Leg Work

Reshaping the Future of Hepatitis C Research One Protein at a Time

By Eric Sauter

If you ask Tim Tellinghuisen, a lot has changed in the field of virology over the last few years, specifically in the study of the hepatitis C virus (HCV), his own professional area of interest. Tellinghuisen, a pioneering Scripps Research scientist and assistant professor in the Department of Infectology at Scripps Florida, has been around long enough to see most of it.

"I started working on HCV before you could replicate a virus in a cell culture," he said recently, "and before you could replicate RNA in a cell culture. So in the last seven to eight years, we've made great strides in understanding HCV biology."

Tellinghuisen himself has participated in that explosion of knowledge. Last March, he and his colleagues found a way to disrupt hepatitis C virus (HCV) production in the early stages, offering up an important research tool that may point toward new ways to disrupt the replication process itself, which would be a medical coup of sizable proportion.

The study, which appeared in the March 7, 2008 edition of the journal PLoS Pathology, was Tellinghuisen's second major accomplishment last year. In April, he received a prestigious Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseaseaward, one of only 14 research scientists from the United States and Canada selected for the $500,000 grant, which will be paid out over the next five years.

The PLoS study described how mutations of the viral NS5A phosphoprotein were able to disrupt virus particle production at an early stage of assembly. NS5A has long been suspected as a regulator of events in the HCV life cycle, but the exact mechanism was unclear.

NS5A is comprised of three compactly folded regions of roughly 50 to 300 amino acids in length—three domains. While scientists understood that domains I and II are required for RNA replication, NS5A domain III, unnecessary for RNA replication, remained a mystery.

To help solve the mystery, Tellinghuisen and his colleagues cut out a coding sequence of approximately 15 amino acids from domain III and created a viral clone, transcribed the cloned RNA, purified this infectious RNA, and produced a liver cell line with the HCV proteins from the new RNA genome.

While Tellinghuisen found no observable defect in RNA replication, he did find that no infectious viral particles were released from the cells. Later studies indicated that the amino acid deletion changed a phosphorylation signal that controls the switch from RNA replication to virus particle assembly; the signal is linked to an inhibited cellular kinase that reduces infectious virus production without modifying RNA replication.

"These data provide the first evidence for a function of domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV," Tellinghuisen said.

Interruption Is Only the Beginning

This discovery, hailed by Charles M. Rice, head of the Center for the Study of Hepatitis C at Rockefeller University, as "a spectacular advance," put Tellinghuisen and his colleagues on a longer and more complicated path—trying to figure out exactly where these molecular events were actually happening.

"We really didn't know where the defect was," he said, "so we infected a group of cells—big Frisbee dishes full of cells—and took them to Jennifer Busy, head of proteomics at Scripps Florida. We asked her which proteins stuck to NS5A in the competent assembly stage (when an infectious virus is assembled and released from the cell) and which proteins didn't stick during incompetent assembly (when no infectious virus is produced). We found a number of differences between the two stages. The majority of those that stuck to NS5A were host proteins involved in membrane trafficking inside the cell. This makes sense because replication occurs in one part of the cell and assembly occurs in another, so you have to have something to provide transportation."

Although Tellinghuisen still isn't certain what happens during this process, data suggests that host proteins are involved in viral production. So now he's hoping to narrow the field down to the host proteins that are the most critical for viral production, which might eventually help drive another spike into the reproductive potency of hepatitis C.

Times have changed.

"There's a lot more we can do in the laboratory now than nine or ten years ago," Tellinghuisen said. "We can take infected cells and remove host proteins involved in replication, we can do large siRNA and cDNA screens to look at what's actually required for authentic viral replication. We've solved the crystal structures of HCV proteins. We know more about viral translation, and we're getting a clear picture of how RNA replication occurs mechanistically and what proteins are required from the host and from the virus."

Interestingly, this explosion in knowledge about HCV and its inner workings has occurred in almost perfect parallel with Tellinghuisen's own career.

Born in Lacrosse, Wisconsin in 1969, his family eventually settled in New Hampshire, where Tellinghuisen grew up. His father, a pharmacist by trade, was also something of an entrepreneur and moved to the Granite State to start his own pharmacy.

Tellinghuisen started out as a painter (the artistic kind) and then fell into science once he realized that job opportunities in the fine arts were severely limited.

"I enrolled in the University of Massachusetts as a liberal arts student," he said. "But I needed a job so I did work-study washing dishes in one of the laboratories. One day, one of the scientists said, 'You know, you're good at washing dishes. How would you like to try cloning?'"

Six months later he changed his major to biology, departing in 1994 with his master's degree. While a lot of graduates headed straight to the biotechnology wonderland of Boston's Route 128, Tellinghuisen went to Purdue instead, gaining his Ph.D. in 2000 and doing postdoctoral work at Rockefeller University in New York.

"Rockefeller is an amazing place to do science," he said. "It's a lot like Scripps Florida. We're focused on research, with no real distractions."

Tellinghuisen is especially grateful for the time and space to focus on viruses.

"I've always been attracted to viruses because they're interesting tools to study cell biology," he said. "Just look at the things that have come out of viruses—you can lay the entire revolution in molecular biology at the feet of virology. Much of the early mechanistic understanding of cancer comes from virology. Much of our knowledge of innate immunity and quite a bit of antibody-based immunity comes from the study of viruses."

The Frankenstein Question

Tellinghuisen's enthusiasm for the hepatitis virus is part of this long and winding road that starts with the classic question. Can a virus ever be considered alive? In any fashion whatsoever?

"This is the classic argument that virologists and microbiologists have—what is alive? Without a host, HCV is just a mix of proteins, RNA, lipids, and some sugar. They are obligate parasites because they have to kidnap things from the host to survive, flourish, and spread. This makes viruses very different than things that I would consider alive"

As for their origins, Tellinghuisen grins and points a thumb backwards.

"They've been around for a long, long time—probably billions of years. There are viruses that have evolved to infect organisms representing all of the kingdoms of life on earth, suggesting viruses have been around for a long time. Viruses also have incredible variation in the type of genetic material used to make their genomes, and their strategies of genome organization and replication are incredibly diverse. This level of diversity suggests a very long evolutionary track. I think viruses are a hangover from the early days of biology, and they've survived and flourished because they have small genomes and huge mutation rates making them incredibly adaptable. If you can get drug resistance in 24 hours form HCV in the laboratory, imagine what you could do in a few billion years."

HCV, which is treatable to a certain degree, remains a hideously problematic virus for millions of people worldwide, although Tellinghuisen believes that its days as a global scourge just might be coming to a well deserved close.

Tellinghuisen says he's taking his research down to the level of very specific kinds of questions that might help in the effort to control the virus, such as why the virus needs a particular protein to function. It's a path that he hopes to continue on as he moves into his laboratories on the new Scripps Florida campus.

"They're great facilities," he said. "They're going to improve our output scientifically."

He's looking forward to the effort.

"The great thing is we get paid to think about these things," he said. "That's quite a luxury, to get paid to think about really hard problems that might some impact on human health. It doesn't make my job feel like work."

Send comments to: mikaono[at]scripps.edu

[At Scripps Florida,] we're focused on research, with no real distractions," says Assistant Professor Timothy Tellinghuisen.