Silvia Vilarinho1 and Richard P. Lifton1,*
Departments of Genetics and Internal Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA
This year’s LaskerDebakey Clinical Medical Research Award honors Ralf Bartenschlager, Charles Rice, and Michael Sofia, pioneers in the development of curative and safe therapies for the 170 million people with hepatitis C virus infection.
The liver plays a major role in energy homeostasis, produces essential plasma proteins, and mediates detoxification and excretion of metabolic products. As a result, loss of liver function is lethal within days. Despite a remarkable capacity for regeneration, continuing insult leads to chronic disease, resulting in replacement of hepatocytes by fibrotic tissue. This results in high venous pressure,
shunting blood through esophageal veins and resulting in varices that are prone to catastrophic rupture, and in fluid accumulation in the abdominal cavity (ascites) which frequently becomes infected. Additionally, loss of metabolite detoxification results in encephalopathy. Causes and Impact of End-Stage Liver Disease The major causes of end-stage liver disease (ESLD) are viral infection with hepatitis B or C and excessive alcohol consumption. ESLD accounts for 700,000 deaths per year world-wide (Xu et al., 2016) and, in the United States (U.S.) alone, results in $4 billion in health care expenditures and $11 billion in lost productivity and quality of life. Liver transplantation can restore hepatic function in suitable patients; the 2012 Lasker Clinical Award recognized Thomas Starzl and Roy Calne for their development of liver transplantation. In the 1970s, the viruses causing hepatitis A and hepatitis B were identified. The development of serologic tests for hepatitis B led to its elimination from transfusion products, and highly efficacious vaccines were developed that prevent infection. Baruch Blumberg received the Nobel Prize in 1976 for his work on hepatitis B, and Maurice Hilleman received the 1983 Lasker Public Service Award for development of the hepatitis B vaccine. Discovery of HCV and Its
Prevalence Serologic testing showed that hepatitis A and B viruses together explained only 50% of viral hepatitis because plasma from patients with non-A, non-B hepatitis, after passage through a fine filter, could transmit hepatitis to chimpanzees. This led to the identification, in 1989, of hepatitis C virus (HCV), an enveloped plus strand RNA virus constituting a distinct genus of Flaviviridae. HCV encodes a single open reading frame of 3,000 amino acids; this primary translation product is processed to produce ten mature proteins, including amino-terminal structural proteins and, distally, proteins required for pro-protein processing and replication (Figure 1).
The discovery of HCV allowed development of screening tests for chronic infection, resulting in elimination of HCV from transfusion products and recognition that 170 million people worldwide, including 3.2 million in the U.S., have chronic HCV infection, resulting in 350,000 deaths per year. HCV is predominantly transmitted via intravenous drug use and re-use of needles in medical care. Following acute infection, which is typically asymptomatic, 75%–85% of subjects develop chronic infection, and 20%–30% of these progress to cirrhosis within 20–30 years. Once cirrhosis is established, each year 1%–5% develop hepatic decompensation (ascites, variceal hemorrhage, and/or hepatic encephalopathy) and/or hepatocellular carcinoma (Hajarizadeh et al., 2013) (Figure 1). In the U.S., HCV is the most common cause of liver transplantation and liver cancer, and HCV causes more deaths annually (20,000) than HIV-1 (12,000). For their discovery of HCV, Michael Houghton at Chiron and Harvey Alter at NIH received the Lasker Clinical Award in 2000. Challenges to HCV Prevention and Treatment Efforts to develop an effective HCV vaccine have not as yet been successful, in part, because HCV is highly heterogeneous, comprising six major genotypes (1 to 6), with numerous subtypes (e.g., 1a, 1b, etc.) and quasispecies. Genotype 1 is the most prevalent, but non-genotype 1 HCV collectively causes more than half of chronic HCV. The scope of the vaccine challenge is underscored by the fact that individuals who have spontaneously cleared HCV infection can be reinfected. These observations, along with the high burden of currently infected subjects who will progress to liver failure, have motivated efforts to develop efficacious therapeutics. Initial therapies were not specifically targeted to HCV. In 1991, the Food and Drug Administration (FDA) approved use of a-interferon, which produced sustained virologic response (SVR; no
detectable virus 12 weeks after therapy, evidence of cure) in only 5%–10% of patients. The addition of ribavirin, a guanosine analog with activity againstmanyRNA and DNA viruses, increased SVR to 40% following 48 weeks of treatment and was approved by the FDA in 2001. Unfortunately, this treatment regimen was poorly tolerated due to significant side effects, such as flu-like symptoms, depression,and bone marrow suppression, requiring dose reduction or drug discontinuation. Moreover, treatment needed to be tailored to specific viral and host genotypes. Despite these limitations, patients cured of infection showed markedly reduced progression of liver failure, need for transplantation, and death (Morgan et al., 2010), providing strong incentive to develop improved therapeutics. Development of Subgenomic HCV Replicons Efforts to develop antivirals specifically targeting HCV gene products were greatly hindered by the inability to propagate virus or replicate the HCV genome in cell culture, requiring low-throughput testing in chimps or humans. The breakthrough overcoming this limitation came from two of this year’s Lasker Awardees, Charles Rice at Rockefeller University
and Ralf Bartenschlager at University of Heildelberg. Considering the lack of infectivity of presumed full-length HCV genomes, Rice noted that the reported 30 end of the viral genome was unusually short, and lacked structural features involved in replication of related viruses. His group purified viral RNA from infected patients, ligated oligonucleotides to the 30 end, and used a complementary primer for cDNA synthesis, followed by PCR to amplify the complete viral 30 end. The results demonstrated a previously missing 98-base extension of the HCV 30 terminus, including a hairpin in the last 50 bases (Kolykhalov et al., 1996). Moreover, this sequence was highly conserved among different HCV genotypes, supporting its functional importance. Similar results were obtained independently by Kunitada Shimotohno of the Chiba Institute of Technology (Tanaka et al., 1996). Rice and colleagues then showed that now full-length HCV transcripts, when injected
into the livers of chimpanzees, produced hepatitis, with seroconversion and production of virus containing RNA identical to the injected RNA (Kolykhalov et al., 1997). This provided formal proof that HCV alone can produce hepatitis and reinvigorated efforts to produce a robust cell culture model of HCV infection. Building upon these efforts, Bartenschlager and colleagues (Lohmann et al., 1999), using methods previously applied to bovine diarrhea virus, produced subgenomic fragments of HCV that excluded genes encoding amino-terminal structural proteins but included a selectable marker linked to viral RNA-dependent RNA polymerase, viral proteases, and NS5A (a gene of then-unknown function). Introduction of these subgenomic constructs into a hepatoma cell line demonstrated their replication, albeit in only 1/106 cells, suggesting that host cell factors inhibited replication. A subsequent study from the Rice lab produced similar sub-genomic replicons from a different HCV genotype (Blight et al., 2000). In a clever extension of this experiment, they isolated clones of cells expressing the selectable marker and sequenced the replicated viral RNA. They found 9 independent clones with mutations that clustered within a 30 codon segment of the viral NS5A gene and a 47 amino acid deletion in a downstream segment of NS5A required for interferon’s ability to inhibit viral replication. They inferred that NS5A plays a role in viral replication and that host gene products inhibit replication via effects on NS5A. Importantly, by introducing NS5A mutations into subgenomic replicons, they dramatically increased the fraction of cells supporting viral RNA replication to >10%.
These subgenomic replicons were highly useful in enabling detailed characterization of aspects of HCV biology. Yet more critically, they provided for the first time the essential substrate for cellbased screening for effective inhibitors of HCV replication, a critical asset for drug development.