Viruses, Paul Ahlquist says, are the hot rods of the living world—simple yet supercharged. Despite their bare-bones genomes, they can rev up host cells to make thousands to millions of viral copies. It's not surprising, then, that viruses cause some of the world's most menacing diseases, including AIDS, SARS, Ebola, hepatitis, and influenza. Moreover, they trigger 10–15 percent of all cancers. To better deal with such infections, scientists need to learn how viruses take host cells hostage and how that process can be stopped. "If you understand the underlying mechanisms, you can rationally design interventions for more effective and diverse therapies," says Ahlquist, who studies the molecular underpinnings of viral reproduction in several RNA and DNA viruses.
One of Ahlquist's first major contributions was to develop broadly applicable ways to study viruses whose genes are fashioned from RNA. In most organisms, genes are made of DNA and are transcribed into RNA molecules that serve as templates for protein synthesis. Most viruses don't toe this line, because they use RNA for both genes and templates. But the best tools for manipulating and studying genes work only with DNA.
Beginning in the 1980s, Ahlquist and his coworkers devised ways to make and use functional DNA copies of RNA genomes, first in the test tube and then in cells. "Perhaps because of the success of these approaches," he says, "many objectives that had to be accomplished that were significant technical challenges at the time are now taken for granted by our students." But the advent of the techniques broke a major research barrier, enabling Ahlquist and others to explore the replication of RNA viruses.
Another challenge was to determine how such viruses interact with cells. Since they have just a smattering of genes, they must persuade the genes of host cells to do most of their work. "What you are studying in a viral infection is not so much the virus as the infected cell," Ahlquist says. "You have to get at what it is on the cell side that is being utilized."
To speed up work on such host factors, Ahlquist devised a way to infect yeast cells with RNA viruses. Then he systematically looked at every yeast gene to see which ones these viruses use. "Yeast reproduces most of the major functions of animal and human cells but has a tremendously simpler genome," Ahlquist says. "And there are much more powerful and rapid techniques for manipulating it."
Using yeast, Ahlquist's group studied positive-strand RNA viruses, which include viruses that cause the common cold, as well as liver cancer, West Nile disease, SARS, and other serious diseases. These viruses have a single strand of RNA, called the positive strand (because it can be used as messenger RNA), that is copied inside a host cell into a negative strand (the complementary strand to the positive strand). Like a queen bee, the negative strand has only one job in life: to make thousands and thousands of offspring (positive strands).
The group filled in the details. When a positive-strand RNA virus enters a cell, it latches onto an internal membrane and indents it to create a lightbulb-shaped compartment. The bulb's neck remains open to the cytoplasm. Inside the compartment, the virus makes a few negative strands of RNA, which remain sequestered with a viral enzyme that uses them as templates to make more positive strands of RNA. The resulting army of positive strands moves out of the lightbulb to the cytoplasm, where the cell's protein-making machinery uses some strands as templates for making viral proteins. Others are wrapped in certain of those proteins to create virions that will infect other cells.
This system is ingenious, Ahlquist says, partly because ensconcement within the lightbulb appears to prevent other parts of the cell from seeing double strands of viral RNA, which form inside the lightbulb when the complementary negative and positive stands cling to each other. Double strands of RNA don't normally occur in cells and would trigger antiviral responses if they could be found. But while the lightbulb protects viral RNA from the rest of the cell, it also allows the small molecules needed for RNA synthesis to enter and permits new positive strands to leave.
While Ahlquist was studying these processes, he had a revelation. "The structure and assembly of the replication complexes of these RNA viruses seemed shockingly similar to the ways that some other viruses make their infectious virion particles," he remembers thinking. This was surprising because it suggested relationships among three viral groups normally considered distinct—the positive-strand RNA viruses, the double-stranded RNA viruses, and the retroviruses. For example, HIV (a retrovirus) integrates its genetic material into the host cell's genome so that when the cell divides, the daughter cells are already infected.
The similarities uncovered by Ahlquist and his coworkers suggest that these three groups, of the six known groups of viruses, may well have evolved from a common ancestor. "This also has substantial implications for [viral] control, because if there are similar underlying features and processes, there is some hope for developing strategies for interfering with those processes that may be broad-spectrum, or at least generalizable," Ahlquist says. "We find that quite exciting."
Ahlquist is currently studying how several RNA and DNA viruses, ranging from laboratory models to human tumor viruses, replicate themselves and cause disease. He's using a variety of systems, including yeast, Drosophila cells, and human cells and tissues.