Profile of Charles M. Rice

The notoriety of hepatitis C belies its breathtaking toll. That is partly because many of the more than 4 million people in the United States infected with hepatitis C virus (HCV) do not look or feel sick. Until, that is, their livers succumb to cirrhosis or cancer, says Charles Rice. Rice, a professor of virology at The Rockefeller University and a member of the National Academy of Sciences, has championed the development of an animal model for hepatitis C, a goal that has by turns tantalized and tormented researchers for decades. The lack of a suitable model has hampered the development of an outright cure or a protective vaccine for this worldwide scourge. Combining creativity and perseverance, Rice has gamely addressed the goal, closing in on an animal model for the disease while unearthing a wealth of insights into the workings of HCV.

Charles M. Rice.

The only child of an insurance claims adjuster and a housewife, Rice was born in Sacramento, California. Rice developed a fondness for animals at a young age. “I had dogs as brothers and sisters, so I was naturally attached to animals,” he says. Later, that fondness matured into passion as Rice decided to study veterinary medicine at the University of California, Davis. As so often happens to scientific trajectories, however, Rice's career path took a turn when a beginners’ biology course, taught by the man who would become one of his mentors, drew him toward basic science. “Dennis Barrett was a fantastic teacher. The course got people thinking about things deeply,” Rice says. Barrett's influence was bolstered by a laboratory stint in chemistry during which Rice studied how sea squirts—denizens of the ocean floor whose delicate bodies are cloaked in colorful tunics—extract whopping amounts of vanadium from seawater. “That gave me an appreciation for the amazing biology of the natural world,” Rice says.

It was Barrett's suggestion to attend the renowned physiology short course at the Marine Biological Laboratory at Woods Hole, Massachusetts, that sent Rice down the road to basic research, however. “That was an amazing summer experience because I was boxed up with a bunch of scientifically minded folks, and I got lab experience in everything from biophysics to biochemistry to immunology,” Rice says. “More importantly, I learned what scientists were really like, and I enjoyed what I learned,” he adds. So much so that a year after graduating with a bachelor's degree in zoology in 1974, Rice returned to Woods Hole to be a teaching assistant in the course. In the fall of 1975, Rice enrolled in a graduate program in biochemistry at the California Institute of Technology, where Barrett had been a graduate student years earlier.

At the California Institute of Technology, Rice joined the laboratory of virologist James Strauss, who studied how RNA-containing viruses, such as those that cause encephalitis, polyarthritis, yellow fever, and dengue fever, multiply inside their hosts. Beginning his graduate work at the dawn of recombinant DNA technology, Rice applied then-novel techniques in cloning and nucleic acid and protein sequencing, perfected at the California Institute of Technology by molecular biology powerhouses Leroy Hood and Tom Maniatis, to unravel the genetic makeup of the mosquito-borne Sindbis virus, which can cause fevers marked by joint pain, rashes, and malaise in people. Rice helped to deduce the sequence of the virus’ structural proteins, paving the way for later studies aimed at stopping such viruses in their tracks (1). Rice's findings on the genome of Sindbis virus fueled interest among researchers to develop ways to manipulate and unravel the genomes of other infectious viruses. As the field gained steam, Rice partnered with Henry Huang, a postdoctoral researcher in Hood's laboratory, to develop ways to produce RNA viruses from DNA intermediates, providing a way to perform genetic analysis on pathogenic viruses. Rice graduated with a PhD in biochemistry in 1981 and decided to stay in the Strauss laboratory for postdoctoral studies, growing, purifying, and studying a strain of yellow fever virus. “When we cloned and sequenced the viral RNA, it became clear that the yellow fever virus was remarkably different from Sindbis virus,” Rice says. Those findings, reported in Science in 1985, helped establish flaviviruses, a group that includes yellow fever, dengue, and West Nile viruses, as a separate family of viruses on the tree of life, underscoring the power of genetic analysis in unraveling how viruses evolve (2). More importantly, the effort uncovered the genome sequence of one of the most effective vaccines known to humankind; the strain of yellow fever virus that Rice used for his studies was the one used to make the yellow fever vaccine. Rice's contributions to viral genetics also resulted in a personal payoff in 1986, a faculty position at Washington University School of Medicine in St. Louis. The same year, Rice won a prestigious Pew Charitable Trust scholarship for young biomedical researchers.

New Virus, New Direction

At Washington University, Rice's exploration of the Sindbis and yellow fever viruses culminated in an important 1989 paper in the now-defunct journal The New Biologist, describing how to produce infectious flavivirus RNA in the laboratory, a step pivotal to studying the pathogens (3). Shortly thereafter, Rice received a telephone call from researcher Stephen Feinstone, who had spent years studying viral hepatitis at the US Food and Drug Administration. Aware of the power of Rice's technique to manipulate RNA viruses, Feinstone suggested that Rice modify his strain of yellow fever vaccine to produce a vaccine for hepatitis C, a disease that was rapidly gaining scientific interest thanks to the 1989 discovery of the causative agent HCV by researchers at Chiron Corporation, now part of Novartis. Elegant in theory, the idea faced formidable practical challenges, not least of which was that the molecular biology of HCV was largely a black box. “That led me to start a pilot project in the lab, thanks to the Pew funding, to define the basic biology of the HCV genome. That was of particular interest to me because HCV was a reasonably close relative of the viruses I had been studying,” Rice says.

The discovery of HCV led to a handful of diagnostic tests for the disease, but the virus resisted researchers’ attempts to grow it in the laboratory, hampering genetic studies, not to mention the development of a vaccine, which remains elusive. “At the time, there was no cell-based assay to evaluate prevention or therapy for HCV,” Rice says. In 2005, Japanese virologist Takaji Wakita, German virologist Ralf Bartenschlager, and two American teams, one led by Rice, broke new ground in HCV research when they showed that a strain of the virus isolated from a Japanese patient with fulminant hepatitis, an uncommon acute form of hepatitis, could be forced to replicate in human liver cancer cells grown in laboratory dishes, allowing researchers to probe the entire life cycle of the virus. That groundbreaking finding crowned nearly a decade of spadework by Rice and others. In 1997, Rice produced the first infectious clone of HCV that could be used for studies in chimpanzees, the only other animal species that the virus infects (4, 5). “That was an exciting finding because it meant you could produce HCV in chimpanzees and then use it to see what kind of cell lines might allow replication of the virus in the lab,” Rice says. Extending Rice's finding, Bartenschlager and University of Heidelberg virologist Volker Lohmann devised a method, dubbed the HCV subgenomic replicon system, in 1999 that allowed the replication of HCV RNA in human liver cancer cells in the laboratory. “Bartenschlager's replicon was a landmark discovery in its own right,” Rice says, “but the frequency with which you could initiate viral RNA replication was low, making it difficult to carry out genetic studies.” So Rice devised a workaround: He used more permissive liver cancer cell lines and identified mutations in the HCV genome that helped the virus adapt to growth in the laboratory. “The mutations increased the ability of the viral RNAs to transduce these permissive cells by almost 50,000-fold. To this day, we don't understand how these mutations work,” Rice says. Yet the discovery breached an important barrier to exploring the life cycle of the virus. It paved the way for the 2005 breakthrough, which, for the first time, allowed Rice and others to produce viral RNAs that would not only replicate in laboratory-grown cells but produce filterable infectious virus, Rice says.

In 2001, Rice moved to New York to accept a faculty position at The Rockefeller University at the behest of Columbia University virologist Stephen Goff, who recommended Rice for the position of scientific director of the first hepatitis C research center in the northeastern United States. The center combined the expertise of researchers and clinicians at The Rockefeller University, New York Presbyterian Hospital, and Weill Medical College of Cornell University. “I had always viewed New York as a concrete jungle and avoided it on most occasions,” Rice recalls. Nevertheless, on the urging of Rockefeller molecular biologist James Darnell, whom he had long idolized, Rice decided to move eastward.

Quadruple Whammy

The ensuing years at The Rockefeller University saw a spate of discoveries from Rice's laboratory on the replication, assembly, and infection cycle of HCV (6). Notable among those findings was the identification of a protein required for the entry of the virus into liver cells. Dubbed claudin-1, the protein, which is found at tight junctions between epithelial tissues in the liver, joined a previously established duo of host membrane proteins, called CD81 and scavenger receptor B1, as HCV's human handhold. Liver cells lacking any of the three proteins failed to take up the virus (7). “The finding stemmed from our efforts to develop a mouse model for the disease,” Rice says, emphasizing that the exclusivity of the virus in infecting chimpanzees and people severely hamstrings researchers hoping to probe its biology. Before techniques to produce infectious HCV in laboratory-grown cells became widespread, researchers routinely used so-called “HCV pseudoparticles,” which were composed of modified retrovirus particles in which the envelope proteins were derived from HCV. Rice and his postdoctoral researchers noticed that the pseudoparticles failed to infect human liver cells when claudin-1 was missing from the host cell membrane.

The discovery of claudin-1 did not complete the story of HCV entry into human liver cells, however; the virus still balked at some cell types bearing all three proteins,—CD81, scavenger receptor B1, and claudin-1—suggesting that the trio was not sufficient for entry. “That was when postdocs Matthew Evans and Alexander Ploss, who was heading our animal model development efforts, entered the picture. Ploss suggested that we screen for the missing factors required for virus entry in mouse cells,” Rice says. “You have to get lucky with that sort of experiment, and it takes a risk-taker to pursue it,” he adds. Chance favored Ploss, and the venture bore fruit. In a 2009 Nature paper, Ploss, Evans, Rice, and others described the identification of a human protein, dubbed occludin, that turned out to be the last of the four proteins that the virus needed to enter mouse and human cells, providing a foundation on which to build an animal model for HCV (8).

Because the entry proteins act as sentinels escorting the virus into human cells, the reasoning goes, blocking their function might be a way to thwart virus entry into the liver cells of infected people. That said, Rice cautions that claudin and occludin play important roles in cell physiology, possibly limiting their potential as drug targets for hepatitis. “You don't want to be dissolving tight junctions in your liver, and occludin is fairly ubiquitous in the body, too,” Rice says. That is partly why HCV drugs based on blocking claudin or occludin, although conceivable, are hardly around the corner. “We first need an animal model to do any kind of therapeutic intervention study,” Rice says. “With the chimpanzee being the only model, you can do only limited kinds of experiments,” he adds. Further, the small number of chimpanzees available for such experiments, not to mention their prohibitive cost, restricts researchers’ ability to gather data to measurable statistical satisfaction. Add to these problems the growing opposition to primate research in many countries, and the need for a small animal model for hepatitis C turns ever more pressing.

Hunt for an Elusive Model

To help address the need for an animal model, Rice struck up a partnership with Massachusetts Institute of Technology tissue engineer Sangeeta Bhatia, who leapfrogged to biotechnology's forefront by creating 2D and 3D environments mimicking human livers to culture liver cells in laboratory dishes. “Sangeeta's cell culture system helped the cells retain their liver-like phenotype outside the liver,” Rice says. Lofty yet realistic, Bhatia's goal is to grow artificial livers in the laboratory for people in need of transplants. Her 2D “micropatterned coculture technique” has already helped Rice uncover a wealth of insights into HCV pathogenesis, however, and has set the search for a small animal model apace. “I'd heard that Sangeeta was talking at a liver disease symposium and called her up to see if she was interested in collaborating with us. The goal was to see if her liver cocultures would support HCV infection and replication,” Rice says. Shuttling between Boston and New York, the researchers created, thanks to a National Institutes of Health grant earmarked for risky, high-stakes research, a small-scale cell culture system that not only supported HCV infection but provided a window into the molecular modus operandi of the virus in liver cells (9). As reported in a 2010 paper in Nature Biotechnology, Rice and Bhatia engineered a fluorescent reporter that illuminated liver cells upon HCV infection, providing a potential means to test drugs that block virus entry (10). “The technique allowed us to determine which cells in the culture were infected with the virus,” Rice says. Extending those efforts, Rice's group now uses laser capture microdissection, a technique that allows them to airlift single infected cells from a carpet of cells growing on dishes, to perform detailed analysis. More importantly, Rice and Bhatia are trying to modify the cell culture system to build tiny livers in the laboratory that could be implanted in mice with muted immune systems, inching closer to the elusive animal model. “If we can create these implantable livers in ways that would preserve their function and support HCV infection, that will allow us to study the virus in a context that would somewhat mimic an infected person,” Rice says. “Also, the effort is a fantastic illustration of groups with complementary expertise coming together to create something that is likely to advance the field,” he adds. To recapitulate human HCV infection truly in preclinical models, however, researchers need an animal host with a working immune system, largely because the pathogenesis of the virus is partly mediated by host immunity. “That's the ultimate goal we're working on, which would also be important for studying diseases like AIDS and malaria,” Rice says. To that end, Rice speculates, induced pluripotent stem cells might someday help to generate bespoke human livers and immune systems to be blended in mouse models.

With lofty goals looming on the distant horizon, Rice says he plans to stay in the laboratory for many years to come. “I get paid for doing something that's a passion, not a job. So, I'd like to see our work pay off for people with hepatitis C,” he adds.