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Unformatted text preview: The Unmet Challenges
Some 1.8 percent of the U.S. adult population are infected with the hepatitis C virus, most without knowing it
by Adrian M. Di Bisceglie and Bruce R. Bacon A s recently as the late 1980s few people other than physicians had heard of hepatitis C, a slowly progressing viral infection that over a couple of decades can lead to liver failure or liver cancer. Today the condition is widely recognized as a huge public health concern. Some 1.8 percent of the U.S. adult population, almost four million people, are infected with the hepatitis C virus, most of them without knowing it. The virus is one of the major causes of chronic liver disease, probably accounting for even more cases than excessive alcohol use, and is the most common reason for liver transplants. Some 9,000 people die each year in the U.S. from complications of the infection, a number that is expected to triple by 2010. Information about the incidence of hepatitis C in other countries is less reliable, but it is clear that the virus is a major public health problem throughout the world. Physicians, historians and military leaders have long recognized hepatitis— inﬂammation of the liver— as a cause of jaundice. This yellow discoloration of the whites of the eyes and skin occurs when the liver fails to excrete a pigment called bilirubin, which then accumuThe Unmet Challenges of Hepatitis C Copyright 1999 Scientific American, Inc. of Hepatitis C
lates in the body. In recent decades, however, the diagnosis of hepatitis has progressively improved, and physicians can now distinguish several distinct forms. At least ﬁve different viruses can cause the condition, as can drugs and toxins such as alcohol. Researchers ﬁrst studied viral hepatitis in the 1930s and 1940s in settings where jaundice was common, such as prisons and mental institutions. They identiﬁed two distinct forms with different patterns of transmission. One was transmitted by contact with feces of infected individuals and was called infectious hepatitis, or hepatitis A. The other appeared to be passed only through blood and was termed serum hepatitis, or hepatitis B. An important development occurred in the 1950s, when researchers devised tests for liver injury based on certain enzymes in blood serum. When liver cells— known as hepatocytes— die, they release these enzymes into the circulation, where their concentrations can be easily measured. Elevated serum levels of alanine aminotransferase (ALT) and, especially, aspartate aminotransferase (AST) became recognized as more reliable signs of liver trouble than jaundice. (In addition to hepatitis, some uncommon inherited metabolic diseases can cause elevated liver enzymes.) There things stood until Baruch Blumberg, working at the National Institutes of Health, made a breakthrough in the mid-1960s. Blumberg identiﬁed the signature of a viral agent, now known as hepatitis B virus, in the blood of patients with that disease. Blumberg’s discovery won him a Nobel Prize and allowed researchers to develop reliable blood tests for the virus. A decade later Stephen M. Feinstone, a researcher at the same institution, identiﬁed a different viral agent in the stool of patients with hepatitis A. This work led quickly to the development of tests that accurately detect antibodies to hepatitis A virus in the blood of those infected. Hepatitis had long been a signiﬁcant risk for recipients of blood transfusions and blood products. As many as 30 percent of patients receiving a blood transfusion in the 1960s developed elevated levels of ALT and AST, or even jaundice,
The Unmet Challenges of Hepatitis C some weeks later. Workers had suspected an infectious agent was responsible. When the new tests for hepatitis A and B became available in the 1970s, researchers soon found that a substantial proportion of cases of post-transfusion hepatitis were caused by neither of these two viruses. The new disease was labeled “non-A, non-B” hepatitis. Most investigators expected that the agent responsible for these cases would soon be discovered. In reality, it took nearly 15 years before Michael Houghton and his colleagues at Chiron Corporation, a biotechnology company in Emeryville, Calif., ﬁnally identiﬁed the hepatitis C virus, using samples of serum from infected chimpanzees provided by Daniel W. Bradley of the Centers for Disease Control and Prevention. Hepatitis C accounts for most cases of viral hepatitis that are not types A or B, although a few result from other, rarer viruses. The Needle in an RNA Haystack THE CULPRIT? Electron micrograph shows what is thought to be hepatitis C virus particles inside a cell vesicle. Scientific American October 1999 81 Copyright 1999 Scientific American, Inc. PHOTOGRAPH BY TONY STONE IMAGES; PHOTOMANIPULATION BY JANA BRENNING (opposite page); YOHKO K. SHIMIZU National Institutes of Health (inset) H epatitis C virus proved difﬁcult to identify because it cannot be reliably grown in cell cultures, and chimpanzees and tamarins appear to be the only nonhuman animals that can be infected. Because both species are very expensive to use in research, only small numbers of animals can be employed. These obstacles, which still impede the study of the virus, explain why it was the ﬁrst infectious agent discovered entirely by cloning nucleic acid. The Chiron researchers ﬁrst extracted RNA from serum samples strongly suspected to contain the unknown viral agent. A chemical variant of DNA, RNA is used by many viruses as their genetic material. RNA is also found in healthy cells, so the problem was to identify the tiny fraction corresponding to the unknown viral genome. The Chiron workers used an enzyme to copy multiple fragments of DNA from the RNA, so that each carried some part of its genetic sequence. Next, they inserted this “complementary DNA” into viruslike entities that infect Escherichia coli bacteria, which induced some bacteria to manufacture protein fragments that the DNA encoded. The researchers grew the bacteria to form colonies, or clones, that were then tested for their ability to cause a visible reaction with serum from chimpanzees and a human with non-A, non-B hepatitis. The hope was that antibodies in the serum would bind to any clones producing protein from the infectious agent. Out of a million bacterial clones tested, just one was found that reacted with serum from chimpanzees with the disease but not with serum from the same chimpanzees before they had been infected. The result indicated that this clone contained genetic sequences of the disease agent. Using the clone as a toehold, investigators subsequently characterized the remainder of the virus’s genetic material and developed the ﬁrst diagnostic assay, a test that detects antibodies to hepatitis C in blood. Since 1990 that test and subsequent versions have allowed authorities to screen all blood donated to blood banks for signs of infection. The antibody test soon showed hepatitis C to be a much bigger threat to public health than had generally been recognized. A remarkable feature— one that sets it apart from most other viruses— is its propensity to cause chronic disease. Most other viruses are self-limited: infection with hepatitis A, for example, usually lasts for only a few weeks. In contrast, nearly 90 percent of people with hepatitis C have it for years or decades. Few patients know the source of their virus, but on direct questioning many recall having a blood transfusion, an episode of injection drug use or an injury from a hypodermic needle containing blood from an infected individual. About 40 percent of patients have none of these clear risk factors but fall into one of several categories identiﬁed in epidemiologic studies. These include having had sexual contact with someone with hepatitis, having had more than one sexual partner in the past year, and being of low socioeconomic status. Whether hepatitis C is sexually trans- How the Hepatitis C Virus Was Discovered CHIMPANZEE WITH NON-A, NON-B HEPATITIS RNA EXTRACTED FROM CELLS DNA COPY MADE FROM RNA DNA INCORPORATED INTO BACTERIOPHAGES R mitted is controversial. Instances of transmission between partners in stable, monogamous relationships are rarely identiﬁed, and the rate of infection in promiscuous gay men is no higher than in the population in general. These observations suggest that sexual transmission is uncommon, but they are hard to reconcile with the epidemiologic ﬁndings. The paradox has not been resolved. Some patients who deny injection drug use may be unwilling or unable to recall it. Others might have been infected from unsterile razors or tattooing instruments. Shared straws put into the nose and used to snort street drugs might also transmit the virus via minute amounts of blood. Slow Progress LAURIE GRACE esearchers identified the hepatitis C virus by making DNA copies of RNA from the cells of infected chimpanzees.They cloned the DNA by using bacteriophages to carry it into bacteria.Colonies were then tested with serum from infected chimps. One produced an immune reaction,indicating it carried viral genetic sequences. — A.M.D.and B.R.B. T he discovery of hepatitis C virus and the development of an accurate test for it mark an important victory for public health. The formerly substantial risk of infection from a blood transfusion has been virtually eliminated. Moreover, the FIBROUS TISSUE FIBROUS TISSUE SURROUNDING BILE DUCT AND BLOOD VESSELS
ELIZABETH M. BRUNT Saint Louis University CHRONIC INFLAMMATORY CELLS rate of infection appears to be dropping among injection drug users, although this may be because anti-AIDS campaigns have discouraged sharing of needles. Yet hepatitis C still presents numerous challenges, and the prospects for eradicating the virus altogether appear dismal. Attempts to develop a vaccine have been hampered because even animals that successfully clear the virus from their bodies acquire no immunity to subsequent infection. Moreover, millions of people who are chronically infected are at risk of developing severe liver disease. The mechanism of damage is known in outline. Viral infections can cause injury either because the virus kills cells directly or because the immune system attacks infected cells. Hepatitis C virus causes disease through the second mechanism. The immune system has two operating divisions. The humoral arm, which is responsible for producing antibodies, appears to be largely ineffective against hepatitis C virus. Although it produces antibodies to various viral components, the antibodies fail to neutralize the invader, and their presence does not indicate immunity, as is the case with hepatitis B. It seems likely that hepatitis C virus evades this defense through its high mutation rate, particularly in regions of its genome responsible for the manufacture of proteins on the outside of the virus to which antibodies might bind. Two such hypervariable regions have been identiﬁed within the so-called envelope regions of the genome. As many as six distinct genotypes and many more subtypes of the virus have been identiﬁed; numerous variants exist even within a single patient. In contrast to the humoral arm, the cellular arm of the immune system, which specializes in viral infections, mounts a vigorous defense against hepatitis C. It appears to be responsible for most of the liver injury. Cytotoxic T lymphocytes primed to recognize hepatitis C proteins are found in the circulation and in the liver of chronically infected individuals and are thought to kill hepatocytes that display viral proteins. Fortunately, liver tissue can regenerate well, but that from hepatitis patients often contains numerous dead or dying hepatocytes, as well as chronic inﬂammatory cells such as lymphocytes and monocytes. Long-Term Consequences I f hepatitis persists for long enough— typically some years— the condition escalates, and normally quiescent cells adjacent to hepatocytes, called hepatic stellate cells, become abnormally activated. These cells then secrete collagen and other proteins, which disrupt the ﬁne-scale structure of the liver and slowly impair its ability to process materials. This pathology is known as ﬁbrosis. Stellate cells are similar in origin and function to the ﬁbrosis-producing cells found in other organs, such as ﬁbroblasts in the skin and mesangial cells in the kidney. They store vitamin A as well as produce the liver’s extracellular matrix, or framework. It is likely that many of the processes that initiate the ﬁbrotic response in the liver occur in these other tissues as well. If ﬁbrosis progresses far enough, it results in cirrhosis, which is characterized by bands of ﬁbrosis enclosing nodules of regenerating hepatocytes. Progression is BILE DUCT CIRRHOTIC NODULES LIVER TISSUE from patients with hepatitis C often shows fibrosis— excess collagen (here stained blue).The top image shows typical mild fibrosis.The bottom image shows cirrhosis, a more serious condition in which fibrotic tissue surrounds regenerating nodules of hepatocytes; chronic inflammatory cells are also visible.
The Unmet Challenges of Hepatitis C 82 Scientific American October 1999 Copyright 1999 Scientific American, Inc. BACTERIOPHAGES INFECT E. COLI BACTERIA BACTERIAL COLONIES SEPARATED SERUM FROM CHIMPANZEE WITH NON-A, NON-B HEPATITIS IS ADDED; COLONY CONTAINING VIRAL SEQUENCES PRODUCES VISIBLE REACTION FURTHER STUDIES faster in people over age 50 at the time of infection, in those who consume more than 50 grams of alcohol a day, and in men, but cirrhosis can result even in patients who never drink alcohol. Fibrosis and cirrhosis are generally considered irreversible, although recent ﬁndings cast some doubt on that conclusion. About 20 percent of patients develop cirrhosis over the ﬁrst 20 years of infection. Thereafter some individuals may reach a state of equilibrium without further liver damage, whereas others may continue to experience very slow but progressive ﬁbrosis. End-stage liver disease often manifests itself as jaundice, ascites (accumulation of ﬂuid within the abdomen), bleeding from varicose veins within the esophagus, and confusion. Hepatitis C infection has also come to be recognized as a major indirect cause of primary liver cancer. The virus itself seems not to put people at increased risk, but cirrhosis induced by the virus does. Cirrhosis is responsible for almost all the illness caused by the hepatitis C virus. Although a small proportion of patients recollect an episode of jaundice when they probably acquired their infection, chronic hepatitis C is often asymptomatic. When symptoms do occur, they are nonspeciﬁc: patients sometimes complain of vague feelings of fatigue, nausea or general unwellness. The insidious nature of the condition is probably another reason why hepatitis C remained undiscovered for as long as it did. The disease plays out over decades. An aspect confounding investigators is that not all infected individuals react in the same way. Some may carry the virus for decades without signiﬁcant injury; others experience serious damage within only a few years. Liver transplantation can save some end-stage patients, but the supply of human livers available for transplant is woefully inadequate. Researchers are
The Unmet Challenges of Hepatitis C therefore working intensively to develop treatments that will eradicate the virus in patients. The ﬁrst therapeutic agent shown to be effective was alpha interferon, a protein that occurs naturally in the body. Interferon appears to have a nonspeciﬁc antiviral action and may also enhance immune system activity. The drug is generally given by subcutaneous injection three times a week for 12 months. Only 15 to 20 percent of patients, however, exhibit a sustained response, as deﬁned by the return of ALT and AST to normal levels and the absence of detectable hepatitis C RNA in serum for at least six months after stopping treatment. Why treatment fails in most patients is essentially unknown, although some viral genotypes seem to be more susceptible to interferon than others. Last year the Food and Drug Administration approved another drug, ribavirin, to treat hepatitis C in conjunction with interferon. Ribavirin, which can be swallowed in pill form, inhibits many viruses. Interestingly, though, it appears to have no effect against the hepatitis C virus by itself and is thought somehow to enhance interferon’s effects on the immune system. Interferon and ribavirin given together for six to 12 months can expunge the virus in about 40 percent of patients, and clinical workers are now studying how to maximize the beneﬁts from these two agents. Long-acting forms of interferon that require administration only once a week are one focus of interest. A new drug is now being tested in small numbers of patients. Vertex Pharmaceuticals in Cambridge, Mass., is investigating a compound that inhibits a human enzyme called ionosine monophosphate dehydrogenase. The hepatitis C virus relies on this enzyme to generate constituents of RNA. No results from these trials are yet available. In the absence of medications capable of dependably eliminating the virus, the NIH recently embarked on a study to determine whether long-term administration of alpha interferon can slow liver damage in patients who fail to clear the virus. And we and other researchers are studying the simple expedient of taking a pint of blood from patients on a regular basis. This treatment reduces the amount of iron in the body, a manipulation that can reduce serum ALT and AST levels. Whether it slows liver damage is still uncertain. Targeting the Virus T he best prospects for future treatment for hepatitis C appear to be agents targeted speciﬁcally against the virus, just as successful treatments for HIV target that agent. With that goal in mind, researchers have elucidated the structure of the hepatitis C virus in detail. Its genetic material, or genome, consists of a single strand of RNA. In size and organization the genome is similar to that of yellow fever and dengue fever viruses; hepatitis C virus has therefore been classiﬁed with them as a member of the family Flaviviridae. Enzymes in an infected cell use the viral RNA as a template to produce a single large protein called a polyprotein, which then cleaves to yield a variety of separate proteins with different functions. Some are structural proteins that go to form new viral particles; others are enzymes that replicate the original infecting RNA. At either end of the genome are short stretches of RNA that are not translated into protein. One of these terminal regions seems to prompt infected cells to manufacture the viral polyprotein; it is an important target for diagnostic assays. The other appears to play a role in initiating the replication of viral RNA. The structural proteins include the
83 Scientific American October 1999 Copyright 1999 Scientific American, Inc. How the Hepatitis C Virus Reproduces Itself
CELL MEMBRANE H KEITH KASNOT; SOURCE: CHARLES M. RICE Washington University School of Medicine epatitis C infection starts when viral particles in the circulation find their way to susceptible cells,particularly hepatocytes.A viral protein called E2 appears to facilitate entry by latching onto a specific receptor.On entering, the virus loses its lipid coat and its protein envelope, freeing the RNA cargo. Enzymes in the cell then use this RNA as a template to make a large viral protein,the polyprotein. It is cleaved into a variety of small proteins that go on to form new viral particles and help to copy the viral RNA. The original RNA is copied to yield a “negative-stranded” RNA that carries the inverse, or complement, of the original sequence. This serves as a template to make multiple copies of the original RNA, which are incorporated into new viral particles, along with structural proteins, at a body called the Golgi complex.Complete viral particles are eventually released from the infected cell, after acquiring a lipid surface layer. Recent studies suggest that a patient produces as many as 1,000 billion copies of hepatitis C virus a day, most of them from — A.M.D. and B.R.B. the liver. 1 Virus binds
to receptor, enters cell inclusion 3 RNA directs cell to make polyprotein HUMAN LIVER CELL 2 Viral RNA reaches cell interior core protein, which encloses the RNA in a viral particle within a structure known as the nucleocapsid, and two envelope proteins that coat the nucleocapsid. The nonstructural proteins include a viral protease responsible for cleaving the polyprotein, as well as other enzymes responsible for chemically readying the components of viral RNA (triphosphatase), for copying the RNA (polymerase) and for unwinding the newly manufactured copy (helicase). The protease and helicase enzymes have been well characterized and their detailed three-dimensional structure elucidated through x-ray crystallogra- phy, necessary ﬁrst steps for designing drugs to inhibit an enzyme. Several drug companies, including ScheringPlough, Agouron Pharmaceuticals, and Eli Lilly and Vertex Pharmaceuticals, are now studying potential hepatitis C protease or helicase inhibitors. Clinical trials are probably only a few years away. Another viral enzyme, the polymerase, is also a possible target. Whether the virus will evolve resistance to such agents remains to be seen. Developing anti–hepatitis C therapies may be about to get easier. Three months ago Ralf Bartenschlager and his colleagues at Johannes-Gutenberg Universi- HEPATITIS C VIRUS GENOME consists of a single RNA gene plus two terminal regions. The gene encodes a polyprotein,which subsequently cleaves to form a variety of smaller proteins. Some of these are used to make new virus particles; others are enzymes that help to replicate the viral RNA for inclusion into new viruses.
Core Envelope 1 Envelope 2 Nonstructural 2 Nonstructural 3 Nonstructural 4 ty in Mainz, Germany, published details of an RNA genetic construct that includes the regions coding for the virus’s enzymes and reproduces itself in liver cancer cell lines. This construct may prove valuable for testing drugs targeted at these enzymes. Another possible therapeutic avenue being investigated is disruption of the process that activates hepatic stellate cells and causes them to instigate ﬁbrosis. This mechanism is known to involve cytokines, or signaling chemicals, that cells in the liver called Kupffer cells release when they are stimulated by lymphocytes. Turning this process off once it has started should prevent most of the untoward consequences of hepatitis C infection. Some workers are trying to develop therapeutics aimed at the short terminal regions of the virus’s genome. One idea,
Nonstructural 5 Terminal region GENE PROTEIN PRODUCTS Core protein Envelope proteins Protease/ helicase RNA polymerase 84 Scientific American October 1999 The Unmet Challenges of Hepatitis C Copyright 1999 Scientific American, Inc. LAURIE GRACE Terminal region 8 New viral particles with lipid outer layer are released 4 Polyprotein cleaves 5 Viral enzymes copy
negative-stranded RNA from original GOLGI COMPLEX 7 RNAs gain protein coat as viral precursors 6 Enzymes make multiple copies of original RNA from negative-stranded RNA NUCLEUS being pursued by Ribozyme Pharmaceuticals, is to develop therapeutic molecules that can cut speciﬁc constant sequences there. Ribozymes, short lengths of RNA or a chemical close relative, can accomplish this feat. The main challenge may be getting enough ribozymes into infected cells. Delivering adequate quantities of a therapeutic agent is also a problem for some other innovative treatment concepts, such as gene therapy to make liver cells resistant to infection, “antisense” RNA that can inhibit speciﬁed genes, and engineered proteins that activate a cell’s self-destruct mechanism when they are cleaved by the hepatitis C protease. All these attempts to counter hepatitis C are hampered by a serious shortage of funds for research. The amount of federal support, considering the threat to millions of patients, is relatively small. We are conﬁdent that much improved therapies, and possibly a vaccine, will in time be available. An expanded research program could ensure that these developments come soon enough to help patients SA and those at risk. The Authors
ADRIAN M. DI BISCEGLIE and BRUCE R. BACON are physicians specializing in hepatitis C. Di Bisceglie received his medical training at the University of the Witwatersrand in South Africa. Before joining Saint Louis University School of Medicine as associate chairman of internal medicine, he was head of the liver diseases section at the National Institutes of Health. His research interests include viral hepatitis and primary liver cancer. Bacon is director of the division of gastroenterology and hepatology at Saint Louis University School of Medicine. He completed his medical training at Cleveland Metropolitan General Hospital. His research has focused on iron metabolism in the liver. Both Di Bisceglie and Bacon are associated with the American Liver Foundation: Di Bisceglie as medical director and Bacon as a member of the board of directors. Further Reading
The Crystal Structure of Hepatitis C Virus NS3 Proteinase Reveals a Trypsin-like Fold and a Structural Zinc Binding Site. Robert A. Love et al. in Cell, Vol. 87, No. 2, pages 331–342; October 18, 1996. Management of Hepatitis C. National Institutes of Health Consensus Development Conference Panel Statement. In Hepatology, Vol. 26, Supplement No. 1, pages 2S–10S; 1997. Interferon Alfa-2b Alone or in Combination with Ribavirin as Initial Treatment for Chronic Hepatitis C. John G. McHutchison et al. in New England Journal of Medicine, Vol. 339, No. 21, pages 1485–1492; November 19, 1998. Molecular Characterization of Hepatitis C Virus. Second edition. Karen E. Reed and Charles M. Rice in Hepatitis C Virus. Edited by H. W. Reesink. Karger, Basel, 1998. Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line. V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann and R. Bartenschlager in Science, Vol. 285, pages 110–113; July 2, 1999. The Unmet Challenges of Hepatitis C Scientific American October 1999 85 Copyright 1999 Scientific American, Inc. ...
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