Innate Immunity Up Close

By Jason Socrates Bardi

Biology enthusiasts take note: the best way to view a Georges Seurat painting is in an art gallery where you can stand both near and far. Looking from across the room, you can take in Seurat's overall impression—for instance, strollers and sunbathers on a Sunday afternoon in 1880s France. But close to a Seurat painting, you can examine his pointillist technique and see the individual specks of color that combine to form the overall impression.

In the gallery of human health, scientists would similarly like to be able to stand back and see the broad physiological picture of the human body and also move in to examine the close-up molecular details—all the interacting systems of proteins, genes, and other flecks of "paint" that together make up what we recognize as biology.

Therein lies the challenge.

Take the innate immune system, for instance. It's easy to understand the immune part of the innate immune system. The innate immune system acts as a first line of defense against viral or bacterial invaders that make it past the physical barriers of the skin and mucous membranes. However, understanding the system part of the innate immune system has not always been so easy.

We know that the innate immune system is comprised of a collection of cells and proteins designed to deal with pathogens, but we do not necessarily understand how all these players interact with one another. Nor do we necessarily know all the players on the field.

Now a team of scientists led by Juan Carlos de la Torre and Dong-Er Zhang at The Scripps Research Institute have elucidated some of these important molecular details of the innate immune response to viral infections. The story involves an enzyme called UBP43 and a small protein called ISG15.

Immune System Responds by Making Ubiquitin-Like Proteins

ISG15 has been something of a mystery to biologists, and nobody has known what role it plays in the cell. However, scientists have reasoned that ISG15 is involved in the innate immune response because its expression is tied to an innate immune system protein that the body produces upon detecting the presence of a virus.

When a mammal such as a human or mouse becomes exposed to a virus, a number of different types of cells in the body express and secrete antiviral proteins called type-1 interferons—so named because they interfere with viral replication in infected cells and block the spread of virus to uninfected cells.

Interferons accomplish this by initiating a number of different complicated signaling pathways that turn on the expression of a number of different genes and antiviral proteins—for instance, RNAse enzymes that have the ability to chew up viral RNA.

However, interferon also induces the expression of other proteins, and in the last several years, scientists at Scripps Research and other institutes have been elucidating the human "interferon susceptible" genes and pathways involved in this interaction. One of these pathways involves the ubiquitin-like protein ISG15, which is known to be rapidly induced when organisms are exposed to type-1 interferons.

ISG15 is called ubiquitin-like because it is similar in sequence to the increasingly famous protein ubiquitin. (This year's Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose, who worked out ubiquitin's role in mediating protein degradation in the cell).

In the cell, ubiquitin is the molecular equivalent of those bright orange tags that highway patrol officers slap on the windows of abandoned cars on the side of the highway that give towing crews free leave to haul them away. Ubiquitin is a small 76-amino acid protein that gets attached to other proteins in the cell that are no longer needed. These unneeded proteins, once tagged with ubiquitin, will then be transported to the proteasome of the cell where they are broken into their constituent amino acids, which can then be recycled.

Because ISG15 is so similar to ubiquitin, scientists have assumed that it acts similarly, becoming attached to its targets when expressed. And because ISG15 is expressed in response to type-1 interferons, scientists have also assumed that this attachment of ISG15 to its targets—whatever they may be—is one way the innate immune system clears the body of viral infections.

Now, in an upcoming issue of the journal Nature Medicine, Zhang, de la Torre, and their colleagues describe part of the ISG15 pathway, which includes the protein UBP43.

Knockouts Don't Lie

Zhang, an associate professor in the Department of Molecular and Experimental Medicine's Division of Oncovirology. Her group has spent years studying the ISG15 pathway. They discovered the UBP43 (USP18) gene and identified the UBP43 protein as a protease that removes ISG15 tags from ISG15 modified proteins. More recently, using a genetic "knockout" approach, they created mice that have no ability to produce functioning UBP43 proteins. Without UBP43, these mice lost normal balance of protein modification by ISG15, which has provided a nice model to study UBP43 and ISG15 modification in innate immure responses. Recently, Zhang's group established various collaborations to study the responses of these knockout mice in interferon signaling, viral infection, and bacterial infection.

Associate Professor de la Torre, who is a member of Scripps Research's Department of Neuropharmacology, has been studying the effect of viral infections on the body for several years. He began collaborating with Zhang because he recognized that her knockout model would allow them to examine the question of whether UBP43 was involved in the innate immune defense. They would determine this by exposing her knockout models to a pathogen he was studying called lymphocytic choriomeningitis virus (LCMV).

When normal murine models are given an intracerebral exposure to LCMV, the effect is severe and always lethal. The infection causes a robust inflammatory response in the brain, and the animals die within six to eight days.

However, when Zhang, de la Torre, and their colleagues treated the knockout models with LCMV, all of them survived—much to their surprise. "This was shocking," says de la Torre.

Upon closer inspection, they found that the course of infection was initially the same in the normal and knockout models, and that on day two, both the normal and the knockout models had similar levels of detectible virus in their brains. But then things radically changed. By day five, there was a tremendous difference in viral load, and the normal animals had a significant level of virus while the knockouts had virtually none.

The lower amounts of virus in the knockout models resulted in less brain inflammation and a much greater chance of survival, making the knockouts more resistant to the infection. In this case, lower levels of UBP43 and higher levels of ISG15 seemed to be a great advantage.

While they do not yet understand all the pointillistic details, Zhang and de la Torre hypothesize that since the mice are hypersensitive to interferon treatment when they lack of UBP43, a lower than normal dose of interferon induces a more vigorous and longer than usual innate immune response in the knockout models, enabling the models to overcome the infection.

This is significant because it demonstrates that the ISG15 pathway plays a real role in the innate immune system, and it details how UBP43 is part of this pathway. Interferon expression leads to ISG15 expression and its conjugation or attachment to other proteins. Balancing this ISG15 conjugation is UBP43, which deconjugates or removes ISG15 from its target.

There is still much basic science that needs to be done. For example, there is no clear target candidate for ISG15 conjugation. Not knowing what critical targets ISG15 attaches to is naturally barrier to understanding its physiological effect.

"Is this a general finding that applies to all viruses?" says de la Torre. "We don't know. The most important thing for the future is to understand the details."

The research article, "Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection" by Kenneth J. Ritchie, Chang S. Hahn, Keun Il Kim, Ming Yan, Debralee Rosario, Li Li, Juan Carlos de la Torre, and Dong-Er Zhang was published by Nature Medicine as an advance online publication November 7, 2004. See: http://dx.doi.org/10.1038/nm1133.

This work was supported by the National Institutes of Health.

Send comments to: jasonb@scripps.edu

Associate Professor Juan Carlos de la Torre has been studying the effect of viral infections on the body for several years. Photo by Kevin Fung.

Associate Professor Dong-Er Zhang and her group discovered the UBP43 (USP18) gene. Photo by Jason S. Bardi.