By David N. Leff
If a confirmed smoker suddenly feels a strong physical aversion to cigarettes, his contrarian symptom may well denote the onset of hepatitis C virus (HCV) infection. That anti-tobacco reaction is an aspect of anorexia - a loss of appetite for food in general - that's typical of HCV's initial inroad.
According to World Health Organization estimates, some 170 million people worldwide harbor HCV infection - but many of them aren't aware of their viral invader, which in the bodies of 70 percent can lie latent and silent for decades. This 170-million body count is four times greater than that of global HIV infections.
Twelve years ago, when researchers first distinguished hepatitis C from the A and B varieties of the virus, they discovered that the blood-borne C pathogen was infecting large numbers of hemophiliacs. These patients had unwittingly acquired HCV from contaminated blood transfusions - along with HIV, the AIDS virus. Nowadays, donor blood is screened for both viral impurities.
Though less lethal than AIDS, HCV accounts for 80 percent of all liver transplants performed in the U.S. - and 8,000 to 10,000 deaths a year. One percent of the population - over 2 million people - are HCV-positive.
The drug therapy of choice for treating HCV infection is interferon combined with ribavirin - a potent antiviral. But that choice is a narrow one. The combo cures a scant 20 percent. And one in four of the hapless 80 percent develop cirrhosis of the liver - often a penalty of alcoholism - which can go on to liver failure and hepatocellular carcinoma. And unlike hepatitis A and B, there is no vaccine to protect against hepatitis C infection.
Unlocking HCV's Chastity Belt
HCV is a positive-sense, single-stranded RNA virus, 9.6 kilobases in length. Genotypically, it's kin to the yellow fever, dengue fever and West Nile viruses, among others. Just how the hepatitis C virus dodges destruction by the human immune system, while settling in to long-term cellular housekeeping, is HCV's well-kept secret - thus frustrating laboratory research and vaccine development. Its stubborn strategy is to refuse being replicated in vitro. A paper in the current issue of Science, dated Dec. 8, 2000, purports to unlock that viral chastity belt. Its title: "Efficient initiation of HCV RNA replication in cell culture."
The article's senior author is molecular virologist Charles Rice, until recently at Washington University in St. Louis. Its lead author is postdoctoral research associate Keril Blight. These two co-inventors have set up a small, limited-liability company in St. Louis, Apath by name. It will make available their patent-pending replicon system, which they call their "Blazing Blight" technology, aimed at an anti-HCV vaccine some day.
As reported in a "News of the Week" commentary accompanying Rice's paper in Science, "Asked whether Apath would seek restrictions on academics' use of the new technology, Rice said he does not want to do anything that would 'impede academic research.'" Apath, he intimated, may request 30-day prepublication review, and coordination of intellectual property rights.
As Science recounted, he and his co-authors fashioned a virus-like "replicon" that efficiently produces authentic HCV proteins - keys to a vaccine. For openers, they modified earlier work by virologist Ralf Bertonschlager at the University of Mainz, Germany. (See BioWorld Today, July 2, 1999, p. 1.)
The Washington University team hunted in HCV's well-hidden genome for genetic mutations that might enhance the pathogen's productivity. They came up unexpectedly with nine such sequencing spelling errors, clustered in the virus' nonstructural NS5A gene. The protein it expressed conferred increased viral replicative ability - 20-fold, 300-fold and 400-fold - in vitro.
One of the nine mutations in particular - a single amino-acid substitution - increased the viral RNA levels in 10 percent of transfected hepatoma (liver cancer) cells. A 10th alteration was the deletion of 47 amino acids encompassing the HCV's resistance to interferon. Many of the 10 mutant RNAs reproduced thousands of times more effectively in cultured cells than did the unmutated RNA of the native virus.
The co-authors suggest that human liver cells in culture might contain a protein that interacts with part of NS5A to prevent HCV from multiplying. When mutations alter this protein, the interaction fails, and replication proceeds on the pathogen's business unhampered. This concept, they intimate, might lead to a long-sought vaccine target.
A Road To Drug Discovery
Rice recently moved from St. Louis to New York, as head of the Rockefeller University's Laboratory of Virology and Infectious Diseases. "For the first time," he commented in a press statement, "powerful genetic and genomics approaches can be used to unravel the molecular details of hepatitis C virus replication, and its interaction with host cells. We hope that this technology will speed up both fundamental research and drug discovery."
Blight commented, "This is a strong and workable system that we can use to learn how this poorly understood virus causes disease, and to develop drugs against it."
Their paper concludes, "Our findings highlight an important constellation of adaptive mutations in NS5A, identify an ISDR [interferon sensitivity determining region] deletion mutant that may represent a starting point for the design of live, attenuated vaccines, and importantly, establish a robust system for future genetic and functional analyses of HCV replication."