You now work on HCV. So when did you switch back?
After my PhD, I went off to do a postdoc in Madison, Wisconsin, with Paul Ahlquist, who was studying the model positive strand RNA viruses: Flock House virus and brome mosaic virus. They're really fantastic in that you can do all kinds of things with them that you can't do with other viruses, but I missed studying medically important viruses. Right around this time, Ralph Bartenschlager's group, who were working on HCV, made a breakthrough by developing replicons—a system for studying intracellular aspects of HCV replication.
Charlie had moved to Rockefeller [University] while I was in Madison, and he started working on these HCV replicons. He and I talked and decided that I should come back to his laboratory and work on developing the cell culture system further—the replicon system allowed us to study intracellular viral replication, but not the entire viral life cycle.
How do replicons differ from the normal HCV?
Replicons are positive strand RNAs, like the full virus, but they only encode a subset of the genes—the ones we think are important in RNA replication. They lack the structural genes that go on to make virus particles.
So what did you do with these replicons?
HCV has an extraordinary genetic diversity because of its error-prone replication, and most replicons can replicate in culture because they accumulate adaptive mutations. When I came back to Charlie's laboratory, we wanted to study full-length viruses in culture, but when we put the existing replicons into the full-length genome, we didn't get viral production. We worked on the hypothesis that the adaptive mutations in the replicons were inhibiting virus production.
Around this time, another laboratory described a replicon called JFH that didn't need adaptive mutations to grow. We thought, “Aha, maybe if we make a full-length genome out of this replicon, it will make an infectious virus.” So we contacted the laboratory, they sent us the replicon, we reconstructed a full-length genome, and lo and behold, it produced virus particles. We went on to show that the reconstructed virus was infectious in animals, so we knew it was bona fide.
Hang on, how does this reconstructed virus differ from the wild type?
That's a really good question. The JFH replicon was derived from a virus that came from a Japanese patient with fulminant hepatitis (that's where the name came from). But according to the researchers who derived the replicon, the original virus wouldn't grow in culture. The JFH replicon seems to be an unusual isolate that replicates without adaptive mutations.
Given HCV's genetic diversity, I'm sure there are additional virus strains out there that will also efficiently replicate in culture, we just haven't found them yet. So for the moment, reconstructed JFH strains are our best system for studying the complete life cycle of the virus.
So what have you learned about the virus's life cycle, using this construct?
When we first grew the virus, we looked at its buoyant density, or how well it floats, to learn about the basic physical properties of the virus. We found that HCV has two forms—a heavier form that's not very infectious, and a light infectious form. This form has an unusually low buoyant density, which is usually a sign that the virus is interacting with lipids.
Lipid droplets (green) are sites of HCV (red) assembly. Lindenbach expects to learn more about both viruses and their host cells thanks to live cell imaging studies.
Now we're interested in understanding, what exactly is in these particles? How does it contribute to the infectivity of the virus? And how are the different virus particles made? We think that the more buoyant form of virus might be coating itself with serum lipoproteins, to help mask it from the host immune response. But that's still an emerging concept.