino acids form the structure Colonno is after. His lab is now looking for a corresponding structure on the cell's protruding receptor- the sites that interact with the attachment points of both the virus and Colonno's mouse antibody. If Colonno is successful, he will have found the secret of how the common cold virus invites itself into our cells-and maybe how to lock it out.
Pioneers like Colonno and Rossmann have, in fact, journeyed to the limits of biotechnology. Their skill in determining the virus's structure, and their ability to describe its interaction with human cells, so far have outpaced their ability to interfere with the infection itself.
"But look how far we've come," exclaims Colonno. "It's a virus that we really knew very little about, if anything, just a few years ago. Now we know the atomic structure-and it's a leading model for other virus research. We know exactly where antibodies bind to it. We've described the molecular interactions of the virus and its receptors on the cell.
"A cure for the common cold isn't realistic because by the time you've got a cold it's too late to stop the infection. But preventing colds is another matter. There's real hope now that this will happen."
Peter Radetsky teaches science writing at the University of California at Santa Cruz and is working on a book about viruses.
While Rossmann and Colonno are investigating the structure of the rhinovirus, others are trying to determine just how it makes its way to our nose in the first place. If they discover that, they may be able to prevent the common cold from ever gaining entry.
Prominent among these transmission detectives are two friendly rivals, back Gwaltney of the University of Virginia and Elliot Dick of the University of Wisconsin. Perhaps predictably-nothing about the common cold seems straightforward- these experienced researchers do not always agree. Dick contends that for the most part rhinoviruses float from nose to nose suspended in moisture droplets carried through the air. Gwaltney says that while-this may well be so, he has shown that they can also spread from hand to hand and, because of our propensity to touch our face, from there to the nasal passage.
Gwaltney first arrived at his hand-to-hand hypothesis in the 1970s when he did experiments showing that cold sufferers who rub or blow their nose can deposit rhinoviruses on the things they touch-on doorknobs, counters, telephones, magazines, books, and so on. The virus can live for hours on these surfaces; when healthy people come along and open a contaminated door or pick up a contaminated phone, they may also pick up cold viruses and unwittingly inoculate themselves by touching their nose or rubbing their eyes. Gwaltney recently showed that dabbing the hands with a virus-killing iodine solution prevents transmission and self-inoculation and reduces the incidence of colds by as much as 40 percent. He is now testing a new disinfectant hand lotion that's less harsh than iodine and doesn't stain the skin brown.
Dick's research doesn't jibe with this at all. In 1986 he brought together 60 avid card players for a 12-hour poker game. Twenty were suffering particularly messy colds. Of the remaining healthy players, half wore either large plastic collars around their neck or rigid braces on their arms, preventing them from touching their face and thus spreading the virus by hand. The other half were not encumbered by restraining devices. But almost as many restrained as unrestrained players came down with colds, suggesting that the virus had spread through the air.
Dick then asked 12 healthy individuals to play another marathon game using decks that had just been handled by heavy cold sufferers in a separate room. The cards were sodden, permeated with nasal secretions, he recalls. "They were a mess-it was the grossest kind of thing." But not one of the 12 developed colds. The study argues against hand-to-hand transmission in favor of an aerosol spread.
Dick has also experimented with viricidal tissues-Killer Kleenexes, as they're sometimes called-which, he claims, prevent the spread of colds by catching and killing viruses before they escape into the air. (Gwaltney, while he sees similar results when the tissues are tested in the lab, says they don't work nearly as well in real-life situations.) Now Dick plans to test air-filtering systems that suck viruscontaminated air away from healthy people and circulate fresh air in its place. "We're going to see if we can stop transmission by simply moving air around a room," says Dick. "If the virus does spread through the air, it's the obvious thing to do."
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