The Cell Detective
Hidde Ploegh's lab uncovers how viruses silence cells--and disarm the immune system.
By Katherine Bourzac, SM '04
Your cells are little chatterboxes that can't keep a thing to themselves. They narrate their day-to-day activities for all to hear--every ache and pain or coming and going. With cells, everything is on the surface.
And it's a good thing, too, because the immune system, like an overprotective parent, needs to hear exactly what's going on to make sure we are safe. Cells' preferred method of communication is to display molecular flags on their membranes. Such flags let the immune system know if a cell has been infected by a virus or has turned cancerous. But some viruses can gag cells so that the immune system has no idea what's happening.
Hidde Ploegh, an MIT biology professor and member of the Whitehead Institute for Biomedical Research, wants to know how they do it. In his lab, researchers are zeroing in on the tactics that viruses and bacteria use to silence cells. "We think that by inspecting these viruses [and bacteria] closely, we can get a glimpse not only at their evasive functions but also at the workings of the healthy immune system," says Ploegh.
The cells of the immune system include many kinds of killer, memory, and chatterbox cells connected through complex communication networks. The deadliest human diseases--including tuberculosis, HIV, and cancer--are very good at hiding their presence from the immune system. Exactly how they do this is not well understood: there are many places in the networks where a disease could disrupt or destroy a signal.
Ploegh is particularly interested in herpesviruses--a large, ancient family of viruses, of which eight infect humans--because many of them can stop the communication process before it starts by preventing chatty cells' flags from going up. "This family has evolved a bag of tricks with which they frustrate this whole process," he says.
What Ploegh finds especially fascinating about herpesviruses is that unlike most other disease-causing microbes, once they infect you, they never go away. "What these viruses have learned," he says, "is not only how to infect the host and hide within it but also how to reactivate from their latent state," causing fever, sores, and other symptoms sometimes years and years later. "The virus comes out of hiding in the face of an immune system that already knows about its presence, and it can still come out on top and be transmitted to the next host. That's a pretty remarkable set of strategies."
In particular, Ploegh has focused on a herpesvirus called human cytomegalovirus (HCMV), which is so prevalent that 50 to 80 percent of Americans harbor it by the age of 40. Infected people usually have no symptoms, but the virus can cause eye inflammation, liver failure, and death in people with compromised immune systems, such as AIDS patients.
Ploegh's research has shown that HCMV is among the herpesviruses that can cloak themselves by preventing the cells they infect from displaying their molecular flags to the immune system. All human cells, whether infected or not, ordinarily display on their surfaces constantly rotating samples of the proteins being made inside. Immune-system cells known as killer T lymphocytes circulate through the blood and the lymphatic system to "read" these samples. If a cell is displaying a snippet of a protein not normally made by healthy cells--like a cancer protein or a viral protein--the killer T lymphocytes wandering by will detect it and kill the cell. "You might consider this the early-warning system by which the T lymphocyte knows what's going on deep inside a cell," says Ploegh. "If the virus could disarm that early-warning system, it would be temporarily invisible to killer T cells."
Ploegh's group discovered that that's exactly how HCMV operates: it targets the protein that carries snippets of other proteins up to the cell surface for display. The carrier protein, called MHC class I, functions as the cell's flag bearer. It hangs around the place in the cell where proteins are made and destroyed, grabbing onto whatever snippets it finds and hoisting them to the cell surface. Researchers in Ploegh's lab have isolated a cluster of HCMV genes that destroy or detain MHC.
This work is giving immunologists a peek into herpes*viruses' bag of tricks--and illuminating the quotidian activities of normal human cells. "The virus has hijacked what we now believe is an essential pathway for protein quality control," Ploegh says. Cells are very careful when copying their DNA, because any mistakes will be passed on to future generations. But protein production is sloppy, with an error rate of about 10 percent. "That garbage needs to be cleaned out of the cell," Ploegh says; indeed, "part of the process of synthesis is also the breakdown of misfits." Biologists aren't sure how misfit proteins are recognized, but once they are, they are given a pass that lets them into a proteasome, which Ploegh compares to a "meat grinder." After exiting a proteasome, minced proteins, be they viral or the cell's own, pass into the compartment where MHC lies in wait, whereupon it rushes them to the surface for inspection by T cells.
Ploegh and his students studied two HCMV genes in human cells and showed that either one can disrupt the flag-bearing process. "Rather than work with infected cells, you can simply install in your cell this single gene and see the entire pathway unfold," he says.
Joana Loureiro, a graduate student from the University of Lisbon in Portugal who is conducting her doctoral research in Ploegh's lab, is studying one of these genes, called US2 (the other, called US11, has similar effects). Using a technique called pulse-chase, Loureiro can track MHC, US2's victim. First, she inundates, or "pulses," human cells with radioactively labeled protein building blocks--so many that the MHCs made during the pulse period will predominantly be radioactive and trackable. Then she "chases" the first set of building blocks with a second set that's unlabeled. Loureiro can thus track a group of proteins made only during a certain period. This lets her see the timing of events: in the presence of the viral US2 protein, MHC pokes out of its usual compartment and is then chewed up by the cell's meat grinder.
HCMV has other ways of disabling MHC--for example, proteins that simply drag it down like an anchor so that it cannot reach the cell surface. But Ploegh says the "most spectacular example" of the virus's ingenuity is the one Loureiro is studying, in which the virus turns the cell's own quality-control machinery against itself. The Ploegh lab has shown that HCMV can disable MHC in literally minutes; the infected cell simply has no time to send out a warning to the immune system.
HCMV may be mimicking a normal protein-processing mechanism in organisms from yeast to humans. Yeasts, those simplest of fungi whose genetic workings have proved very much like our own, contain genes that define pathways similar to those used by US2 and US11 in human cells. This similarity, Ploegh says, suggests "a link that can be made between escape from immune [system] detection by viruses and very basic, normal pathways." He adds, "We think the pathways used by US2 and US11 are emblematic of how your typical mammalian cell deals with protein garbage." By studying how native human proteins help the viral protein destroy MHC, Loureiro hopes to uncover how the normal pathway works.
Lisa Kattenhorn, a Harvard Medical School graduate student in Ploegh's lab, discovered another cloaking mechanism. In order to be displayed at the cell surface by MHC, viral proteins must first go through a proteasome's grinders. But to get into a proteasome, the proteins need a special pass called ubiquitin; without this control, any and every protein could go into the grinder, and the cell would eventually die. Kattenhorn found that one of the first proteins to enter a cell during infection by a herpesvirus is an enzyme that can remove ubiquitin from viral proteins. No ubiquitin means no viral-protein fragments for MHC to display, so the infection is likely to be invisible to killer T cells. All herpesviruses have this enzyme, so Kattenhorn hopes her work might lead to a broadly applicable therapy.
Though Kattenhorn is a virologist, her work relies on probes made by chemists in Ploegh's lab. Howard Hang, a chemistry postdoc working with Ploegh, describes these probes as "bait for pulling out proteins" so they can be examined in detail. Each probe has the equivalent of a fishing fly that entices proteins to bite, as well as a "line" that can be used to retrieve them. Kattenhorn's probes, for example, use ubiquitin as bait to attract the enzymes that remove it.
These chemical probes work well in studies of the parts of the cell undermined by herpesviruses, but they cannot be used in live cells, Hang says. The probes are too big to enter and exit intact cells, so the cells must be pulverized before they're examined. Hang is designing a smaller, more flexible probe to do live-*imaging studies of Salmonella bacteria in action. In the cells it infects, Salmonella somehow fends off proteases, enzymes that, like proteasomes, break down proteins. It thus prevents its telltale proteins from reaching the cell surface and being seen by T cells. But Salmonella has to be intact and alive to pull off this feat.
Ploegh is using other kinds of live-cell imaging techniques to study the interconnections between the various branches of the immune system. He is also investigating immune-system cells that operate on a more general level than killer T cells do. Rather than responding to a particular strain of E. coli or to herpes simplex virus type 1, these cells recognize threats in very general categories--bacterium, virus, or fungus--and act fast. Textbooks make sharp distinctions between these two branches of the immune system, but "in real life they are intimately connected," says Ploegh. "They function on a continuous spectrum."
During an infection, a microbe tries to multiply, and its host tries to destroy it. "There you have the beginnings of a protracted battle," says Ploegh. In studying the war plans of herpesviruses and other microbes, Ploegh says, he's looking, not for a way to cure a specific disease, but for a better understanding of how the immune system works. And that understanding will better prepare us to combat any disease.
