By Dean A. Haycock

Special To BioWorld Today

Efforts by the immune system to counter the effects of pathogenic bacteria and viruses often are compared to war. In this familiar and often apt analogy, the burning fever and physical pain that accompany a short-term infection might be compared to a conventional war. But the struggle between the immune system and invading pathogens also has an equivalent to guerrilla war. On the microscopic battlefield, it begins when viruses retreat into cells and hide there, waging a long-term, low-intensity campaign that can lead to defeat for the host as surely as can an all-out ¿hot¿ war.

The viruses that cause AIDS and hepatitis B and C engage in such guerilla warfare. These agents of persistent infection pose serious long-term health risks. Efforts to figure out how these viruses manage to go undercover, evade the immune system, survive and, too often, prevail, may receive a boost with the publication of an article in the Oct. 12, 1999, issue of the Proceedings of the National Academy of Sciences. Titled ¿Inhibitory role of the host apoptogenic gene PKR in the establishment of persistent infection by encephalomyocarditis virus in U937 cells,¿ the paper describes a new model system for studying persistent viral infections. Allan Lau, associate professor of pediatrics at the University of California, San Francisco, and his co-authors have succeeded in converting a normally cell-destroying encephalomyocarditis virus (EMCV) infection into a persistent infection. Such a model offers the opportunity to study the adjustments made by viruses and their host cells in the course of the transition from acute to chronic infection.

The model appears to represent a novel and promising approach for studying persistent infections. Bryan Williams, chairman of the department of cancer biology at the Lerner Research Institute of the Cleveland Clinic Foundation in Ohio, commented on the paper for BioWorld Today: ¿There are some long-established models that have really looked at RNA viruses. They have been around for probably 25 to 30 years now, but the mechanisms by which those models were established were never really thoroughly investigated, at least not using modern technology in terms of recombinant DNA and so on. So it is hard to know the basis of some of those models. Some of these viruses are important human pathogens, particularly the mosquito-borne viruses which have been recently in the news. Some of these can establish persistent infections in mouse cell cultures, but they really haven¿t been studied in a modern context.¿

New Virus Model Lets Cells Survive

Lau told BioWorld Today, ¿In our case, we are using a highly cytolytic virus. Normally, this virus kills all cells it infects. It is an encephalitis and myocarditis virus. It is very potent in most cells. It affects human cells and is a model for studying encephalitis. The fact that we can turn this into quite a relatively benign infection in which the cells survive is, in its own right, unique. In that regard it is the first model of its kind. We are using a highly cytolytic virus and making it become a persistent and chronic infection.¿

Lau and his colleagues were able to turn the EMCV into a persistent infection by repressing the expression in cultured promonocytic U937 cells of an enzyme referred to as PKR, a double-stranded RNA-dependent protein kinase. The protein plays a role in a type of programmed cell death (apoptosis, or ¿cell suicide¿) that cells infected with viruses such as EMCV or treated with tumor necrosis factor often undergo. Such cellular self-sacrifice is the final strategy a cell invokes to limit a viral infection.

¿The link between virus infection and apoptosis has been an intriguing one,¿ Williams said. ¿It wasn¿t known until relatively recently that different viruses can cause this form of programmed cell death. This particular mechanism is addressed in this paper.¿

By decreasing the expression of PKR, the researchers thought they were setting the stage for enhanced viral replication. But, surprisingly, when the cells were prevented from self-destructing, they instead became carriers of the virus. And in time, the EMCV becomes less infectious and the host cells experience changes and grow more slowly.

Repressing PKR Expression May Foster Persistance

Williams says the finding was unexpected. ¿It has been known for some time that this kinase that Dr. Lau has been working on, PKR, is a target for inhibition by different viruses, but it was suspected that the viruses really ablated the antiviral effects of PKR. This allows for a lytic infection. What he has shown here is that by repressing PKR expression ¿ so that is not by knocking it out completely ¿ viruses may be able to establish a persistent infection. I think that is an interesting, different mechanistic twist to the story.¿

The authors suggest that other viruses such as those that cause hepatitis and AIDS may use a similar mechanism to establish persistent infections.

They suggest three practical uses for their model: 1) Identifying and targeting agents used by viruses to inactivate PKR with the hope of stopping persistent infections, 2) Developing a screen to identify naturally occurring antiviral agents, and 3) Testing drugs that activate PKR in combination with a broad-spectrum, multi-subtype interferon with the hope of increasing the effectiveness of antiviral agents.

¿In step one, we are looking at ways to activate the enzyme so it will not allow persistent infection,¿ Lau told BioWorld Today. ¿So far, there is no small molecule to perform this work, to activate PKR directly, but certainly PKR is regulated by interferon. That is why I mention the phrase broad-spectrum, multi-subtype interferon. In the current market, interferon is a single-subtype molecule, but in nature we have at least 12 different kinds of interferon subtypes. So we need a broader spectrum for that.¿

Williams believes the model may be particularly promising for studying HIV infection. He said, ¿As Dr. Lau proposes, if he can establish a persistent HIV infection in a cell culture system, that would be useful for antiretroviral drugs.¿

Collaborations with potential biotech partners are still under discussion, according to Lau, whose research was supported in part by funds supplied by the Rhone-Poulenc Group/Pasteur Merieux Connaught, of France, and the department of pediatrics at the University of California, San Francisco.