Potential Breakthroughs in Hepatitis C Research

Jules Levin
NATAP
www.natap.org

Three articles in the current Science are being heralded as reporting groundbreaking findings in the development of finding hepatitis C treatments and in understanding why interferon doesn't work well for everyone. Certain genotypes (1a and 1B) may either have a predisposition to not responding well or develop interferon resistance quickly. The 2nd potentially major discovery is the ability of the authors in the 2nd article to duplicate HCV in cell culture. The inability to do this previously severely limited the capacity to screen potential drug candidates.Here are excerpts from the articles in the current Science issue.

First, here's what the Philadelphia Inquirer said about these research findings--

Ever since the virus was identified in 1989, nobody has been able to grow it in the laboratory, a fundamental problem that has hampered the development of effective antiviral drugs and a vaccine to prevent infection.

Scientists still can't grow the virus itself, but German investigators said today that they have been able to genetically engineer a portion of the hepatitis C virus and get it to reproduce in large numbers in human liver-cancer cells in the laboratory.

What is growing in the lab is just a small portion of the genetic material that makes up hepatitis C. But it has an important function: It carries the code for the virus to make certain enzymes it needs to survive.

For that reason, the newly developed cells may give scientists a rapid way of reviewing existing drugs to see if one might attack the virus by blocking those enzymes.

"The cell-culture system we established is a breakthrough in the hepatitis C field and hopefully for the development of efficient therapies," said Ralf Bartenschlager, a virologist at the University of Mainz, who headed the study.

Because scientists have been unable to culture the virus in the laboratory, relatively little has been known about how hepatitis C grows, infects human liver cells, and leads to disease. But the feat announced by the German team could begin to unravel those secrets.

Bartenschlager said his laboratory's next step would be to find a way to engineer a larger portion of the virus so that it more closely mimics what happens during an actual infection with hepatitis C.

In December, the US government licensed the first combination therapy against hepatitis C. It uses interferon plus a drug known as ribavirin. The combination is effective in about 40 percent of patients.

Excerpts from Science--

The similarities between the HCV field today and HIV research in the 1980s are striking. "There's so much you can learn from HIV," says David Thomas, a Johns Hopkins University clinician who studies and treats both viruses. Like their counterparts studying AIDS in the early 1980s, HCV researchers still can't grow the virus in laboratory cultures, and they don't know precisely how it infects a cell. They also have but foggy notions about the timeline between infection and illness, the so-called "natural history" of the disease. Currently available drugs, like early AIDS therapies, have serious toxicities and fail in most people--and no one knows for sure why some people respond to treatment and others do not. Nor have vaccines lived up to early hopes; just like HIV, HCV mutates rapidly, creating a swarm of different viruses in each infected person that can thwart antibodies easily. And, reminiscent of the struggles over patents on AIDS tests, lawyers from companies making diagnostics and drugs are firing salvos at each other over HCV patents.

Chiron Corp., discoverer of the hepatitis C virus, has aggressively defended its HCV-related patents, filing suit against Murex, Organon Teknika, and Hoffmann-La Roche, charging the companies with selling HCV blood tests without paying licensing fees. Last July, it went after four companies involved in HCV drug research. Some researchers say the lawsuits are having a chilling effect on the field. Some companies have stopped their research and other work in the hepatitis C area for fear of lawsuits from Chiron.

HCV is not, of course, HIV. The hepatitis virus does not splice itself into the genes of a host, which means it may be easier to eradicate from a person's body. Indeed, some people become infected for several weeks and then naturally clear HCV from their bloodstream. HCV also does not target and destroy the immune system, and it may not cause clinical symptoms for decades in most of the people who become chronically infected. And, unlike HIV, HCV is rarely transmitted sexually; it seems to require direct blood-to-blood contact. Still, differences aside, HIV holds up an interesting mirror to the young HCV field, where "I don't know" remains the most common answer to a question.

With an estimated 170 million people infected worldwide, hepatitis C virus (HCV) has emerged as a major public health problem. One of the greatest barriers to HCV research has been the lack of a reliable cell culture system for studying viral replication Lohmann et al. (reported in current Science) have produced selectable RNA replicons from the HCV genome and found that these RNAs replicate stabley and to high levels in transfected human hepatoma cells. This experimental system should allow detailed molecular studies of the HCV life cycle and testing of new antiviral therapies. At present, interferon (IFN) is the most common therapy for HCV, but many viral isolates are resistant to it. Taylor et al. (in current Science) show that the HCV envelope protein E2 contains a sequence identical to the phosphorylation sites in the IFN-inducible kinase PKR and the translation initiation factor eIF2a, a target of PKR. In cultured cells, E2 inhibited the kinase activity of PKR and blocked its ability to suppress protein synthesis and cell growth.   Thus, the E2-PKR interaction may be one of the strategies by which HCV circumvents IFN's antiviral function.

This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon.

These study results suggest that the E2 protein of HCV genotype 1 binds to and inhibits PKR in vitro and does so in mammalian cells and yeast because of sequence homology to the PKR and eIF2 phosphorylation sites. These effects correlate with the relative resistance of HCV genotype 1 to IFN. Thus, HCV may have evolved a two-level attack, namely NS5A and E2, on PKR to interfere with IFN action. Although the NS5A-PKR interaction may contribute to development of resistance during IFN therapy, the E2-PKR interaction may account for the intrinsic IFN resistance of HCV genotypes 1a and 1b. The combined effects of NS5A and E2 may explain why most HCV infections become persistent. Resistance to interferon may develop more easily. Another potential outcome of PKR inhibition is the promotion of cell growth, which may contribute to HCV-associated hepatocellular carcinoma.

The PKR-inhibitory activity of HCV E2 protein in mammalian cells is enhanced by the presence of the leader sequence, suggesting that E2 and PKR likely interact with each other at the ER. An ER-resident eIF2 kinase (PERK) has recently been described, which shares homology with PKR and recognizes the same substrate, eIF2. This may provide another target for E2.

The sequences of the PKR-eIF2 phosphorylation homology domain (PePHD) of the more IFN-resistant HCV genotypes (1a and 1b) more closely resemble the sequences of PKR and eIF2 than do the corresponding sequences of the less resistant HCV genotypes (2a, 2b, and 3a) B). This sequence is highly conserved within each genotype.

"The consequence of the genetic diversity of HCV is a virus that has the ability to escape the immune surveillance of its host, leading to a high rate of chronic infections and a lack of protective immunity in individuals exposed repeatedly to the virus," said Robert Purcell, an HCV researcher at the National Institutes of Health

This second article defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.

It's not money, however, that hepatitis C researchers mention when asked what the field needs most. As Frank Chisari, a leading hepatitis immunologist at The Scripps Research Institute in La Jolla, California, puts it, "We desperately need a culture system." To date, no one has been able to grow HCV reliably in a laboratory culture of cells, a lack that has slowed critical studies of everything from drugs to vaccines to basic understanding of the viral life cycle. The show stopper at the recent NIH conference on HCV were the reports in this Scienceissue of a new HCV culture system.

Developed after 5 years of effort by Ralf Bartenschlager and colleagues at the Johannes-Gutenberg University in Mainz, Germany, the culture system does not actually grow HCV itself. Rather, Bartenschlager's group engineered a stretch of DNA that contains the mirror image of a portion of HCV's RNA. Bartenschlager injected this "replicon," which codes for HCV's nonstructural proteins but not its core or surface proteins, into immortalized human cell lines. The replicon then copied itself to high levels, which he showed both by polymerase chain reaction assays and by measuring viral proteins.

"It's a groundbreaking study," says NIDDK's Jake Liang, who with Hoofnagle co-organized the NIH conference. "People have to be cautioned--this is not productive infection. It does not generate virus. Still, it's a major step in the right direction." Stanley Lemon of the University of Texas Medical Branch at Galveston adds that "if these results hold up, they'll be enormously useful for drug screens."

Because the replicon does not produce whole viruses and their attendant envelope proteins, however, researchers cannot use it to determine how HCV infects cells--a critical question that has been frustratingly difficult to answer. As a team led by Sergio Abrignani of Chiron's Siena, Italy, branch reported in the 30 October 1998 Science, one of HCV's surface proteins binds to a cell surface receptor called CD81. But the group did not show that HCV used the receptor to infect cells, and many researchers suspect that it is only part of the story. "CD81 is very intriguing, but no one has proven that it is needed for entry," says Charles Rice, a prominent HCV molecular biologist at Washington University School of Medicine in St. Louis, Missouri.