| |
Complete Replication of Hepatitis C Virus in Cell Culture
|
| |
| |
The researcher and co-discoverer Charles Rice (Rockefeller University, NYC) of this new cell culture system says in his article below, which follows Cohen’s review:
".......A major obstacle to understanding the virus life cycle and to developing improved therapeutics is the inability to efficiently grow HCV in cel culture......There is an urgent need for improved HCV drug therapies .......In summary, we describe a full-length genotype 2a HCV genome that replicates and produces virus particles that are infectious in cell culture. This system lays a foundation for future in vitro studies to examine new aspects of the virus life cycle and to develop new drugs for combating HCV..... , we refer to this cell culture-produced virus as HCVcc .....Dose-response experiments showed that IFN_ inhibited HCVcc RNA accumulation in infected cells....... BILN 2061 (hep c protease inhibitor), SCH6, and PI-1 all inhibited HCVcc RNA accumulation.... In addition, a nucleoside analog inhibitor of the NS5B RNA polymerase, 2'Cmethyladenosine (24), was found to inhibit HCVcc replication..... Thus, HCVcc infection can be inhibited by IFN_ and several HCV-specific antiviral compounds. The specificity of these latter compounds further shows that HCVcc infection leads to authentic replication in target cells, and demonstrates that this infectious system may be useful for testing current and future antiviral compounds."
Culture Systems for Hepatitis C Virus in Sight at Last
Jon Cohen
The inability to grow this widespread pathogen in laboratories has delayed the development of more-effective drugs and a vaccine.
Since the discovery of hepatitis C virus (HCV) in 1988, researchers have made remarkable headway against this liver-destroying pathogen, which infects a staggering 170 million people around the world. Scientists have delineated in fine detail the interaction between HCV, the liver cells it targets, and the immune system. In many people, the drug cocktail of interferon-alpha and ribavirin eliminates the virus. Studies have also clarified that there are six major genetic families of HCV and that current treatments work better against some so-called genotypes than others. But a fundamental roadblock has stymied scientific progress: HCV has stubbornly refused to grow in laboratory cell cultures--until now.
Two labs using overlapping but unique approaches this week published evidence online of cell culture systems that can grow relatively high levels of HCV; the virus, in turn, can establish new infections.
HCV researchers are ecstatic. "This is really a great advance," says Michael Gale Jr., who studies HCV at the University of Texas Southwestern Medical Center in Dallas. Gale notes that the new culture systems will finally enable researchers to study critical aspects of the viral life cycle such as cell entry, replication, and packaging into new virus particles, "each of which presents a novel drug target," says Gale. More precise targets, in turn, may yield more effective drugs than interferon-alpha and ribavirin, which work by unclear, nonspecific mechanisms and fail in a substantial fraction of patients. They are expensive, too, and require a year of injections, so they are of little use in developing countries, where the disease is most prevalent. The culture systems also promise a better way to judge the power of experimental vaccines.
This accomplishment is the result of a friendly but fierce competition between the two groups--one led by Frank Chisari of the Scripps Research Institute in La Jolla, California, and the other by Charles Rice of Rockefeller University in New York City--who shared many critical materials and ended up crossing the finish line neck and neck. As the Chisari team describes in the 6 June Proceedings of the National Academy of Sciences (PNAS) Early Edition, it designed the culture system by using an unusual isolate of HCV and manipulating an immortalized liver cell line. And Rice's group reports online in Science this week (www.sciencemag.org/cgi/content/abstract/1114016) similar results achieved by exploiting the same HCV isolate and a closely related cell line.
Six years ago, Ralf Bartenschlager and colleagues at the Johannes-Gutenberg University in Mainz, Germany, laid the foundation for an HCV culture system in a report in Science. Bartenschlager's group engineered a small stretch of RNA that codes only for HCV's nonstructural proteins; specifically, the enzymes the virus uses to replicate. This "replicon"--so named because it carries all the information needed to copy itself--replicated to high levels when researchers artificially infected, or transfected, a cell line. But the system had two serious limitations: The replicon replicated efficiently in very few cells in culture, and it could not create a whole, infectious version of HCV.
Bartenschlager (now at the University of Heidelberg) and, separately, Rice recognized that high-level replication might occur only in replicons that mutated and adapted to the cells. Both groups then demonstrated that by introducing mutations into the replicon, they indeed could vastly increase replication efficiency.
Rice and co-workers further increased efficiency by improving the cell line. The replicons apparently only copied themselves in a subpopulation of the cells in culture. So the researchers transfected cells, identified the subpopulation that best supported the replicon, and then used interferon- to eliminate it. By "curing" the cultured cells, they created a new, optimized cell line.
All the researchers needed next was to engineer a complete viral RNA that used the replicon with the adapted mutations as its backbone. "We hit the wall," says Rice. These engineered HCVs replicated in the improved cell lines and also made proteins, but they did not form a new virus that could establish infections. For unknown reasons, the adaptive mutations in the nonstructural genes reduced the infectiousness of the complete virus.
The race took an odd twist in 2001. Takaji Wakita of the Tokyo Metropolitan Institute for Neuroscience in Japan and his colleagues published a report in the Journal of Medical Virology describing an intriguing HCV isolate from an HCV-infected patient who developed fulminant hepatitis and then, oddly, cleared the virus. Wakita's group soon showed that a replicon made from the nonstructural regions of this virus worked in cell lines about as well as replicons that had adaptive mutations. Both the Chisari and Rice labs owe much of their success to clones of Wakita's isolate. "Without his clone, nothing would have happened," says Chisari.
Wakita first teamed up with Bartenschlager, but they had little success. Rice decided to create a chimeric virus that used Wakita's replicon for the nonstructural region and RNA from a different HCV from the same genotype for the rest of the genome. As Rice and co-workers show in their Science Express report, this chimeric virus produces high levels of infectious virus when transfected into the optimized cell line they had made earlier.
Chisari collaborated directly with Wakita and used a complete clone of the virus isolated from the patient. "There's something special about that clone," Chisari says. Rice also supplied Chisari with the optimized cell line, which Chisari's group attempted to improve one more time by transfecting and curing it again. In PNAS Early Edition, Chisari, Wakita, and co-workers report that when the clone was put into this cell line, it produced roughly equivalent levels of infectious HCV as did Rice's chimera.
What is it that makes Wakita's isolate so special? "That's the main question in our laboratory," says Wakita, who has another paper in press at Nature Medicine that describes how he and Bartenschlager ultimately succeeded, too. The secret must reside in the nonstructural region of that isolate, the only common part of the viruses used by each lab, says hepatitis researcher Robert Purcell of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland. "Eventually that will be sorted out because each gene will be sorted out," says Purcell, who plans to put Wakita's clone into chimpanzees to better understand the relationship between the virus in vitro and in vivo. (Rice also plans to test his chimeric virus in chimps.)
A key limitation of the current advance is that both Wakita's clone and Rice's chimera only contain RNA from one of HCV's six genotypes, which are geographically distributed around the world. "I think this is the long-sought-after culture system, but it's far from as good as we would like it to be," says Chisari. "That's the next frontier," agrees Rice. But for the time being, Chisari, Rice, and other hepatitis researchers at the front of the pack see far more opportunities than limitations.
Complete Replication of Hepatitis C Virus in Cell Culture
Science Magazine June 9 2005
Brett D. Lindenbach 1, Matthew J. Evans 1, Andrew J. Syder 1, Benno Wolk 1, Timothy L. Tellinghuisen 1, Christopher C. Liu 2, Toshiaki Maruyama 3, Richard O. Hynes 2, Dennis R. Burton 4, Jane A. McKeating 5, Charles M. Rice 1*
1 Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
2 Howard Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Present address: Alexion Antibody Technologies, San Diego, CA 92121, USA.
4 Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
5 Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA; Present address: Division of Immunity and Infection, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, UK.
Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 105 infectious units/ml within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-{alpha} and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.
Hepatitis C virus (HCV) is a major cause of chronic liver disease, with over 170 million persistently infected individuals worldwide (1). HCV-associated liver disease frequently progresses to cirrhosis, which can lead to liver failure and hepatocellular carcinoma. Current drug therapies are often poorly tolerated and effective in only a fraction of patients; there is no vaccine for HCV. A major obstacle to understanding the virus life cycle and to developing improved
therapeutics is the inability to efficiently grow HCV in cel culture.
HCV is an enveloped, positive-sense RNA virus of the family Flaviviridae. Naturally occurring variants of HCV are classified into 6 major genotypes. The 9.6 kilobase genome encodes one large polyprotein that is processed by viral and cellular proteinases to produce the virion structural proteins (core and glycoproteins E1 and E2) as well as nonstructural (NS) proteins (p7 through NS5B) (Fig. 1A). Subgenomic RNA replicons have been adapted for efficient RNA replication in the human hepatoma line Huh-7 and other cultured cells (2-5). However, full-length genomes containing cell culture-adaptive mutations do not produce infectious virus particles in culture and are severely attenuated in vivo (6-8). This led us to hypothesize that mutations that enhance RNA replication may have deleterious effects on virion production. To test this idea, we utilized a genotype 2a subgenomic replicon, SGR-JFH1, that efficiently
replicates in cell culture without adaptive mutations (4). Fulllength chimeric genomes were constructed by using the core-NS2 gene regions from the infectious J6 (genotype 2a) and H77 (genotype 1a) virus strains. Both full-length
chimeras and the subgenomic RNA were competent for RNA replication, as seen by the accumulation of NS5A protein and viral RNA 48 hours after RNA transfection into the Huh-7.5 cell line. As expected, mutation (GND) of the
NS5B RNA polymerase active site destroyed the ability of FL-J6/JFH to replicate. Within transfected cells, both full-length genomes expressed core, E2, and NS5A. As expected, SGR-JFH1 expressed NS5A but not core or E2. While =30% of cells were productively transfected with FL-J6/JFH, FL-H77/JFH, or SGR-JFH1 RNA, >95% of FL-J6/JFH-transfected cells were positive for NS5A
by 96 hours (fig. S1). This suggested that FL-J6/JFH spread within the transfected cell cultures.
To test whether infectivity could be transferred to naive cells, we clarified conditioned media from these cultures by centrifugation (9), filtered the supernatants (0.2 mm), and incubated them with naive Huh-7.5 cells. NS5A expression could be transferred by the FL-J6/JFH-transfected culture
media, but not by media from cells transfected with FLH77/JFH or SGR-JFH1. Interestingly, the amount of FL-J6/JFH RNA released into the transfected cell culture media exceeded that of the other RNAs by a factor of >200, and only FL-J6/JFH produced an extracellular form of core. Given that the infectivity of the
genotype 2a chimera is filterable and is associated with the release of HCV RNA and core protein, we refer to this cell culture-produced virus as HCVcc. The ability of the genotype 1a/2a chimera to replicate but not spread suggests that
interactions between the structural and nonstructural gene products may be important for HCVcc formation, as has been observed for other members of this virus family (10, 11).
Limiting dilution assays for NS5A expression in electroporated cells showed that 30.3 ± 9.5% (n=6) of cells were productively transfected with FL-J6/JFH, and of these, 55% produced infectivity that was detectable upon transfer to naive cells. Thus, FL-J6/JFH RNA transcripts were highly infectious and formation of HCVcc did not depend on the emergence of rare variants. Limiting dilution assays were also used to quantitate the amount of HCVcc infectivity between samples as median tissue culture infectious units per ml (TCID50/ml). The TCID50 is the dilution that infects 50% of replicate cell cultures (9). Following an eclipse phase (≥9 hours), FL-J6/JFH infectivity could be detected in the media by 18 hours post-transfection, and it continued to accumulate until 48 hours. Interestingly, FL-J6/JFH (H2476L), which contained a weakly adaptive mutation in NS5B (4), showed slightly delayed growth kinetics but also peaked to similar levels by 48 hours. Viruses could be serially passaged, infecting 50-90% of cells within 5 days after two rounds of passage at low multiplicity of infection (MOI of 0.1-1.0). The expression and subcellular localization of core, E2, and NS5A within FL-J6/JFH-infected cells was shown to be consistent with what was previously seen in subgenomic replicon-bearing cells (supporting online text). Taken together, these measurements show that HCVcc replication is
robust and occurs with kinetics similar to those of other Flaviviridae.
A classic method in virus identification is to neutralize the suspected virus with specific antisera. As shown in Fig. 2A, an E2-specific human monoclonal antibody neutralized HCVcc infectivity in a dose-dependent manner, while an
isotype-matched control antibody had no effect on HCVcc titer. These data affirm the viral nature of HCVcc infectivity and show that E2 is essential for virus entry.
HCV E2 has been shown to bind to the cellular surface protein CD81 (12), which is an essential coreceptor for the entry of HCV glycoprotein-pseudotyped retroviruses (HCVpp) (13-15). We found that HCVcc infectivity could be
blocked with a soluble recombinant form of the CD81 large extracellular loop. To further examine the role of CD81 in virus entry we utilized HepG2 cells, which lack CD81 expression but are capable of supporting HCV RNA replication (3). As seen in Fig. 2C, normal HepG2 cells were not infected by FL-J6/JFH, while CD81-expressing HepG2 cells were infected under the same conditions, albeit with reduced efficiency (=850-fold less than Huh-7.5 cells). These data confirm that interactions between E2 and CD81 are important for HCV entry.
The physical nature of HCV particles has been difficult to study in the absence of an infectious culture system. In density gradients, clinical isolates of HCV exhibit a broad distribution and unusually low buoyant densities [reviewed in
(16)]. These properties have been partly explained by the interaction of HCV with serum components such as immunoglobulins and B-lipoproteins. We examined the profiles of RNA and infectivity associated with HCVcc particles by equilibrium sedimentation through 10-40% iodixanol, an iso-osmotic gradient material. A series of controls confirmed that this method accurately measured the buoyant density of HCVcc (supporting online text). HCV RNA was broadly distributed through the top of the gradient, with a peak in fractions 16 and 17 (1.13-1.14 g/ml) and was not found beyond fraction 20 (1.18 g/ml). HCVcc infectivity was also broadly distributed among fractions 1-15 (1.01-1.12 g/ml), and infectivity was not seen beyond fraction 18 (1.17 g/ml). Surprisingly, fractions 16 and 17, which
contained the highest levels of HCV RNA, had little infectivity associated with them. The specific infectivity of a virus preparation relates the amount of infectivity to the total number of virus particles or genomes in the preparation.
Interestingly, a plot of HCVcc specific infectivity vs. buoyant density indicates that the most infectious material is at 1.09-1.10 g/ml, which is similar to the peak of infectivity (1.09-1.11 g/ml) previously seen in chimpanzees (17). In
contrast, RNA-containing material with a buoyant density of 1.14 g/ml (fraction 17) had a low specific infectivity, with =300,000 RNA molecules per infectious unit. Many groups have reported an HCV RNA peak near this density (18-20),
although infectivity could not be assessed. Thus, HCVcc exhibits physical properties similar to what has been previously described for natural isolates of HCV.
There is an urgent need for improved HCV drug therapies. The current standard treatment, pegylated interferon-alpha (IFN_) and ribavirin, leads to a sustained response in only =50% of genotype 1-infected patients. We examined the
ability of HCVcc replication to be inhibited by IFN_ and other antiviral compounds. Dose-response experiments showed that IFN_ inhibited HCVcc RNA accumulation in infected cells with a median effective concentration (EC50) of 1 IU/ml. We also tested three HCV-specific inhibitors of the NS3 serine protease for their effects on HCVcc infection. As seen in Figs. 4B-D, BILN 2061 (21), SCH6 (22), and PI-1 (23) all inhibited HCVcc RNA accumulation in the sub-mM range. In addition, a nucleoside analog inhibitor of the NS5B RNA polymerase, 2'Cmethyladenosine (24), was found to inhibit HCVcc replication in the low nM range. Thus, HCVcc infection can be inhibited by IFN_ and several HCV-specific antiviral compounds. The specificity of these latter
compounds further shows that HCVcc infection leads to authentic replication in target cells, and demonstrates that this infectious system may be useful for testing current and future antiviral compounds.
In summary, we describe a full-length genotype 2a HCV genome that replicates and produces virus particles that are infectious in cell culture. This system lays a foundation for future in vitro studies to examine new aspects of the virus life
cycle and to develop new drugs for combating HCV.
|
|
| |
| |
|
|
|
