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Alice S. Huang and David Baltimore are Professors at the California Institute of Technology, Pasadena, where David is the President.
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Coming of Age of Animal Virology
Virology's history began a century ago; viruses, including bacteriophage, have been the source for much of today's molecular biology
Alice S. Huang and David Baltimore
As participants in the last 40 years of research on animal virology, we have watched the field go from just growing and identifying animal viruses to a highly sophisticated branch of science. In fact, viruses, including bacteriophage, are the source for much of today's molecular biology. This personal and, of necessity, somewhat limited record reflects on how far animal virology has come and where it still needs to go.
Virology's history begins a century ago, with the discovery of plant viruses in 1898 and of animal viruses soon thereafter. Within a decade, the tumor-causing ability of viruses was established. There ensued a period of discovery of new viral agents and linkages of agents with diseases in animals and humans.
Visionaries Recognized Profound Implications of Viral Size
The definition of a virus remained operational even following World War II. Simply, a virus was any infectious agent that passed through a filter that caught bacteria. Nonetheless, as early as 1927, visionaries such as Hermann Muller recognized that the small size of viruses had profound implications. There was simply not enough room inside a virus for much more than its genetic material. Of course, no one then knew what genetic material was.
Most virology until the 1950s focused on laboratory animals such as mice, chickens, and ferrets. A pathfinding exception was the work of John Enders and his colleagues at Harvard Medical School who, in 1949, adapted poliovirus to grow in cell culture. This achievement not only revolutionized the production of vaccines, but it set the path for the biochemical analysis of this and other viruses.
Harry Eagle, then at the National Institutes of Health (NIH), took cell culture a step further by finding a relatively simple medium in which to grow HeLa cells. Then these cells could be used as hosts for poliovirus, allowing James Darnell, Bill Joklik, and Leon Levintow to begin to analyze the virus life cycle.
Animal Virology Seemed Primitive in the 1960s
When we entered the field of animal virology in the early 1960s, much about this field was still primitive. Laboratories studying viruses were extremely clean but mainly empty because most of the work went on in small rooms that were kept sterile by ultraviolet lights when unoccupied. Much of the laboratory work was repetitive, involving the maintenance of host cells needed for propagating the viruses. There were endless subdivisions of cells in monolayer cultures into daughter cultures, or the preparation of primary cells from minced tissues such as 10-day chick embryos or the hindlimb muscle of mice.
Considered lucky at that time were those investigators who used ascitic tumor cells as hosts for growing viruses. All one of them had to do was inoculate the tumor cells into the abdominal cavity of mice and then watch those mice grow fat with the ascitic fluid packed solid with cells. In England, such cells were used as hosts for mouse picornaviruses, enabling a very active group of researchers to study viral physiology. These efforts soon led to discovery of double-stranded RNA molecules.
Following World War II, bacteriophage immediately became an object of active research, with the celebrated Phage Group, led by Salvador Luria and Max Delbrück, particularly productive during the 1950s. Surprisingly, however, this research on bacteriophage did not setthe issues for animal virology research of the early 1960s. Indeed, reworking in animal viruses the experimental paradigms of phage did not appear a worthwhile endeavor.
Instead, the central role of RNA in such viruses proved key to what made them so interesting. Many animal viruses have RNA as their genetic material, whereas in 1960 no RNA bacteriophages were known. Although such phage were found within a few years, by that time the animal virus field had momentum of its own. Moreover, the RNA phage proved not to be a good model for much of what is interesting about RNA animal viruses. Both of us subsequently devoted considerable efforts to understanding RNA animal viruses.
Early Quantitative Methods Proved Valuable
During the mid-1950s, Marguerite Vogt and Renato Dulbecco, working in a basement laboratory at the California Institute of Technology, developed the basic plaque assays for measuring viruses quantitatively. Although details varied from one system to another, the basic approach involved counting plaques that formed within a monolayer of cells that was covered with agar-containing medium.
Not all viruses cause distinct plaques of killed cells. Some, such as the herpes simplex virus, cause cells to fuse, forming syncytia. Other animal virus-infected cells form giant cells, whereas some RNA tumor viruses cause infected cells to form foci of rapidly growing, randomly oriented piles of tumor cells on a background of relatively uniform monolayer cells.
Each of these characteristics led to a distinctive type of focus assay. These assays were much easier to use and provided more reliable quantitative measurements of viruses. Soon, historic assays, including one that entailed inoculating the chorioallantoic membranes of embryonated chicken eggs, were abandoned.
Other, even simpler approaches for tracking animal viruses were coming into use. For instance, Julius Youngner at the University of Pittsburgh devised a method that relied on diluting virus inocula and infecting cells in microwell plates. Virus growth was detected by examining the color of the phenol red pH indicator in the medium bathing the cells: orange meant healthy growing cells, whereas pinkish purple meant virus-infected, nonmetabolizing cells. Although this endpoint assay was not as precise as a plaque assay, it was very easy to use and gave a rapid readout, and thus was soon widely used by researchers working in the vaccine field.
Broad-Based but Naive Searches for Antiviral Agents
During this same period in virology, many researchers shared the naive belief that with good, quantitative assays, it should be easy to find effective chemical inhibitors of viruses. Pharmaceutical companies set up huge screening programs, but almost no antiviral compounds for human use were ever found.
With such good assays, this failure was surprising. However, the chemical libraries then under scrutiny derived mainly from natural microbial sources and thus probably were inappropriate starting materials. In other words, this earlier failure to find microbial inhibitors of animal viruses seems to reflect the underlying fact that animal viruses do not ordinarily compete with fungi and bacteria in natural settings. Later, investigators began to find useful antiviral compounds only when the molecular biology of the viruses was understood well enough to provide more appropriate leads.
Virology Research Builds Momentum
Animal virology was a wonderful field for young scientists to enter during the 1960s. Although it was not yet a mainstream of molecular biology, career opportunities were ample, and there was plenty of money to support research. From the 1930s until a polio vaccine became available in the mid-1950s, virology was largely supported by the National Foundation for Infantile Paralysis, also known as the March of Dimes.
It is hard to remember that before World War II most scientific research in the United States was supported by charitable organizations like the Rockefeller and Carnegie foundations. In the 1960s, as the March of Dimes shifted its focus from polio to research on inherited developmental defects, funding from the federal government ramped up, with NIH generously supporting the growth of research in virology. Indeed, during this period, NIH extramural officers often vied to support the best stable of investigators, taking personal pride in the successes of their charges and making sure that bureaucracy did not get in the way of good science.
Research in animal virology was propelled by the advent of easy-to-care-for suspension cell cultures and the introduction of specific radioisotopic precursors for tagging macromolecules, thus permitting investigators to focus on newly synthesized proteins and nucleic acids in virus-infected cells. Also, new centrifugation procedures were developed for purifying viruses readily from cellular debris. The first electron micrographs of viruses were made in the 1940s, but by the 1960s the electron microscope had become a powerful tool of the field.
For many years, the Academic Press Journal Virology published most of the seminal reports in this field. However, in 1966 ASM established the Journal of Virology under the leadership of Robert R. Wagner, then at the University of Virginia, Charlottesville.
During this early period, researchers in virology got together in two series of meetings. The Pocono Virology meetings in the winter were known fondly as the GinsWash meeting to honor their main organizer, Harry Ginsberg. This meeting was the direct precursor in the mid-1960s of the summer Gordon Conference on Animal Viruses and Cells, which yearly alternated between focusing on one or the other of its dual themes. The second series of meetings, held in New York City and supported by the Hartz Mountain company, came to be known as the Bird Seed Meeting because the company was a supplier of feed for pet birds. The sponsors created the first prize in the field, The Gustav Stern Award in Virology, named for the company's founder.
Early on, Two Main Scientific Focuses in Animal Virology
The early successes in animal virology came in two general areas: understanding how viruses program cells to become factories for viral reproduction and the early glimmerings of understanding how viruses commandeer the growth control pathways of cells to cause tumors. These developments during the 1960s, brought animal virology from a research backwater to a prominent subdiscipline of molecular biology.
The tumor virologists and the cytocidal virologists (those interested in non-tumor viruses) tended to be quite separate in their interests, which limited the communication between these two groups. Although the annual Gordon conference brought them together, even then members of the two groups typically had little to say to one another.
The tumor virologists mostly focused on DNA tumor viruses, and their interest was largely molecular genetics. Because those virologists who focused on RNA tumor viruses were mainly focused on disentangling the fundamental from the phenomenological, few others had the patience to concentrate on their arcana. Meanwhile, the cytocidal virologists were mostly concerned with RNA viruses, although a few hardy souls began to take on the relatively huge herpes and vaccinia DNA viruses
The history of tumor virology traces to the early part of the century, when pioneers in this field isolated many viruses that caused cancer, most of which turned out to be RNA-containing retroviruses. Not until the late 1950s did Sarah Stewart and Bernice Eddy identify polyo- mavirus, a remarkable agent that rapidly causes many different tumors in mice. Noting that this virus contains only a little DNA in its genome, researchers recognized that the key to how this virus causes cancer lies in several thousand nucleotides.
Such findings led James Watson in 1959 to say that the secret to cancer should soon be unraveled because there it lay, isolated for study in polyomavirus. That secret turned out to be much more challenging to understand than that early genetics-based calculation suggested.
Molecular Biology of Viruses Brought Surprises
Based on previous work, we knew that poliovirus encodes all its proteins in one large piece of RNA. Nonetheless, it came as a surprise to learn that this RNA molecule is translated into one long polyprotein that subsequently is cleaved into smaller functional proteins.
With that background, we were amazed to find that such matters are handled differently by vesicular stomatitis virus (VSV). Its long genomic RNA gives rise to a series of mRNA molecules, each of which encodes a fraction of the information of the genome. Even more surprising, the VSV mRNAs are complementary to the larger genomic RNA contained in the virus particle, thus making the genomic RNA antisense or negative-stranded.
That finding raised the perplexing question of how the VSV mRNAs are synthesized, a prerequisite for the infection cycle. Since animal cells were not known to contain RNA-dependent RNA polymerases (and we had searched for one), one possibility was that this virus carries its own polymerase among its structural proteins when it enters a cell.
A casual discussion David had with our MIT colleague Salvador Luria about the possibility of such a polymerase within VSV yielded a compelling case for doing experiments to look for the enzyme. By early 1970, the VSV virion- associated RNA-dependent RNA polymerase was identified based on unambiguous radioisotopic incorporation into RNA complementary to the genomic RNA. These were simple and rapid experiments; from discovery to publication was about 3 months.
This discovery was rapidly extended to other enveloped viruses like Newcastle disease virus. Others had earlier discovered virion-associated polymerases in vaccinia virus and reovirus, leading to the generalization that viruses with unusual transcriptional strategies carried their own enzymes into cells as part of the incoming virus particle so that they could synthesize their own nucleic acids.
Reverse Transcriptase Discovery Soon Follows
This generalization was part of the background leading to discovery of viral reverse transcriptase later in 1970. Howard Temin, a prominent and brilliant tumor virologist, developed the idea that, in infected cells, a DNA molecule might carry the genetic information of such viruses, implying a transfer of that information from the viral RNA to DNA. Despite inhibitor experiments supporting this idea, it did not gain wide acceptance.
We thought that RNA tumor viruses might also contain the enzymes needed for the genetic information transfer that Temin's work suggested. With help from George Todaro, then at NIH, we obtained sufficient amounts of a murine virus to do enzyme assays indicating significant catalytic activity. Moreover, this viral polymerase resisted the antibiotic actinomycin D, suggesting that it was RNA-directed rather than DNA-directed.
The next step in linking this putative RNA-dependent DNA polymerase activity to the virions involved banding the virions in a sucrose gradient and testing for activity in each fraction collected from the gradient. This crucial experiment was left half completed for much of a week when MIT students and faculty voted to strike in protest of the U.S. invasion of Cambodia. Eventually the frozen gradient fractions were assayed and it was clear that the virus and polymerase activity peaks coincided.
Before submitting the results for publication, we learned that Howard Temin and his postdoctoral fellow Satoshi Mizutani also had found polymerase activity associated with a chicken RNA tumor virus. The editors at Nature agreed to accept the two manuscripts for back-to-back publication and dubbed the new enzyme reverse transcriptase-viruses carrying it became known as retroviruses.
Reverse Transcriptase Discovery Leads in Many Directions
The organizers of the annual symposium held at Cold Spring Harbor invited representatives from the two laboratories to describe our reverse transcriptase findings in early June. Sol Spiegelman from Columbia University listened to the presentations, immediately drove back to his lab in Manhattan, and next morning announced that he had confirmed the results. Although other participants at that meeting appreciated the implications of reverse transcriptase for RNA tumor viruses, there was then no evidence that retroviruses cause tumors in humans.
Thus, the potential importance of this enzyme in human cancer or, more generally, for furthering our understanding of the molecular biology of cancer was far from the minds of the scientists present at that symposium. Moreover, during this earlier phase of molecular biology research, no one thought to apply for a patent covering commercial rights for use of reverse transcriptase. Thus what became a key tool of researchers doing basic molecular genetics and applied biotechnology remained unencumbered by commercial restrictions or licensing agreements or royalty payments (much to the dismay of the involved universities).
Many more advances came from the study of animal viruses: oncogenes, splicing, transcription factors, signal transduction proteins, and a plethora of variations on the basic patterns of molecular trafficking in cells. When techniques became available to dissect the big DNA viruses, researchers identified important protections against immune attack along with other insights that illuminate functions of the immune system.
In the very early 1980s, when reports of a lethal immunodeficiency disease began circulating, the news at first generated deep fears because no one knew what was causing the disease. Many hypotheses were entertained, some quite bizarre. Luckily it was only a few years before the right answer emerged, that AIDS is caused by a retrovirus, human immunodeficiency virus (HIV). In fact, the reverse transcriptase in the viral particle provides the signal that was used to isolate the virus for the first time. If AIDS had appeared just 15 years earlier, the tools needed to find the virus would not have been available.
Today AIDS is a scourge of the whole world, with 5.8 million new HIV infections in just the last year. It has devastated the economies and life of many African countries and promises to do the same in parts of Asia. Our principal hope to control this epidemic is to develop a vaccine. However, HIV has many defenses against immune attack, making the design of an effective vaccine a formidable challenge.
Anticipating Virology's Future
Virology is a work in progress. With much left to learn even about extensively studied viruses, we are also certain to be challenged by new, emerging viruses.
At first, virologists focused on the essential interactions of viruses with cultured cells in which they are grown. However, the strategies of viruses are often focused on interactions with systems of the host animal, notably the immune system. The present and future of research in virology involves unraveling the many details of the virus-host relationship. We are likely to be dazzled over and over by the subtlety of the mechanisms that have evolved both to replicate viruses and to defend against host responses. In some cases, we will see new examples of viral mimicry of cellular functions. Meanwhile, intracellular locations of viral activities and associations of viral proteins and nucleic acids with cellular counterparts will continue to illuminate details of cellular organization and regulation. Animal models of many human viral diseases still are needed to help elucidate the paths of viral dissemination and specific interactions of certain cells or organs with viruses.
In agriculture and as an object of public health concerns, viruses are generally portrayed negatively. However, a major development of contemporary virology is efforts to harness viruses for benefit- for instance, by engineering viruses as vectors in gene therapy procedures. Such opportunities were recognized 30 years ago when viral integration was discovered. This phenomenon reflects the ability of some viruses to add genes to host cells. Recombinant DNA methods developed during the past 20 years provide many of the tools needed to control this inherent viral capacity.
Retroviruses were the first viral class to be harnessed for this purpose, but this capability now is being extended to adenoviruses, parvo-viruses, herpesviruses, and even flaviviruses. Lentivirus vectors, under development more recently, may have even better characteristics for certain gene therapy applications. Despite its promise and the intense research efforts under way for the past decade, however, no gene therapy application is ready for routine use in humans. Perhaps sometime during the first decades of the 21st century, gene therapy will enter the armamentarium of medical practice.
It is ironic that having dissected viruses down to their molecular sequences, the formidable task of curing most viral diseases remains. Indeed, the greatest triumphs have been in viral identification and vaccine development. However, new methods of intervention and of vaccine design are in the offing. Antiviral chemotherapy has been a marvel against HIV, showing that molecular approaches can produce effective drugs. We can certainly go further, integrating our knowledge of the molecular events with modern drug screening methods. The knowledge that viruses mutate rapidly means that multiple drug therapy will probably always be needed.
Personal Reflections after Several Decades in Virology
We got together shortly after our graduate training. David arrived at the Salk Institute in 1965, when it was housed in temporary wooden buildings; it was so undeveloped that lab personnel removed a rattlesnake from his basement laboratory. Alice came to the Salk Institute as David's second postdoctoral fellow; after one year we moved to MIT-driving across the country together in an MG Midget. During our graduate work, we had separately focused on different RNA viruses-for David, a relative of poliovirus and for Alice, vesicular stomatitis virus (VSV). At Salk, we worked on poliovirus, and then on both VSV and poliovirus after moving to MIT.
In her thesis work, Alice verified a long-held concept: namely, that viruses spawn incomplete particles that can interfere with the growth of the wild-type virus. In later discussions, we recognized that a similar phenomenon applies to many other viruses and thus generalized the notion with the term, defective interfering (DI) particles. Although we thought that DI particles might become an important therapeutic agent for limiting viral growth, that expectation has yet to be met.
Meanwhile, we feel deeply lucky to have had the opportunity to watch the emergence of this field from its very rudimentary beginnings and to see it develop into a mature and highly effective science. However, the challenge of emerging viruses, most prominently HIV, reminds us that our work is not complete. Virology and immunology-those twins of offense and defense-are generally in a fine balance, but the story of HIV shows that the balance can be tipped against the immune system by a particularly cunning agent. It requires application of the intelligence of the scientific community to regain the balance we have historically enjoyed.
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