Viral survivor: new studies suggest how Epstein-Barr virus infects and persists.

A staple of the Cold War espionage novels that used to populate best-seller lists was the sleeper agent. In a typical scenario, a Russian would sneak into the United States and live discreetly for years. Then, after getting a signal from Moscow to carry out an assassination or another nefarious task, the agent would emerge as a ruthless killer, and the good guys would try to stop him. Think of Epstein-Barr virus as a microscopic sleeper agent.

Usually picked up in early childhood, it infects almost all adults. However, most don't know they have it. Among the few who know are teenagers and others who come down with the usually mild condition known as mononucleosis. Spread by saliva, the energy-sapping state is also known as the kissing disease. When not causing illness, the virus seems to reside quietly inside B cells, the immune system's antibody-making factories.

"The natural reaction of the public is that it's a pretty benign virus," notes Paul Lieberman of the Wistar Institute in Philadelphia. "The worst-case scenario, one imagines, is mononucleosis, so why worry about it? Basically, the pharmaceutical industry feels the same way."

Yet under certain circumstances, Epstein-Barr virus is far from benign. When roused from its quiescent state, the virus can trigger the cells it infects to grow wildly, producing several kinds of cancer (SN: 2/18/95, p. 104). Epstein-Barr virus appears to cause at least two malignancies common in specific locations: Burkitt's lymphoma, a B cell cancer frequently found in Africa, and nasopharyngeal carcinoma, a tumor in the back of the throat that's the most common cancer among men in many parts of Asia.

Studies have also linked the virus to other lymphomas in people with a weakened immune system and gastric carcinoma--a cancer of the lining of the stomach. Some scientists are even building a case that the virus plays a role in breast cancer.

"Clearly, the virus is a major contributing factor in a number of diseases," says Lieberman.

Recently, several research groups have reported findings that may help explain how Epstein-Barr acts as a viral sleeper agent. For example, some scientists have found a mechanism by which Epstein-Barr virus may sneak into a body. Other investigators have demonstrated that the virus exploits a set of B cells, known as memory B cells, in which it can persist for years. And finally, biologists identified cellular molecules that the virus co-opts to protect itself during its long stay in a host's cells.

SWITCH HITTER Epstein-Barr virus clearly has a propensity for B cells. Mononucleosis results when the virus sweeps through the body's B cell population, triggering other immune agents, so-called T cells, to kill off the infected cells. As a result of this civil war within the immune system, people develop sore throats, fatigue, and other symptoms.

Although B cells are the main reservoir of the virus, they may not be its entry point. Some scientists point to epithelial cells, which line cavities and exposed surfaces of the body. The virus "causes a lot more epithelial tumors than B cell tumors," notes Lindsey Hutt-Fletcher of the University of Missouri in Kansas City. Nasopharyngeal and gastric carcinomas, for instance, are cancers of epithelial cells.

There's been a controversy for many years about whether the virus infects healthy epithelial cells or just cancerous and precancerous cells. There are good reasons to think that healthy epithelial cells aren't natural targets. For one, it's hard to infect them in lab dishes. Moreover, it's rare to find an epithelial cell that contains the infectious agent in a healthy person carrying the virus or even someone with mononucleosis.

Hutt-Fletcher and her colleague Corina M. Borza recently proposed that the Epstein-Barr virus initially infects epithelial cells but then switches into a form that prefers to infect B cells. "Our findings lend support to the hypothesis that infection of an epithelial cell is normally a transient event," they conclude in the June Nature Medicine.

Their conclusion is based on some unexpected observations. In laboratory dishes, epithelial cells are a bit more easily infected by Epstein-Barr virus that was first grown inside B cells than by virus previously grown in epithelial cells. Even more dramatic, Hutt-Fletcher and Borza found that Epstein-Barr virus grown inside epithelial cells is 30 to 100 times more efficient at infecting B cells than is virus grown in B cells themselves.

Molecules on the virus' surface that the virus uses to gain access to cells might explain this phenomenon. To infect epithelial cells, Epstein-Barr virus seems to depend upon a complex of two proteins, gH and gL. But to get into B cells, the virus needs a third protein, gp42, in the complex. The virus typically has both types of complexes on its surface, but their ratio influences the virus' preference for one cell or another. Borza and Hutt-Fletcher discovered that Epstein-Barr virus growing in epithelial cells has more gp42-containing complexes than virus grown in B cells has. That factor, they say, may explain why the epithelial cell-derived virus more easily infects B cells.

The researchers traced the difference in viral surface complexes to the observation that B cells make immune molecules called class II MHC proteins. The viral protein gp42 binds to these molecules when they're on the surface of B cells. But if an infected B cell is building new copies of Epstein-Barr virus, gp42 also can bind to class II MHC molecules inside the cell. Consequently, gp42 molecules get diverted from viral surfaces, yielding a viral population that tends to have more of the simpler gH-gL complexes. This doesn't occur in epithelial cells because they don't make class II MHC molecules.

"So, virus coming out of an epithelial cell has a lot more gp42, which makes it better at infecting a B cell," says Hutt-Fletcher. That extra gp42, however, interferes with the process of infecting other epithelial cells.

The investigators suggest the following life cycle for Epstein-Barr virus. A person gets infected when virus from someone else's saliva makes its way into epithelial cells lining the throat. After a brief bout of replication, the new viruses are ready to infect nearby B cells and spread throughout the body. Later, virus growing in B cells is shed into the infected person's saliva, where it's ready to start the cycle again in someone else's epithelial cells.

"If that virus goes on to a new individual, the virus should be well-equipped to infect an epithelial cell," says Hutt-Fletcher.

Epstein-Barr virus researcher Bill Sugden of the University of Wisconsin-Madison calls Hutt-Fletcher and Borza's observations "absolutely fascinating." Still, he remains wary because their data rely upon virus growing inside cells in lab dishes.

"I don't know what [the new work] is telling us about the lifecycle of the virus inside us," he says.

HARMFUL MEMORIES David Thorley-Lawson of Tufts University School of Medicine in Boston focuses on how Epstein-Barr virus persists in the body rather than how it infects a person initially. In work over the past 5 years, summarized in the October 2001 Nature Immunology, he and his colleagues have demonstrated that the virus endures by taking advantage of memory B cells.

These long-lived, rarely dividing B cells help the immune system remember what microbes it has previously battled and respond more quickly to them than to new invaders. Memory B cells also enable vaccines to protect people for decades.

B cells start out in what immunologists call a naive state. When an infection arouses the immune system, inflammatory chemicals spur naive B cells to mature. These activated B cells can then become plasma cells, which churn out large quantities of antibodies. Alternatively, they become memory B cells and wait for subsequent infections by the microbe that initiated the immune response.

In carriers of Epstein-Barr virus, Thorley-Lawson and his colleagues have found that the virus resides in memory B cells. There, the virus essentially shuts down, making few, if any, proteins that would betray its presence. In memory B cells, says Thorley-Lawson, the virus "can't be attacked by the immune system. It's completely safe."

While Epstein-Barr virus may infect a memory B cell directly, Thorley-Lawson's team has discovered that the virus can apparently build itself a home out of a naive B cell as well. Once inside a naive cell, the virus uses its own genes to transform its host into a memory B cell.

Two other viral genes, which encode the proteins dubbed LMP1 and LMP2, promote the survival of the newly created memory B cells. The immune system has developed safeguards to eliminate unnecessary B cells that might harm the body. If a B cell doesn't get two signals--an antibody-evoking molecule, or antigen, and a signal from T cells--that B cell will commit suicide. Epstein-Barr virus uses LMP1 and LMP2 as a substitute for the two signals, thereby protecting the infected memory B cell, says Thorley-Lawson.

A resting memory cell may offer a nice vacation to a virus, but sooner or later, the virus will make more copies of itself. To do so, it triggers the memory cell out of its quiescent state. "We'd love to know what that signal is. We've been looking really hard and getting nowhere," says Thorley-Lawson.

PROTECTIVE PROTEINS Establishing residence in memory B cells is an excellent way for a virus to persevere in a person, but Epstein-Barr virus must also prevent itself from getting evicted from the cell. Unlike many persistent viruses, such as HIV, Epstein-Barr virus doesn't integrate its genes into host chromosomes. Instead, its DNA forms an independent loop, called an episome, that resides in the nucleus. Whenever an infected cell divides, the episome makes a copy of itself, and each daughter cell receives one.

Most loops of DNA thrown into a cell nucleus will be eliminated within a few days. What protects the episomes of Epstein-Barr virus? A viral protein called EBNA1 and a viral DNA sequence known as OriP offer an answer. Sugden and other biologists have demonstrated that EBNA1 can bind to two regions of the OriP DNA sequence.

When bound to one region, EBNA1 helps initiate replication of the viral episome and the dispersal of the two copies into the daughter cells. When bound to the other region, however, EBNA1 somehow protects the viral DNA from being eliminated by the cell. In fact, gene therapy investigators are considering the possibility of inserting human genes into loops of DNA that contain the OriP sequence and the gene for EBNA1. Such constructs survive for long periods in a host cell, perhaps offering physicians a new means for inserting beneficial genes into patients.

Scientists have suspected that EBNA1 doesn't work alone. Lieberman's group has recently found several human proteins that bind to EBNA1-OriP complexes. The discovery, reported in the March 29 Molecular Cell, hints at an unexpected strategy by which Epstein-Barr virus could protect its genes.

Three of the proteins identified by Lieberman's team are normally associated with telomeres, the protective stretches of DNA at the ends of chromosomes. Telomeres prevent chromosome tips from fusing with one another and may play crucial roles in aging and cancer (SN: 11/25/95, p. 362). They also somehow prevent cells from treating a chromosome tip as a broken chromosome and launching DNA-repair efforts or committing suicide.

Lieberman and his colleagues found that the three telomeric proteins might help Epstein-Barr virus episomes persist in cells. When the investigators inhibited the activity of one of the three proteins, an enzyme called tankyrase, loops of DNA containing OriP didn't survive as long as normal inside cells. When they mutated the DNA sequence of OriP so that another of the telomeric proteins could no longer bind, the DNA loops also had difficulty persisting.

Lieberman and his colleagues then took a close look at the DNA sequence of OriP, and they discovered that it contains stretches similar to telomeres. The DNA of a few other human herpes viruses, the family to which Epstein-Barr virus belongs, also contains telomere-resembling sequences, the scientists note.

"Why these viruses have long stretches of telomeric repeats has never been understood," says Lieberman. "I think at least some of these viruses are using telomeric-binding factors to protect their genomes."

Drugs that interfere with telomere maintenance may rid the body's memory B cells of Epstein-Barr virus, Lieberman speculates. The risk is that such medicines could harm cells in general, since every cell's chromosomes are capped with telomeres.

Lieberman's work "doesn't answer questions. It raises questions," says Sugden. Resolving such questions is a challenge, he points out, because the virus doesn't infect mice or other laboratory animals in which investigators can easily test their theories.

"Epstein-Barr virus has remained somewhat of a backwater because the tumors really associated with it are in Third World countries," Sugden notes. But more and more cancers are being linked to the virus, and if an Epstein-Barr virus connection to breast cancer is ever confirmed, drug companies will quickly take much more of an interest in this stealth virus, he predicts.