About a Virus

By Jason Socrates Bardi

"The first [precept] was never to accept anything for true which I did not clearly know to be such; that is to say, carefully to avoid precipitancy and prejudice, and to comprise nothing more in my judgement than what was presented to my mind so clearly and distinctly as to exclude all ground of doubt."

———Rene Descartes, Discourse on the Method of Rightly Conducting the Reason, and Seeking Truth in the Sciences, 1638

It is the early 1970s and a nurse named Johnny accidentally sticks himself with a needle while doing a routine blood draw, exposing himself to the hepatitis B virus (HBV). There are at this time no screens for the virus, and aside from admonishing himself for his clumsiness and washing the tiny wound with antibiotic soap, Johnny thinks nothing of it.

A few weeks later, while the virus is replicating prolifically in his liver, he transmits hepatitis B to his wife during intercourse. Unbeknownst to either of them, she is already pregnant. And when their baby is born, he is also infected.

The odds, in this fictionalized case, are that Johnny and his wife both fully recover from their infection. Their immune systems mount a strong defense against the virus and it is eliminated from their systems. The odds do not favor their son, however. His immature immune system will most likely not be able to fight off the hepatitis B virus, and it will establish a chronic, lifelong infection in his liver. Like all other chronically infected people, he will have potentially lethal health problems and an enormously increased risk of developing liver cancer.

In the United States, there are 1,250,000 people living with chronic hepatitis B, according to the Centers for Disease Control and Prevention (CDC). The average American’s lifetime chances of being infected by HBV are about five percent, and about five percent of those infections will become chronic.

Globally, the situation is more dire. Nearly half the world’s population lives in areas where more than eight percent of people are chronically infected by hepatitis B. Anyone living in those areas has a greater than 60 percent chance of being infected by hepatitis B virus at some point in their lifetime. Hepatitis B is the leading cause of liver cancer in the world.

“There are 350 million people in the world who are chronically infected with hepatitis B,” says physician Frank Chisari, who is professor in The Scripps Research Institute (TSRI) Department of Molecular and Experimental Medicine.

“My lifelong dream is to contribute to the termination of hepatitis B infection in all those chronically infected people—that has been driving my research throughout my career.”

Cells Can Cure Themselves

Hearing of goals, rather than of accomplishments, is strange from someone who has recently received more than one lifetime achievement award.

A few weeks ago, Chisari and TSRI Chemistry Professor Chi-Huey Wong were elected to the National Academy of Sciences, becoming two of the now 14 investigators at TSRI who have been admitted to this august body. And just days ago, he was elected to the American Academy of Microbiology, the highest honor the American Society of Microbiology bestows upon its members. Both honors recognize the work Chisari has done on hepatitis since coming to TSRI in 1973.

In the last three decades, by studying infections in patients and in a closely related species, and by developing transgenic models to study the HBV immunobiology and pathogenesis, Chisari and his collaborators have characterized the course of HBV infection in the liver, the immune system’s response to the virus, and the mechanisms whereby a chronic HBV infection can lead to liver cancer. In recent years, he and his collaborators completed a comprehensive analysis of the virological and immunological features of HBV infection using liver biopsies and blood samples they obtained from infected subjects every week for six months after inoculation, describing the course of infection with a level of detail that had never before been attempted.

The insights they gained from these studies and their earlier human and transgenic model experiments have revolutionized the way we think the immune system can control a viral infection. Furthermore, they also demonstrated that there’s a dark side to the antiviral immune response, which can produce progressive tissue damage and even trigger the development of cancer when it goes awry.

Hepatitis is caused by one of several evolutionarily distinct viruses (called A, B, C, D and E) that all target hepatocytes, the parenchymal cells of the liver. Hepatocytes are the tiny chemical factories in the liver that produce most of the proteins present in blood, nurturing all the other organs of the body. They also produce bile, a fluid used for digestion of fat in the diet and for the elimination of waste.

Hepatitis B virus is a circular, double-stranded DNA virus just over 3,000 base pairs long belonging to the Hepadnaviridae family. The infectious particle, or virion, contains this tiny genome and a viral polymerase enzyme in a protein capsid shell surrounded by a lipid coat. HBV infection starts when these virions are introduced into the bloodstream through routes that are similar to those used by the human immunodeficiency virus (HIV)—unprotected sex, contaminated needles, and mother-infant transmission.

Once inside the bloodstream, the virions eventually pass through the liver, and the process of disease starts when HBV infects one or more liver hepatocytes. The initial number of cells infected may be small—just a few—but within six to eight weeks, the virus rapidly replicates and can infect every hepatocyte cell in the liver.

Upon reaching this widespread infection, there is a rapid spike of viral DNA in the bloodstream and the initiation of an immune response, which is evident by the appearance of T cells in the liver and a reduction by several orders of magnitude in the amount of virus in the blood.

Most adults who are infected with hepatitis B suffer an acute infection. After the HBV activity peaks, the body mounts an immune response and the virus disappears. The immune response is so effective that the liver goes from having all its hepatocytes infected to having none of them infected.

Over a decade ago, Chisari suspected that the immune system must be using an unexpected way of clearing the virus from infected liver cells during an HBV infection, because in many cases it was clearing the virus without killing off all the infected cells.

For years, scientists had recognized that one of the principal ways that the immune system deals with a viral infection like HBV is to unleash cytotoxic T lymphocytes (CTL), also called killer T cells, which carry a receptor on their surface that specifically recognizes tell-tale viral markers on the surface of infected cells that indicate these “target” cells should be eliminated. CTLs then kill these infected cells by inducing them to undergo apoptosis, the cellular equivalent of suicide. Until recently, however, this destructive process was thought to be the only antiviral mechanism that CTLs had at their disposal.

But Chisari realized that this couldn’t be the primary mechanism for clearing HBV because killing requires direct contact between a CTL and an infected hepatocyte, and there are simply not enough killer T cells to kill off every hepatocyte in the liver. And even if there were, killing your liver is the last thing your body would want to do, because it is impossible to live without this vital organ.

So, in the early 1990s he started looking into the possibility that CTLs might be able to coax infected cells into curing themselves without being destroyed. The mechanism of this clearance occupied nearly a decade of Chisari’s time, and a few years ago he and his colleague Luca Guidotti, an Associate Professor at TSRI, demonstrated that the immune system can indeed help cure infected cells and how this “intracellular effector” function may actually be the primary way that the immune system controls HBV infection—something that took most people by surprise.

This unprecedented concept established a new paradigm in our understanding of the host-virus relationship, and like many revolutionary ideas, was initially met with surprise and skepticism. In the past several years, however, it was independently confirmed for HBV and it has been extended to a number of other infections as well.

The Immune System Helps Cells that Help Themselves

Basically, in addition to the direct killing of infected hepatocytes, the activated killer T cell will start to produce and secrete chemicals, called cytokines, that bind to surrounding cells that are also infected and that carry specific markers to which the cytokines bind.

Once these cytokines bind to an infected cell, that binding event activates genes within the infected cell that produce proteins that intercept the lifecycle of the pathogen, leading to an internal elimination of the virus without destroying the cell. In hepatitis, the primary cytokine that drives this process—which also occurs in other cells that are infected with other pathogens—is called interferon-gamma (IFN-g).

In hepatitis, Chisari and Staff Scientist Stefan Wieland in his group demonstrated that the first defense mechanism of INF-g involves interrupting the assembly of the viral RNA and associated proteins into infectious capsids.

“Assembly of [capsid] is very rapidly abolished by signals that are delivered by IFN-g,” says Chisari. In recent years, he has worked to categorize the molecules that are produced by HBV-infected liver cells after they are activated by IFN-g.

One candidate class he and his postdoctoral fellow Michael Robek have found to be upregulated in response to the cytokines are proteins of the proteasome, the cell organelle responsible for degrading protein in the cell’s cytoplasm. Chisari has demonstrated that treating HBV-infected cells with inhibitors of these proteasome proteins blocks the antiviral activity of IFN-g.

Clearance is not limited to this one mechanism. A second, slower mechanism that HBV-infected cells engage after they are turned on by IFN-g is to remove all the viral RNA from the cell by destroying a cellular protein that protects the viral RNA. Without the protection of this cellular protein, the viral RNA is susceptible to ribonuclease enzymes in the cytosol, which destroy it.

Nor is the production of cytokines during such an immune response limited to one type of immune cell. Multiple cells of the immune system, including cytotoxic T lymphocytes, helper T cells, natural killer cells, macrophages, and dendritic cells all release such cytokines. And when these cells are activated to produce IFN-g in the liver, the infection will be cleared.

“We think this is what happens in most of the acute infections in adult patients,” says Chisari. “[Cytokine-induced viral clearance] is the dominant effector force for the control of HBV infection.”

Not every intracellular event involved in this clearance is known, and a large portion of Chisari’s laboratory is busy mapping all the details. Nevertheless, the usefulness of purging the infection while preserving the integrity of the cells is obvious when one compares acutely infected patients to the more serious, chronic cases: acute infections are rapidly controlled by the immune system and chronic infections are not.


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Professor Francis Chisari hopes to find an answer to chronic hepatitis B infections—a condition affecting millions worldwide. (Photo by Alan McPhee.)