By David N. Leff
Today is Black Friday for the hepatitis C virus (HCV). The current issue of Science, dated July 2, 1999, blows HCV's cover two ways.
One way cracks a key corner of the conundrum as to why interferon, the only drug that treats this sinister virus, fails clinically in four out of five patients. The second revelation solves the long-standing inability of virologists to culture HCV in vitro, which frustrates anti-HCV medical strategies and antiviral drug development. HCV is a single-stranded RNA virus.
HCV is accountable for 80 percent of all liver transplants in the U.S, and 8,000 to 10,000 deaths a year. It infects 170 million people worldwide, 4 million of them Americans. (See BioWorld Today, Oct. 30, 1998, p. 1.)
Just as HIV infects cells of the immune system, HCV targets hepatocytes - liver cells. It's to blame for chronic liver disease and cirrhosis, which often precedes lethal hepatocellular carcinoma. But HCV can lie low in its infected liver cells for years, decades, even an entire lifetime, only to flare up long after initial infection. Much of this transmission used to occur from transfusion with contaminated donor blood. Now a leading culprit is the dirty needle of an infected drug addict.
Early last month, 775 scientists gathered at the National Institutes of Health campus, in Bethesda, Md., to attend the Sixth International Symposium on Hepatitis C and Related Viruses. The program included two speakers, molecular virologist Michael Lai from the Howard Hughes Medical Institute at the University of Southern California, in Los Angeles, and molecular biologist Ralf Bartenschlager, from the University of Mainz, Germany.
Lai reported his discovery of the mechanism by which HCV evades the therapeutic effects of interferon; Bartenschlager described his group's development of the first vitro system to lay bare HCV's life cycle. Both presentations figure in back-to-back articles in today's Science.
Lai's paper bears the title: "Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein."
"HCV is the major hepatitis virus in the world today," Lai told BioWorld Today. "It exists in six genotypes [gene variants] - 1 through 6," Lai said. "Genotype 1 is particularly evident in the U.S. and Europe, also in Asia. Of HCV present in the U.S., 75 percent is genotype 1. The only therapy is interferon, though starting last year a second drug, ribovirin, was added. Unfortunately, HCV genotype 1 is very resistant to interferon. Comparing 1 with variants 2 and 3, in other parts of the world, maybe 40 to 50, even 60, percent of patients will respond to interferon. But less than 20 percent of those infected with genotype 1 benefit from interferon treatment. Therapy - has been a major clinical problem.
"This is where our finding comes in," Lai pointed out." To describe that discovery, he first explained how interferon works - when it works. Interferon [INF] is a normal substance in the body. It's an immune-defense molecule, produced in response to virus infection. Because it's a natural substance, physicians and drug companies have taken advantage of this activity, and turned INF into a drug. When you add INF to the body, it will activate a cellular enzyme called PKR - double-stranded, RNA-activated protein kinase. This PKR will not by itself inhibit protein translation, and thereby inhibit virus replication. So INF works by activating PKR to inhibit protein translation. Thus, the virus will be killed.
"But viruses are smart," Lai observed, "and HCV has developed a strategy to counter the effects of INF. So the way HCV works - and that is our finding - one of its viral envelope proteins, the E2 protein, actually has sequence homology with PKR. As a result, that viral E2 can inhibit PKR. So, when you treat patients with INF, that drug cannot activate PKR. Therefore INF loses its activity. So that is how HCV develops resistance to INF.
"I think that our finding solved a major clinical problem," Lai added, "why genotype 1 HCV is so resistant to INF. So that will allow us in the future to develop new drugs, maybe chemicals or peptides, to counter the effects of E2, and allow INF to work better. But E2 is not the only protein that allows HCV to escape from INF. So, obviously, we need to have more research."
From INF Resistance To Cell Culture System
Bartenschlager was singing from the same hymn book. Describing his in vitro system, he told the HCV Symposium - and subsequently BioWorld Today: "For clinical and basic research, what you can do now is study in much more detail the interaction of virus replication and its effect on the host cell. What is much more interesting, I think, is to look at what interferon does. What you can do now is just add interferon to the cell lines, and see how this affects HCV RNA replication. If you find that it really does affect replication, you can now identify in which way this occurs, and which viral proteins are responsible for HCV's resistance or sensitivity to interferon."
In response to "the great response" his presentation got at the symposium, Bartenschlaer's lab at the University of Mainz is now "filling requests for our in vitro system from both academic and industrial researchers."
He recounted how he and his co-authors devised their HCV-scoping system: "From an infected human liver, we cloned an HCV genome, and took a piece of it which we assumed, by analogy to other viruses, would cause HCV RNA replication. We took out this part and substituted it by a neomycin-resistance gene. The sequence we deleted codes for the structural capsid and envelope proteins that make up the virus particle.
"Then, we took this RNA," Bartenschlager continued, "and put it into human hepatoma cell lines. As the RNA replicated, it also propagated the resistance gene. This meant that every cell supporting replication of this RNA would become resistant to the antibiotic. So, in this way we could select from the millions of cells in culture the few cells that support replication of this HCV RNA, because only those cells express this resistance gene, and get resistant to this antibiotic.
"Now we really can look in a cell line," he pointed out, "and see whether a drug is functional or not, whether replication goes down or not. And you can use this system of course to identify new drugs."