The Backward Enzyme

When geneticist Howard Temin of the University of Wisconsin stepped to the lectern at the Tenth International Cancer Congress in Houston in 1970, he faced a challenging task. The previous speaker and session chair—his former collaborator Harry Rubin of the University of California, Berkeley—had just trashed the hypothesis Temin was about to expound, listing multiple reasons it had to be wrong. But Temin was about to surprise the audience.

He had come up with what one colleague called his “wild idea” about 10 years before, when still a graduate student at the California Institute of Technology, to explain how some viruses commandeer cells. Those viruses contain RNA, not DNA, as their genetic material. Temin postulated that to take over a cell, the viruses first make a DNA version of their RNA, a molecular doppelganger that can function as the cell’s own genes. The notion defied biological orthodoxy. Although researchers knew that cells could fashion RNA from a DNA template, they believed that the reverse reaction did not occur. “He was literally laughed at at scientific meetings,” says John Coffin, a molecular biologist at Tufts University and Temin’s former graduate student. And Temin’s efforts to confirm the hypothesis in the lab hadn’t won over his colleagues. “The experiments he published in the 1960s weren’t wildly convincing,” says cell biologist G. Steven Martin of UC Berkeley.

Most attendees at the 1970 conference probably expected Temin to present yet more lukewarm evidence for what was known as his provirus hypothesis, Coffin says. Instead, Temin reported a startling discovery: The RNA virus he had been studying carried an enzyme, later named reverse transcriptase, that can make a DNA copy of an RNA molecule. The finding wasn’t definitive, but it offered the strongest support yet for Temin’s wild idea.

The case became even stronger just a couple of weeks later when molecular biologist David Baltimore, then at the Massachusetts Institute of Technology, revealed that a different RNA-carrying virus also harbored reverse transcriptase. As more supporting data rolled in, even Temin’s biggest detractors had to admit that he was right and that certain viruses, known as retroviruses, use reverse transcriptase to make DNA duplicates of their RNA genomes. “If ever a man was in a position to say I told you so, it is he,” the journal Nature noted.

For uncovering reverse transcriptase, Temin won the 1974 Albert Lasker Basic Medical Research Award and the 1975 Nobel Prize in physiology or medicine. Baltimore also received the Nobel that year, and his work on the enzyme was one of the accomplishments that earned him the 2021 Lasker~Koshland Award for Special Achievement in Medical Science.

The discovery of reverse transcriptase toppled a central tenet of molecular biology and transformed how researchers think about viruses. But that was just the beginning for the enzyme, now an invaluable research tool. “I don’t think you can go into any lab and not find a tube of reverse transcriptase in the freezer,” says virologist Nicolas Sluis-Cremer of the University of Pittsburgh. Reverse transcriptase acted as a critical way to detect and identify new retroviruses, including HIV. Pharmaceutical companies seized on its discovery, leading to new treatments for HIV infection and hepatitis B. Researchers are testing whether those drugs will work against a range of age-related diseases and even aging itself. To maintain their chromosomes, our cells rely on a version of reverse transcriptase. The enzyme also has shaped our genome by permitting vagrant DNA sequences known as retrotransposons to reproduce. The far-reaching impact of the enzyme makes its identification “one of the most incredible foundational discoveries,” says RNA biologist Kathleen Boris-Lawrie of the University of Minnesota, a former postdoc in Temin’s lab.

Hatching an Idea

Temin’s wild idea came to him during his PhD research on the RNA-containing Rous sarcoma virus, which invades chicken cells and increases their odds of becoming cancerous. Temin and Rubin had developed a new assay that enabled researchers to measure the amount of virus in a sample and isolate individual infected cells. While working on that procedure, Temin noticed that cells infected by the virus were usually round and plump but occasionally took on other shapes. Some were long and tapered at both ends, for instance, whereas others were elliptical. Temin wanted to determine whether the virus was responsible for those differences. He isolated individual Rous sarcoma viruses, allowed them to replicate, and then transferred them to uninfected cell cultures. The viruses stayed true to their origins. Viruses derived from rounded cells caused previously uninfected cells to form spheres, whereas viruses that hailed from elongated cells spurred cells to stretch out. “The present work shows that the virus interacts with the cell so as to influence its morphological character,” Temin wrote in a 1960 paper that summarized his experiments. “Thus, the virus becomes equivalent to a cellular gene controlling cell morphology.”

As Baltimore noted, Temin was committed to the idea that “DNA carries the heredity in cells.” He rejected the possibility that the virus’s RNA drove the alterations in shape. DNA must be the cause, Temin reasoned, even though that conclusion clashed with contemporary thinking in molecular biology. Temin’s making that intellectual leap illustrated one of his scientific gifts, “his ability to extrapolate from very limited data,” Coffin says. “Howard was a very intuitive scientist.”

After finishing his PhD, Temin took a faculty job at the University of Wisconsin in 1960. He spent the next decade performing experiments to prove his hypothesis. But none of those studies yielded the decisive evidence he sought. Temin remained certain that he was right, and “he was a pretty persuasive personality,” notes colleague and friend Bill Sugden, a cancer virologist at the University of Wisconsin. “It would not be trivial to ignore his conviction.” Still, most scientists remained skeptical—as did half the researchers in Temin’s own lab, says Coffin, who was one of the doubters.

Enzyme Hunting
In the late 1960s, findings by other research groups showed Temin and colleagues a new way to tackle the problem. Those scientists, including Baltimore, discovered that some RNA viruses carry enzymes that can make copies of RNA molecules. Temin and his postdoc Satoshi Mizutani wondered whether the Rous sarcoma virus contained a comparable enzyme that could copy RNA into DNA—a possibility they had never thought of testing before. To find out, the scientists used detergent to break open Rous sarcoma virus particles and release their contents. Then the researchers added DNA building blocks. Sure enough, new DNA molecules formed. However, if the team first mixed in an enzyme that destroys RNA, no DNA resulted, suggesting that the RNA was the template for the new DNA.

About the same time, Baltimore was performing a nearly identical experiment. Agnostic about the provirus hypothesis, he decided to test for enzymes in viruses that could produce DNA or RNA molecules. Once he obtained samples of the virus he wanted to study, he took “a few days” to find evidence of reverse transcriptase, he wrote.

Baltimore published his results in the same issue of Nature as Temin and Mizutani. The studies unleashed a flood of experiments by other “Teminizers,” as Nature dubbed them, who bolstered and extended the original findings. For instance, Temin had proposed that the viral DNA copy slips into the host cell’s chromosomes, a mechanism that other scientists confirmed.

In the Lab and the Clinic
Reverse transcriptase soon proved its usefulness for research. “RNA is hellishly unstable. Being able to make a DNA copy is extremely powerful,” Sugden says. By producing durable DNA versions of RNA molecules, reverse transcriptase allowed scientists to clone genes, measure their activity, and detect viral infections. If researchers hadn’t known about the enzyme, “I don’t think we’d have realized the potential of DNA sequencing technology, which has exposed genetic mechanisms of disease and aging,” Boris-Lawrie says.

In addition, Coffin says, the enzyme “was extremely important in telling us how cancer is caused.” Harold Varmus and J. Michael Bishop, both then at UC San Francisco, enlisted reverse transcriptase in studies showing that cancer-causing oncogenes reside within our genome. The pair was probing how the Rous sarcoma virus induces tumors. The researchers isolated the responsible gene and used reverse transcriptase to create a DNA copy of it. That DNA served as a probe, enabling them to scan the genomes of other organisms for equivalent genes. To their surprise, similar sequences were lurking not just in chickens but also in a variety of other species, including humans. Varmus and Bishop shared the 1982 Albert Lasker Basic Medical Research Award and the 1989 Nobel Prize in physiology or medicine for the discovery.

The discovery of reverse transcriptase also spawned drugs to combat HIV, still one of the world’s biggest killers with more than 600,000 deaths every year. Researchers identified HIV as the cause of AIDS in 1983. Four years later, azidothymidine (AZT), a reverse transcriptase inhibitor that thwarts HIV’s replication, received FDA approval as the first drug for treating the illness. Newer reverse transcriptase inhibitors remain standard HIV treatments and are part of some forms of preexposure prophylaxis, or PrEP, a regimen for preventing infection. “You can’t help but underscore how important reverse transcriptase has been for HIV,” Sluis-Cremer says. Although the hepatitis B virus sports a DNA genome and is not a retrovirus, it also responds to reverse transcriptase inhibitors. The mechanism for copying its genome involves an RNA intermediate, and the virus carries its own reverse transcriptase.

At first, researchers thought that reverse transcriptase was a peculiarity of retroviruses. But human cells also depend on the enzyme to protect our telomeres, the structures at the tips of our chromosomes. Telomeres shrink every time a cell divides, and if they become too short, the cell can suffer DNA damage, stop dividing, or die. But to lengthen telomeres, cells deploy an enzyme known as telomerase—whose main component is a type of reverse transcriptase. Elizabeth Blackburn, Carol Greider, and Jack Szostak shared the 2006 Albert Lasker Award for Basic Medical Research and the 2009 Nobel Prize in physiology or medicine for identifying telomerase and uncovering its functions.

The actions of reverse transcriptase also created much of our genome. Only about 2% of the human genome consists of the familiar protein-coding genes. Parasitic DNA sequences known as retrotransposons account for more than 40%. Some retrotransposons, making up about 8% of the genome, are remnants of ancient retroviruses. Those pathogens infected our ancestors at some point in the past and used reverse transcriptase to produce a DNA version of their genome, which then permanently settled in our forerunners’ genomes.

A retrotransposon known as LINE-1 constitutes an even larger share of our genome, about 17%. Although most copies of LINE-1 are inert, others can duplicate themselves and colonize other parts of the genome through a mechanism that relies on reverse transcriptase. The cell’s standard gene-reading enzymes first make an RNA copy of the retrotransposon, which gives the cell instructions for synthesizing two proteins, one of which acts as a reverse transcriptase. That protein then converts the retrotransposon RNA back into a DNA molecule that can insert elsewhere in our genome.

Retrotransposons may have a surprising connection to our health. They become more active as we age and in illnesses such as Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Clinical trials are testing whether existing reverse transcriptase inhibitors are beneficial in such conditions, and drug companies are developing blockers to thwart retrotransposons.

Temin didn’t envision how important reverse transcriptase would turn out to be for science and medicine. But his ability to look beyond conventional wisdom, and his tenacity in pursuing his iconoclastic idea, served as the bedrock for those discoveries. “He carved his own path,” Martin says.

By Mitchell Leslie