Chapter One The Accidents of Research CHILDHOOD ACCIDENT North of Poitiers, France, National Highway 10 emerges from the sharp bluffs of the Clain Valley on its way toward a plateau bordered by small hills. This is the ancient boundary between northern and southern France, not far from the point at which the advancing Saracen invaders were stopped, at the gateway to Queen Eleanor's beloved Aquitaine. A bit farther on, before Châtellerault, the red Roman roof tiles give way to Angevin slate, and the singsong accent of the Poitou region is eclipsed by the purity of Touraine speech. Once past these hills, the highway doglegs down into the valley of what is no longer but a feeble stream, the Auxance. There, well tucked away, lies the village of Grand-Pont. The front-wheel-drive vehicle is rolling along at a high speed, the driver feeling almost surprised at the ease with which the car hugs the road, handling the sharpest curves with no difficulty at all. It is a clear, hot day in midsummer. All of a sudden, behind a bend, a little boy darts out into the road. The driver slams on the brakes. The child collides violently with the hood, rebounds into the air, and lands some ten yards away, at the base of a stone wall at the edge of the road. A truck that flipped over in that same spot a few days earlier left behind some broken bottles. The shards of glass make mincemeat of the little creature, who by now is no longer moving. The blood is pouring from the wounds in his face, head, arms, and legs. Two young girls accompanying the little boy, his cousins, are crying, wondering how they let him elude their grasp. The mother, in a panic, comes running. A passing motorist stops. Together they wrap the little body in a blanket and take him to a clinic in Poitiers. His heart is still beating. After two days in a coma, wavering between life and death, the boy revives. He sustained a slight fracture of the skull, with a bit of blood entering the fluid in the brain. The other wounds are superficial. Their only lasting effects will be permanent scars, in particular a nasty star-shaped hole in the left cheek, which the doctors say will later turn into a dimple and be attractive to women. But there were other scars as well. I can still remember perfectly waking up in the hospital room. Everything was white: the curtains, the walls, the bed frame, the dressings around my limbs and on my head. At last I saw my mother, who with tears in her eyes was watching for the slightest sign of consciousness. I emerged from it all without the slightest recollection of what had happened to me. Why was I there? I wondered. My mother told me about the car and the accident. My memory was a blank: no image or recollection of pain remained in my mind. I remembered only the moments preceding the impact and the conversation I had had with my cousins. Then nothing. But this gap aroused my curiosity. I was five years old. I recovered very quickly. The wounds healed. My dozens of stitches were removed. I was photographed from every angle, in anticipation of the trial, which was held at the court of summary jurisdiction in Poitiers. The driver was charged with reckless driving and speeding. The victim too, however, had been reckless: I had crossed without looking. Moreover, in the courtroom I walked with a determined step, and a murmur of satisfaction rose up from the audience: the child would not suffer any aftereffects. The driver was acquitted. This was in 1938. Two years later there was war and exodus. Bombs fell along the road as we fled the advancing German army. In June 1944, still more bombs fell on Châtellerault, one of which destroyed part of our house; the German soldiers, in disorderly retreat, commandeered my parents' bicycles, though these were quite old. I also remember the unheated classrooms at school, where teachers muffled in overcoats taught us the rudiments of Latin and Greek. I began reading everything I could get my hands on: Balzac and Voltaire, science fiction, the novels of Jules Verne, as well as the municipal library's impressive collection of large, red-and-gold-bound books by a Russian author whose name I forget, in which I passionately followed the heroes in their adventures through the solar system and beyond. In the illustrated magazines whose publication was controlled by the Germans, I followed the stories of the first mail-carrying rockets created by Werner von Braun. Rockets, the sole means of escaping the Earth ... I also read the books my father left lying about. A certified public accountant in town, he used to tinker with electrical sodium cells on weekends, when the weather prevented him from going fishing; in the evenings he used to read and reread popularized scientific books ranging in subject from physics theories to organic chemistry. I too derived benefit from them, accumulating a self-taught body of knowledge that served to feed my nascent scientific curiosity. News of the explosion of the atomic bomb, on August 6, 1945, filled me with terror. It also revealed to me that the physicists of atomic structure and radioactivity had been right. I read with great passion, from its very first issue, a review entitled Atomes, tous les sujets scientifiques d'un nouvel âge (Atoms: All the Scientific Subjects of a New Age) and all the popular scientific studies of the period. I was about fifteen years old at the time. Was I going to become a physicist? An astrophysicist? A theoretician of matter? Or a chemist? After our house was bombed, the town government of Châtellerault put us up in a rather modern, empty house that had been occupied by the local Gestapo. In the cellar, which no doubt had been used for some sinister purpose, I set up a little chemistry laboratory, where I enthusiastically produced hydrogen gas, sweet-smelling aldehydes, and nitro compounds that had the unfortunate habit of blowing up in my face. My parents did not look kindly on these childish games. They would have rather seen me study literature, then take up law, to follow in my father's footsteps. At secondary school, I excelled more in literature and the humanities than in math and science. To have a career as a physicist, I would have to get through the university science colleges. I wasn't ready to hit the books and cram for the baccalauréat exams required for entry into college. And so I settled for the next best thing: biology. That is, "natural sciences," as they called it at the time, and medicine. I should also add that meanwhile a cancer in the digestive tract brought my grandfather a slow, cruel death. The discovery of this mysterious, inexorable illness no doubt figured significantly in my choice of study. POITIERS AND THE SORBONNE: INITIATION INTO RESEARCH After passing my exams before the age of seventeen, I enrolled in the Departments of Medicine--in a preparatory program (PCB, or Physics, Chemistry, and Biology)--and Science at the University of Poitiers. My parents were delighted. They already imagined me as a doctor, well established in the fine town of Châtellerault, living a stone's throw away from their house and ready to come to their rescue in their last days. Unfortunately, I had something entirely different in mind. I wanted only to research and explore the mysteries of nature, life, and our origins. The Science Department at Poitiers was near the medical school. My plan was to pursue, at the same time, a program of medical studies and a bachelor's degree in "natural sciences." The PCB would allow me access to both. I passed the class at the top of the list--only to learn the next day that a ministerial decree had just changed the rules of the game. Now one needed another diploma to continue science studies! Worst of all, one needed to know the rudiments of geology, of which I was utterly ignorant. Not to worry. With the complicity of a geology professor who was also dean of the science faculty, I spent my vacation that year force-feeding myself in geology, learning to recognize the minerals in the department laboratory. In October I obtained the diploma without difficulty. Both paths were now open to me, and thus I began shuttling between the hospital in the morning and my science courses in the afternoon, returning later in the evening to attend the anatomy lectures of a certain professor Jean Foucault, father of Michel. The Poitiers faculty offered a very limited number of specializations. In fact, they taught botany, zoology, and geology, mainly for the purpose of training secondary school instructors instead of researchers. Fortunately, in Pierre Gavaudan, who held the chair in botany, I encountered a passionate master of research. With his small team of assistants, he explored still little known avenues of plant physiology and cellular pharmacology. I myself had begun some modest research in the same areas. With a microscope hooked up to a movie camera, I was able to film infusoria, rotifera, and other creatures inhabiting the local ponds. One phenomenon in particular captured my attention. One day, while observing a fibrous freshwater alga called mesocarpus, I noticed that all its chlorophyll, which was condensed in the form of a rectangular patch called a chromatophore, turned about an axis in the cellular cylinder depending on the intensity of the light. When the light was strong, the chromatophore would show only its edge. Once the light weakened, however, the alga displayed its full surface. This was one of my first moments of wonder at the numerous regulatory mechanisms that make up the life of cells. This phenomenon had been described more than a half century earlier, but its mechanism remained obscure. With the help of differently colored filters, I showed that it was blue light, not the red light absorbed by the chlorophyll, that triggered this rotation: other pigments were therefore involved. The speeded-up film revealed that the rotation of the chlorophyll patch depended on the movements of the cytoplasm. This was my first research work. Needless to say, I was very proud of it. The experiment earned me a degree, in 1953, in natural science. But Poitiers had its limitations. The medical school, at the time, was in fact only a premedical school. The program of studies was only for two years. One had to follow up by going to Tours or Paris. It seemed to me that in the capital the courses in the biological sciences must be more varied, since they were taught by the very cream of French scientists. The physical proximity between the Sorbonne, where the science courses were held, and the medical school would allow me, I thought, to pursue my medical studies and science degree at the same time. I thus decided to go "up" to Paris. The sheer volume of medical students was so great that, to have access to patients, one had to pass two competitive exams: first the nonresidency test, then the residency test. I thus began attending the preparatory lectures for nonresidency, grumbling and stumbling over the "questions" I had to learn by heart. I had an excellent memory, yet my allergy to competitions quickly reappeared--especially since the defense of my little thesis at Poitiers entailed significant bibliographical research in a different subject: the L forms of bacteria. There was no way I could simultaneously carry on this exciting exploration--which among other things brought me for the first time to the library of the Pasteur Institute, the Mecca of microbiology--and frequent the nonresidency lectures, which were becoming more and more demanding. In my poorly heated room on rue Tournefort, the papers on algae and L forms were starting to win out over the mimeographs of anatomy and pathology. What had to happen, happened. I abandoned the nonresidency lectures and the hallowed path of medicine in order to conclude quickly my double thesis, which I went to Poitiers to defend. I then began attending courses in general physiology at the Sorbonne, all the while continuing my training at the hospital in the morning, cutting the medical courses in the afternoon, and then catching up by devouring the mimeographs later on. I learned a great deal in my hospital training, even though it involved very little direct contact with patients. The Sorbonne, on the other hand, profoundly disappointed me. The courses were usually poorly conceived. Even though biology at the time was being totally revolutionized in the United States and England, the university mandarins pretended to know nothing about this. (The Sorbonne at the time clung to outdated notions and to the cult of the great national prophets of the early century.) Indeed, French research had suffered greatly from the four years of isolation brought by the Second World War. Living science is a community activity. Without exchange, it dies. I had a special passion for the physiology of the nervous system. I read and reread the French translations of English writings on the subject. Nevertheless, one must live. The following year, I stumbled into a position as tutorials monitor for this same certificate of general physiology. When in Paris to deliver a lecture at a conference, Pierre Gavaudan introduced me to one of these famous professors, who was in fact looking for an assistant. Thus at age twenty-three, I became an Assistant in Cellular Biology at a laboratory of the Curie Institute, an institute specializing in cancer research and treatment, in Paris. A prestigious center for cancer research, the physics section had hosted the two generations of Curies themselves, and the Radiobiology Department had close ties with the Pasteur Institute. My own department was also connected with the Pasteur Institute as well as with the University, but due to a disagreement among the supervisors, we had no ties with other laboratories, despite the passageways between the buildings. My orientation changed radically at this time. I turned from botany to animal cells and the foot-and-mouth disease virus. In our small laboratory, I familiarized myself with methods dating from before the war, especially the culture of chicken embryo fragments, the favorites of Aléxis Carrel. Then I turned to the suspended culture of mouse spleen lymphocytes. A miserable failure! With good reason: we still did not have adequate growth factors--interleukins--at our disposal, since these weren't discovered for another twenty years. As for foot-and-mouth disease, I was assigned to filming the foot-and-mouth vaccination experiments conducted first in the slaughterhouses of Ivry, then in a fort on the isle of Aix. Though my talents as a filmmaker contributed greatly to my involvement, these experiments too were failures. Meanwhile, the first rumblings of molecular biology were beginning to reach our laboratory. I had passionate discussions with my assistant and colleague, Jean Leclerc. We were also attending the seminars of the molecular biology club organized by Jacques Monod and François Jacob at the nearby Institute of Physico-Chemical Biology. Every evening I would go to Pierre Langlois's cinémathèque; I only had to cross the street. Nineteen fifty-seven was a decisive year for me, determining my vocation as a virologist. Viruses were then known to have two main components: a nucleic acid (RNA or DNA) and proteins. Two teams--one American, headed by H. Fraenkel-Conrat, and one German, that of A. Gierer and G. Schramm--showed independently that for the tobacco mosaic virus the ribonucleic acid alone was sufficient to allow reproduction of the virus in infected leaves. The proteins had no role, other than protecting the viral RNA against destructive plant enzymes. After discovery of "transforming" deoxyribonucleic acid (DNA) by Oswald Avery and elucidation of the double-helix structure of DNA by James Watson and Francis Crick, this was the first demonstration that ribonucleic acid (RNA) also carried important genetic information. The fundamental dogma of molecular biology was beginning to take shape: we knew by then that all genetic information necessary to the synthesis of proteins was contained in the nucleic acids--DNA for cells, RNA for certain viruses. Shortly thereafter, the RNA for a human virus, poliomyelitis, proved equally infectious to culture cells. (The beginning sections of Chapter 3 will describe in greater detail what viruses are and how they work.) Together with Jean Leclerc, I feverishly endeavored to extract RNA from the virus we had ready at hand, the foot-and-mouth virus, or more precisely, from the tissues infected with this virus. And this is exactly what led me to develop an original technique for increasing the penetration of RNA into the cells. It was my first and last research success of the 1950s. My rapport with my supervisor was deteriorating. Viruses seemed to me the simplest of genetic elements; their secrets should therefore be easier to discover and perhaps be of some help in understanding the more complex systems--cells--whose obligatory parasites they were. The laboratory did not offer the modern techniques I needed in order to move ahead. I had to leave. All I had to do, in fact, was knock on the neighboring door, that of Philippe Vigier, who at the time was (already!) working on a retrovirus, the Rous sarcoma virus, which produces cancer in chickens. He introduced me to the director of the Biology Department of the Curie Institute, Raymond Latarjet, who greeted me warmly. How to guarantee my "rescue"? The possibilities were limited. My future at the university was blocked. I had completed my studies in Science and Medicine, but I had no thesis in Medicine. I was counting on making my research on the RNA of the foot-and-mouth virus the subject of my medical thesis. One professor of Medicine, asked to preside over my jury, was a friend of my former supervisor. When learning of my inclination to leave, the supervisor became infuriated and asked the professor of Medicine not to preside over my thesis jury. The professor respected his friend's wishes. Such were the mandarin-like customs of French higher learning in those days. And such they still are today, at times. Fortunately, there were some exceptions. Latarjet found a new president for my jury in the person of a rather nonconformist professor of medicine named Raoul Kourilsky, who understood the problem at once and gave me his approval. But the months were passing, meanwhile. It was already the end of the 1959 academic year. I would have to wait until the November session to defend my thesis. It became clear to me that I had to leave France at once, learn modern virological techniques abroad, and at the same time find a stable situation before leaving. I applied for a post at the CNRS (National Center for Scientific Research) and was accepted; I was awarded a scholarship in an exchange program between the CNRS and the British Medical Research Council, and sent to work in the laboratory of Kingsley Sanders, near London. These were favorable circumstances for me. The CNRS, an organization founded as a kind of counterweight to the university mandarinate, was enjoying renewed favor in the eyes of the incoming government. General de Gaulle's prime minister, Michel Debré, spurred by his father, Robert Debré, and the few molecular biologists in France who mattered, had understood that they had to play the scientific development card in a decisive manner, creating research positions and new institutes and sending young people abroad to study. This was the time of the creation of the General Delegation of Scientific Research, precursor to France's Ministry of Research. Thus I was able to spend more than three years in a British laboratory, thanks to a succession of scholarships. I was, in any case, an exception: most of the scholarships were given for stays at U.S. laboratories. In that early summer of 1960, the horizon seemed to brighten a little. There was only one little problem, a small detail: I didn't speak a word of English. I could read, of course, or rather, I could decipher the English I read in scientific reviews. But that was a far cry from speaking! A BRITISH PERIOD My sojourn was to begin with English lessons at a summer school in Bournemouth. One fine morning in July 1960, I boarded the Dieppe--New Haven ferry with my little car. Once on board, I was already in another world. The coaches of the Paris--Dieppe train were unloading ruddy-faced British passengers on their way back from the beaches of the Côte d'Azur, who rushed forward in disciplined batallions to seize the wooden deck chairs, which they unfolded on the ship's deck, to enjoy a little more of a sun so rare on the other side of the Channel. Once on land, I discovered an exotic country ruled by wind and rain, by tea so strong you had to drown it in milk, and by naturally amiable and helpful natives. A few weeks later, armed with a few rudiments of the language of Shakespeare and a lady friend who was soon to become my wife, I introduced myself to my future supervisor at his Carshalton Laboratory, in the south London suburbs. I had prepared a few English phrases in advance. What a surprise when I heard him answer me in perfect French! Sanders was not your typical Englishman. A Gitanes smoker and black coffee drinker, he also composed operas in his spare time. Along the way he headed a laboratory that at the time was one of the best in virology. It was specifically studying the multiplication of a little virus containing RNA: the mouse encephalomyocarditis virus, which was very harmful to the animal (killing it in forty-eight hours) but of no danger whatsoever to humans. It was no accident that I ended up at this lab. It had already welcomed numerous French researchers among its number, and the Sanders family regularly spent their vacations in France. A perfect, cordial understanding reigned. The atmosphere at the lab was quite relaxed. Kingsley, who lived north of London, took an hour and a half to get to the lab each day. He would arrive around 10:00 A.M. in his little VW bug. At eleven the whole lab would rush to the cafeteria to gulp down the traditional late-morning tea with milk. They were there again at 1:00 P.M., this time for an unvarying lunch, which usually consisted of some meat accompanied by overboiled potatoes followed by a pudding moistened with some custard. Then, at 4 P.M., another tea with milk. At five, laboratory life ceased. Despite this very relaxed rhythm, the research progressed. And there were passionate discussions, too, with Sanders and his assistant, Alberto Visozo, an anti-Franco Spaniard who by now knew all the secrets of English slang and was happy to teach them to me. While working with Kingsley I became fascinated with the question, how does the RNA molecule replicate itself? It took me three years--three years of hard work, Saturdays and Sundays included--to show that, during the reproduction of a virus inside a cell, the RNA also takes the form of a double helix very similar to, though much more rigid than, the famous DNA double helix discovered by Watson and Crick. Paradoxically, this scientific success, which boosted my confidence in my abilities as a researcher, resulted in the dissolution of the laboratory where I was working. Indeed, Sanders was offered and accepted an important post at the Memorial Sloan-Kettering Cancer Center in New York. The researchers of our team scattered, some to the United States, others to various parts of the United Kingdom. As for me, I had my eyes on California. In particular, I wanted to work in the laboratory of Renato Dulbecco, who was studying viruses as the source of cancers in animals. These were rich times for biological research. New institutes were sprouting up like mushrooms. And British virology, unlike the French, boasted numerous important teams, such as that of Michael Stoker and Ian MacPherson in Glasgow, where Renato in the end decided to spend a year on sabbatical. So much for California! My wife and I were off to Glasgow in our little blue car. Glasgow, at the time, had all the characteristics of a nineteenth-century industrial city in decline: mine shafts in the middle of town, and abandoned houses where screeching birds took refuge after dark. Saturday nights, beer and whiskey flowed in abundance in the pubs. The Institute of Virology at the University of Glasgow, was a haven of modernity and warmth for us foreign trainees--especially as our rooms were teeth-chatteringly cold, poorly heated as they were by gas stoves in which one had to deposit a shilling every half hour to keep them running! My greatest problem, however, was an administrative one. The CNRS would only let me stay abroad for three years, and I had already entered my fourth year. Latarjet assigned me to a fictional laboratory in Paris. Officially I was in Paris, while in fact I was a kind of stowaway in Glasgow. But this was the moment that the science committee of the CNRS chose to award me a bronze medal for work on double-helix RNA. Just my luck! The medal was sent to the Curie Institute and then returned to sender with the message "no longer at the address indicated." Confusion. Madame Plin, chief administrator of the CNRS and much feared by the researchers, was in a tizzy. Latarjet mustered up some embarrassed explanations: "Montagnier? Oh yes, he's training in Scotland, you know, way up there, north of England? But he'll be back ... " But what was Montagnier doing in Glasgow? He wasn't wasting his time ... While I was still at Carshalton, Sanders had obtained the BHK line of hamster cells from Michael Stoker and Ian MacPherson, transformed (made cancerous) by the mouse polyoma virus. He wanted to adapt these cells to grow in suspension inside the hamster's (abdominal cavity), so as to produce great quantities without culture. Kingsley had also learned from another virologist, Peter Wildy, that BHK cells could live at the bottom of a tube of agar. He showed me whole colonies produced by these cells in petri dishes containing a nutritive medium gelled by agar. He thought it was due to some unique property of the cells he had adapted to the hamster peritoneum. During the final months of my stay at Carshalton, I also tried my hand at this technique. I proudly showed Kingsley that a variant of BHK cells which became spontaneously cancerous could also grow in agar. Upon my arrival in Glasgow, I spoke to my hosts about this new technique. Why not use it to detect cells that have been newly transformed by the polyoma virus? It would be a precise way to measure this transformation which was equivalent to the capacity of forming tumors in animals. By mistake, I lowered the concentration of agar to the very limit, to form a gel. Then I left for Paris, as Latarjet had informed me of the medal business. A week later, on my return to Glasgow, I looked at the petri dishes. Wonder of wonders! The dishes with hamster cells that had been infected by the polyoma virus displayed magnificent colonies of cancer cells growing three-dimensionally in the agar. I announced the news to MacPherson, who calmly told me that he too, using my technique, had obtained the same results. We decided to put both our names on two publications, one in French and the other in English. It is the latter that is always cited. For the first time we had a precise test, in vitro (that is, growing in a test tube), showing the carcinogenic power of a virus. The immediate application was to demonstrate that DNA extracted from the polyoma virus, in all its molecular forms, was equally capable of transforming cells into a cancerous state. This firmly proved that all the information required to cause cancer was found in the DNA. The variant of hamster cells I had isolated at Carshalton, which also grew in agar, was the exception that proved the rule: that was a case of spontaneous transformation, since those cells were capable of forming tumors in the hamster. A GROWING INTEREST IN CARCINOGENIC VIRUSES OF BIRDS But it was time to go back to Paris. It was spring of 1964 and I was anxious to continue working on the molecular biology of viruses, to attack a retrovirus, the Rous sarcoma virus, with Philippe Vigier, and then to apply the famous agar technique to the detection of carcinogenic viruses in humans. If such viruses existed, they should, in my opinion, be able to bring about the cancerous transformation of human cells, such as embryonic skin cells, and this could be detected by the formation of colonies in agar. This was skipping some steps, it is true, since normal cells go through several stages in their evolution toward a cancerous state, and the growth in agar corresponds to a late stage. Normal human tissues, unlike those of rodents, do not easily generate cells that spontaneously skip a primary stage. Nevertheless, I had all the enthusiasm of a neophyte, and with the teams of Philippe and André Boué, I showed that the Rous retrovirus could also transform human cells and make them grow in a very pure agar without any of the negatively charged polymers normally present in ordinary agar. Quarters were tight: at the Pasteur Pavilion of the Curie Institute, I had only one room and a corridor transformed into an office. For virus detection in human tumors, I was given a little room on the sixth floor of the hospital, across the street from the institute. The experiment was a failure. The human tumor cells, not adapted to in vitro culture, refused to grow. On the other hand, by 1965 things were progressing rapidly with the carcinogenic animal viruses. As for the polyoma virus, I showed, together with Robert Cramer and Raymond Latarjet, that by using ultraviolet and gamma radiation the carcinogenic and infectious abilities of the virus could be separated from each other. The carcinogenic feature was more resistant to radiation. This meant that there existed a cancer "gene" that was a smaller target than the sum of all the genes needed for the replication of the virus. On the subject of retroviruses, we were getting some stiff competition from American teams. Peter Duesberg and William Robinson at Berkeley were the first to isolate the RNA intact from the Rous sarcoma virus; together with Jacques Harel and Joseph Huppert, we obtained the same results a few weeks later. There still remained the mystery of the replication of this large-sized RNA, whose structure was actually made up of subunits, a fact we were to demonstrate in 1969, at almost the same time as Peter Duesberg. Howard Temin firmly maintained, though without convincing proof, that there was an intermediary DNA (DNA existing during an intermediate stage) in the RNA replication process. I was inclined to believe that this RNA replicated itself like other viral RNAs, by forming a double helix of RNA. In the case of the Rous virus, however, the RNA was not infectious. Sophisticated molecular biology techniques were thus necessary to separate these double helices, notably by using the resistance to the enzyme ribonuclease, which can only digest RNA in single strands. I isolated just such a specimen of RNA from chicken cells transformed by the Rous virus, but I found the same thing in the control cells not infected by the virus. These molecules were therefore not specific to the virus; they reflected a purely cellular process. I thus spent several years with Louise Harel (Jacques Harel's wife) analyzing the nature of these double helices, wondering whether they might not reflect the self-replication of some of the cell's messenger RNA, the RNA that is synthesized from DNA and then translated into proteins. More likely they were the result of simultaneous transcription of two strands of the DNA double helix of the chromosomes or of the mitochondria. (Mitochondria are organelles within each cell that serve as its "energy battery.") In any case, the double helices had nothing to do with the retrovirus. That left the hypothesis of an intermediary DNA. We could find no DNA in the viral particles, only RNA. There must therefore have been a specific enzyme capable of making a DNA copy of the viral RNA in the infected cells. An army of researchers and technicians working for the National Institutes of Health (NIH) in the United States were preparing liters of reagents and milligrams of chicken, mouse, and cat retroviruses. This was one of the offshoots of the U.S. effort, launched at President Richard Nixon's initiative, to prove the viral origin of cancer. Underlying this vast program was the simple idea that in humans, leukemias, lymphomas, and sarcomas (three different kinds of cancer) must, like their equivalent forms in animals, be caused by retroviruses. To find these, it was therefore necessary to start with known animal retroviruses. This resulted in the vast production of retroviruses, which in June 1970 enabled David Baltimore, at the same time as Howard Temin, to quickly isolate reverse transcriptase, the enzyme that transcribes RNA into DNA. For my part, however, still beclouded by my theories of self-replicating RNA, I gave little importance to looking for that enzyme, and thus was left behind by this discovery, which shook up the world of molecular biology. The discovery of this enzyme, which is present in viral particles, was not, however, the final word on the research into the replication mechanisms of retroviruses. Its activity had been observed in vitro, but it remained to be proved whether it would function correctly in infected cells and synthesize a true copy of DNA capable of being integrated into the DNA of the cell's chromosomes and reproducing the virus. A Czech émigré couple, Hill and Hillova, who had fled the repression following the Prague Spring and had been welcomed by Joseph Huppert in his laboratory at Villejuif, were the first to prove that infected cells contained an infectious DNA capable of reproducing the virus. Their success was clearly the result of patience and tenacity, since the foci (clusters) of transformed cells showing the presence of the virus only appeared after three months of culture! Nevertheless, few people lent credence to their findings. The discovery of reverse transcriptase would, however, have decisive effects on different areas of biology. Most notably, it would make it possible to synthesize DNA from the messenger RNA of any cellular gene and thus open the way to cloning (making exact copies of) genes. Research into retroviruses implicated in human cancers was also to advance by leaps and bounds, since the enzymatic activity of reverse transcriptase made it possible to detect infinitesimal quantities of virus. Saul Spiegelmann, at Columbia University, and a newcomer to the retrovirus field, Robert Gallo, got into the act in the early 1970s. As for me, I tried to extract the DNA from human tumors and make it enter normal cells, and await the transformation of these cells in agar. In vain, once again! Around this same time I also proposed, at the request of the CNRS, a project for an institute that would bring together the clinic and some research laboratories at Orsay, counterpart to the Curie Institute in Paris. It was rejected. By now I was itching to move on. Stagnation seemed to be settling in. But where to go? It did not take long to tour all the top institutes in France. Two events then determined the course my future would take. The first was the rise to preeminence of Jacques Monod, who became director of the Pasteur Institute in 1971. The second was meeting the "Pasteurians," among them André Lwoff and François Jacob on the occasion of the First International Conference on Cellular Differentiation in June 1971. Together with two young colleagues of mine, Patricia Allin and Dmitri Viza, I organized this conference, held at the Hôtel Negresco in Nice, to bring together all the major scientists in the world working in the field of cellular differentiation, the maturation of cells into physiologically and functionally distinct units. We were untroubled by doubt. Indeed, it was clear that, after the molecular mechanisms of regulation in bacteria had been brought to light, the next great question would be whether the same mechanisms also applied to the cells of higher organisms, from sponges to humans. "What is true for the bacterium is also true for the elephant," Monod used to say, but in fact this glib quip aroused skepticism. Our goal, then, was to bring together scientists who were working on relatively simple systems of differentiation in which the methods of molecular biology could be applied. And one of the subjects discussed was, of course, cancer. A few months later I met with Monod at the Pasteur Institute. We had chosen November 11, a holiday, for the sake of greater discretion. The head of the Virology Department, Pierre Lépine, was retiring. Ellie Wollman and Monod wanted to reorganize and renew the department in its entirety. There was a building, built after the War, that could be renovated thanks to some private funding. I was a little reluctant to leave my friends at the Curie Institute, to whom I was greatly indebted, and to abandon the field of oncology (cancer research), which was not the Pasteur Institute's specialty. But I was, after all, most interested in cancer viruses, so why hesitate? Furthermore, Monod seemed to me a rather enthusiastic director. I accepted his offer. And thus in 1972, the Viral Oncology Unit was created on the first floor of the virus building. The name made it clear what it was about. However, while still at the Curie Institute, I had begun, with two Belgian colleagues, Edward and Jacqueline De Maeyer, to broach another subject: antiviral defenses. STUDYING INTERFERON The oncologist's arsenal of chemicals that inhibit the multiplication of viruses is quite limited. Indeed viruses, which are intracellular parasites, actually use to their own advantage the cell's mechanisms for transmitting its genetic messages. It is therefore difficult to find inhibitors capable of impeding viral synthesis without affecting cellular synthesis. Fortunately, all vertebrates have "invented" an early natural defense which goes into action well before the immune system can respond: interferon. Actually, there are many interferons. They are little proteins that today are classified in the family of cytokines, regulatory molecules that cells exchange among themselves. When a cell is infected by a virus, it produces interferon before dying. In still healthy neighboring cells, this protein generates a signal that calls into action a whole battery of enzymes to curb viral multiplication and, to a lesser extent, cellular metabolism. The latter result is the source of interferon's antitumoral effect, which was so well demonstrated by Ion Gresser at Villejuif. There were at this time a number of excellent laboratories in France working on interferon. The intention was, of course, to use it as a therapy in treating viral illnesses and cancers. Unfortunately, it was very difficult to manufacture. The only attempt to do so, in the 1970s, was made by Finnish scientist Kari Cantell, who for this purpose used white blood cells, by-products of blood donations of the Finnish Red Cross. Given the difficulties, many laboratories thought to clone the interferon gene in bacteria, in order to produce interferon at less cost. We were among those following this course. By 1972, together with Edward and Jacqueline De Maeyer, I had isolated interferon's messenger RNA using a biological method. We were just entering the age of genetic engineering. The Pasteur Institute had a relatively prominent place in this field. It took me no time at all to interest Monod in the potential of these techniques for producing antiviral vaccines. An intense discussion followed, at the Pasteur Institute, reflecting the debate that had previously taken place at the international level. Scientists were beginning to ask themselves if it wasn't perhaps dangerous to release bacteria carrying human genes into the world. The newspapers began talking about the "mad scientists of the Pasteur Institute." In 1974, at the conference held in Asilomar, California, scientists decided to have a moratorium. In actual fact, it was very short-lived, and in the United States remained nothing more than a facade. But we had overestimated the danger, and those colleagues of mine who were most vehemently against my projects were the first to launch into the field in their own right. At the Pasteur Institute, a high-security "P 3 " laboratory (now called BL 3 (for Biosafety Level [sub.3]) was created to prevent manipulated bacteria from escaping. It was called "the submarine" because it was as difficult to enter as a submarine turret. Four genetic engineering units were set up there. I joined forces with Pierre Tiollais, one of the pioneers in this domain. We needed money, however, a lot of money, to analyze thousands of bacterial clones in order to find the one carrying the interferon message amid the multitude of other messages in the cell. It really was like looking for a needle in a haystack. A pharmaceutical company, Laboratoires Roussel offered us a contract that would bring in the necessary money. Management, however, said, "Nyet!" Monod had just passed away, and his successors did not possess the same powers of persuasion. The veto came from very high up, from the government, which was afraid that the Pasteur Institute's technological innovations might fall into the hands of the German firm Hoechst, which had just taken over Roussel. They were unaware that the institute did not have a monopoly on this technology, and that it would soon to be used by thousands of laboratories all over the world. In fact it was the Zurich team of Charles Weissmann, one of my former competitors in the field of viral RNA replication, that came up with the first good clone of human interferon. And that team included some British and a Japanese, and worked in conjunction with a Swiss-American biotechnical company called Biogen. So much for French nationalistic narrow-mindedness! Today the three major types of interferon are cloned, and two of them are industrially produced through genetic engineering. Applications of interferon are not as wondrous as one might have thought, but they do exist, in AIDS treatment as well. Interferon blocks a late stage in the replication of retroviruses-the retrovirus' emergence from the infected cell. When interferon is present, the viral particles are not well formed and cannot detach themselves from the cell wall. Interferon ... retrovirus: the connection between the two elements could not be ignored. THE SEARCH FOR CARCINOGENIC RETROVIRUSES IN HUMANS The search for retroviruses involved in human cancers was running out of steam. How many times had major journals like Nature or Science proclaimed "a great discovery" only to see it later fizzle out like a bad firecracker when the isolated retrovirus proved to be a laboratory contaminant, usually a mouse virus! Not the least egregious of these false hopes was the one announced by Robert Gallo in 1977. A human leukemia virus, HTLV, which Gallo believed he had discovered, turned out to be a mix of monkey retroviruses! Indeed, by the late 1970s most labs became discouraged by this sort of research and reoriented themselves to studying oncogenes, genes controlling cellular reproduction and which, when mutated or expressed in untimely fashion, are the source of many cancers. At first there was the discovery of the sarc gene, capable of making chicken cells cancerous. It was identified in the Rous sarcoma retrovirus, thanks to painstaking work in genetics and molecular biology on the part of Peter Duesberg, Peter Vogt, Dominique Staehlin, and Michael Bishop on both sides of the San Francisco Bay. Sarc is, in fact, similar to a cellular gene present in the chicken genome, which is also represented in every kind of vertebrate, including humans. It is as though the Rous virus had in some way adopted this cellular gene among its own viral genes, while modifying it slightly. This was the start of a fantastic rush among the laboratories to be the first to isolate the oncogenes of the other carcinogenic viruses, about twenty of which had been identified, and later on, other oncogenes not carried by retroviruses. My lab did not enter the race. We preferred to hunt for human retroviruses. In fact, a subtle reorientation had taken place, without my even having noticed at first. My interest in nucleic acids, carriers of genetic information, had been waning in favor of the results of this information's expression: proteins. To make an oft-used analogy, nucleic acids are the recording tape or the score for a piece of music, while proteins are the music itself. On with the music! My research into the mechanism of cancerous transformation had led me, perhaps erroneously, onto a path other than molecular genetics, that of cell membranes and membrane proteins. In the early 1970s, we still knew nothing about the structure of a biological membrane. All we knew was that it was made up of proteins and lipids. But how these lipids were organized among themselves and linked to proteins, that remained a mystery. The answer was found in 1973 by American scientists S. J. Singer and Garth Nicolson. The mosaic-like structure they proposed explained what was already known, and was quickly embraced by everyone: the membrane is made up of a double layer formed by lipids. This double layer, which constitutes a rather fluid medium, receives the transmembrane proteins, which pass through it from one side to the other. Other proteins, embedded farther out in the membrane and carrying sugar chains, steep in the bath of lipids. The membrane's fluidity explains how the proteins, especially those acting as receptors, can join together. Such aggregations, induced when the molecules attach themselves to these receptors, themselves result in a signal being transmitted within the cell. The presence of cholesterol diminishes the fluidity of the membrane and can thus alter these responses. The discovery of the membrane structure seemed to me as important as Watson and Crick's discovery of the structure of DNA. Yet it went relatively unnoticed, most molecular biologists being concerned only with the central memory, DNA. Living beings, however, cannot live without membranes. Electron microscopy enabled us to "see" transmembrane proteins, with the two sheets of the lipid membrane laid open like a book. There was a surprise in store for us. The proteins joined together into particles were three to four times more numerous in cancer cells than in normal ones. Buckling down to isolating them and studying their biochemistry was no small task. The search for human retroviruses well illustrates how dry our work can be. But sometimes when crossing the desert, you arrive at an unexpected oasis. I admit that I stand apart from Robert Gallo on many matters. Nevertheless, we shared one important thing, unbeknownst to either one of us, during the late 1970s: the desperate, despairing search for retroviruses linked to human cancers, in particular, leukemias. Gallo was not a medical doctor, but rather a biochemist by training. He did not join the "retrovirology club" until after the discovery of reverse transcriptase. His limited experience with viruses at that time perhaps explains his misinterpretations and the contaminations that occurred in his laboratory. But his will and the driving sense of urgency he imparted to his collaborators paid off in the end, and led to the isolation of the human T-cell leukemia virus (HTLV), initially from ill-defined tumors. Later, thanks in part to Japanese contributions (in particular by Isao Miyochi and Yorio Hinuma) as well as his own efforts, the causal role of this virus in a rare form of leukemia occurring in southern Japan was established. My approach was a different one. Being well familiar with animal retroviruses (Among other things, I had to cover them in a virology course I taught at the Pasteur Institute), I started by using the animal strains most like human cancers: acute leukemias, sarcomas, and mammary (breast) tumors. The arrival of a new team at my lab, led by Jean-Claude Chermann and specializing in mammalian retroviruses--especially mouse retroviruses, which are quite numerous--enabled us to go further in this direction. Chermann, as well as his assistant, Françoise Sinoussi, and a technician, came from the annex of the Pasteur Institute in Garches (a suburb of Paris), where Louis Pasteur had developed his serums and vaccines and finally passed away. But the search for retroviruses in human cancers still obsessed me. We had isolated retroviruses that induced cancers and leukemias in all mammals, including primates. Why should man, another primate, be an exception? I knew that interferon was a powerful inhibitor of retroviruses and that the interferon system was particularly effective in humans. That might well be the reason we could not isolate any human retroviruses in man: they were perhaps totally inhibited by the interferon produced by the infected cells. Gresser at Villejuff, and Yves Rivière and Ara Hovanessian in my own lab, had shown that one could increase a viral infection and make it fatal to an animal by injecting this animal with an anti-interferon serum, that is, a serum containing antibodies that neutralized the interferon's protective effect. Sometimes, in a chronic infection, the situation was the reverse. Interferon would become harmful, and one would save the lives of the mice by injecting them with this same anti-interferon serum. At Villejuif, Gresser had two sheep that produced this serum when repeatedly injected with human interferon. I resolved to try once again to find human retroviruses, this time with the help of this serum. I made the cell cultures, and Françoise Sinoussi looked for reverse transcriptase. The human leukemias we used came from the Cochin Hospital in Paris, from Jean-Paul Lévy's department. Gresser had generously given me a few milliliters of the precious anti-interferon serum produced by his sheep. For their part, Chermann and Sinoussi tried to test my hypothesis in the system of mouse retroviruses. They demonstrated that a mouse anti-interferon serum--also supplied by Gresser--increased the production of retrovirus by the mouse cells by a factor of 10 to 50. We were very enthusiastic about this. A manuscript was sent to Nature and was rejected on the basis that the mouse interferon used to produce the antiserum was not pure. This seemed unfair, since at that time nobody could produce enough mouse interferon to make a completely pure antiserum! As for human retroviruses, the experiments began in 1977. I wrote them down in a red notebook, the same one in which I later described the isolation of the AIDS virus. One experiment followed another, yielding nothing. At times we did observe some enzymatic activity; unfortunately, it corresponded not to retroviruses but mycoplasmas. (See note I for a brief description of mycoplasmas, which will be discussed in greater depth in Chapter 7.) Finally, in 1979 came the big news of the discovery of HTLV-I by Robert Gallo's team. Gallo himself gave a detailed lecture on the virus at Villejuif; HTLV-I seemed specific to humans, but it had been isolated from a rare cancer, mycosis fungoides. There were many skeptics, given Gallo's past blunders. All the same, what held my attention was his announcement of the isolation, by Doris Morgan and Frank Ruscetti in his laboratory, of a new growth factor they called TCGF (T-cell growth factor), which made it possible to culture normal human T lymphocytes over a long period of time. These lymphocytes might be useful in growing human retroviruses, which would be drawn to them. The HTLV-I virus replicated itself easily in these T lymphocytes in the presence of the growth factor, but it transformed them, so that they gradually had less and less need for this factor to become independent and "immortal"--indeed, this corresponded closely with the initial phase of leukemia in humans. I shared with Robert Gallo the results I had obtained using the anti-interferon serum on mice. A collaboration thus began. Françoise Sinoussi (now Barré-Sinoussi) went into Gallo's laboratory with the mission of transposing the results of the mouse experiment onto the system of a monkey (to get as close as possible to the human model). The point was to see whether the serum would increase the production of a gibbon retrovirus that was chronically infecting some human cells. The results were positive, but the effect was less impressive than with the mouse. Nevertheless, this result prompted me to look for other human retroviruses in cultures of human T lymphocytes with additions of Gallo's TCGF and Gresser's anti-interferon serum. I received from Gallo a bottle containing the culture medium of activated cells including this factor, in an impure state. This enabled me in early 1980 to conduct a number of experiments. But by April 1982 the reagent was used up, and I had to turn to other sources for AIDS viral cultures, which were made in 1983 by Didier Fradellizi at the St. Louis Hospital in Paris. There was a chance that a retrovirus might be involved not only in leukemia, but also in breast cancer. A good viral model of this cancer existed in mice, which have retroviruses that cause mammary tumors transmitted either by heredity or in the milk. As it happens, in the 1970s a number of researchers, including Saul Spiegelmann, believed they had found similar viral particles in women's milk, especially in that of Parsee women. The Parsees are a very closed Indian sect of Persian origin, where strict endogamy is the rule; that is, they can only marry other Parsees. Now, nearly one-quarter of all Parsee women, even young women, have breast cancer. In North Africa there is a kind of breast cancer, called inflammatory, which also strikes young women and has a swift evolution. Thanks to progress in molecular biology, it became possible to reopen this file with much better chances of success. No doubt we would have continued down this path if, in early 1982, the search for yet another retrovirus had not become our chief concern. In any case, we had the necessary technology and training to move on from cancer retroviruses to AIDS retroviruses. But fourteen years later, the breast cancer of Parsee women remains a mystery, and even from that distance, my interest in it has not waned. It was the early 1980s--1983 to be exact. HIV was already growing in my laboratory. I had to abandon all my other research projects since they could hardly move forward while we were refocusing our efforts on the new retrovirus. Our findings on the retrovirus associated with a breast tumor were nevertheless published in high-level cellular biology journals. They went completely unnoticed. The reader, moreover, would be mistaken to think that all laboratory research necessarily leads to publications of varying degrees of importance and fame. In fact, 90 percent of all experiments lead to nothing whatsoever; most of the time some unforeseen technical snag arises, or the initial idea proves faulty. The day-to-day life of researchers consists mostly of disappointments, with the occasional success that allows them to maintain their enthusiasm. One must have the mentality of a gambler or fisherman. As for me, I am only interested in big fish. But they are rather rare. And so my drawers are full of lab notebooks and beginnings of manuscripts that will never be published, unless of course I send them to The Journal of Irreproducible Results . Copyright © 1994 Editions Odile Jacob.
