The 2020 Nobel Prize in Physiology or Medicine was awarded to three scientists, Harvey J. Alter, Michael Houghton, and Charles M. Rice, for their decisive contributions to the discovery of hepatitis C virus (HCV), whose infection causes liver cirrhosis and liver cancer in chronically infected people around the world. HCV has infected over 170 million people worldwide, and approximately 75% of patients develop chronic infection. There is an estimated anti-HCV antibody prevalence of 0.38% to 3.0% in China, with 1b (62%) and 2a (17%) as the dominant genotypes.[2–4] The discovery of HCV paved the way for blood testing and finding effective treatments, now achieving a cure.
Single-stranded, positive-sense RNA viruses include many medical important pathogens such as the pandemic SARS-CoV-2, hepatitis E virus, human norovirus, and flaviviruses, which share a common replication strategy as HCV. Either active antiviral drugs or robust cell culture and animal models are still lacking for some of these viruses. The experiences in HCV studies and the knowledge of developing direct antiviral agents against HCV may inspire the studies of other difficult-to-culture viruses and antiviral drug development.
The discovery of the HCV
In the 1970 s, Alter and colleagues realized that a large number of transfusion-associated hepatitis cases remained undiagnosed after blood tests for hepatitis A virus and hepatitis B virus. They called the “new” disease non-A, non-B hepatitis. The authors further demonstrated that the blood from these hepatitis patients could transmit the disease to chimpanzees and that the unknown infectious agent had the characteristics of a virus. However, a frustrating period of 14 years followed, during which all traditional techniques for virus hunting were put to use, with no success. Michael Houghton and coworkers discovered HCV in 1989 using exclusively molecular biology techniques. They made a cDNA library of nucleic acids from the blood of an infected chimpanzee. Based on the assumption that some cDNA fragments would be derived from the unknown virus and the patient sera would recognize the virus cDNA-coded peptides, the researchers used patient sera to identify phage-displayed viral proteins and phage-packaged viral cDNA fragments. Following a comprehensive search, they identified one positive clone and finally deciphered a nearly complete genome sequence of the virus, a novel member belonging to the flaviviridae. They named it hepatitis C virus. Charles M. Rice and his colleagues provided a final proof that hepatitis C virus alone could cause hepatitis C. They identified a previously uncharacterized region at the 3’-end of the viral genome, which is now known to be important for viral replication [Figure 1A]. After injection of a genetically engineered viral genome into the liver of chimpanzees, the virus was detected in the blood, and chronic disease with pathological changes resembling those observed in humans was confirmed.
After the discovery of HCV, scientists around the world have performed tremendous work to characterize the molecular features of this virus. Its 9.6-kb positive-sense RNA genome encodes a single open reading frame, which is translated into a polyprotein. The polyprotein is co- and post-translationally processed by host proteinases or the viral protease to produce the viral structural proteins (core, E1, and E2) for virion assembly and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B [Figure 1A] (see for review). NS3 contains a protease domain in the N-terminal region, which uses NS4A as a cofactor, and an RNA helicase/NTPase domain in the C-terminal region. NS4B is a multispanning integral membrane protein, and its oligomerization is required for viral replicase assembly. NS5A has a non-enzymatic activity and plays a central role in viral replicase assembly, as well as in virion production. NS5A has an amphipathic helix at the N-terminus, which was suggested to mediate membrane anchoring and was recently found to regulate polyprotein processing and protein-protein interactions within the replicase. NS5B is the viral RNA-dependent RNA polymerase [Figure 2] (see for a review). NS3, NS4A, NS4B, NS5A, and NS5B assemble the viral replicase on the endoplasmic reticulum, forming double-membrane vesicles.[8,9] Within double-membrane vesicles, the viral genome undergoes amplification and is then assembled into virions. Finally, virions are released, together with host lipoproteins [Figure 1B].
Cell culture and animal models of HCV
As replication of HCV in cultured cells remained difficult, researchers developed a strategy of using a consensus sequence of the viral population to initiate viral replication in cultured cells, usually with cellular adaptive mutations [Figure 1B]. The first subgenomic replicon system used a consensus sequence of the con1b genome. Introducing cellular adaptive mutations significantly enhanced the viral replication in cultured cells. The first infectious clone of HCV was generated by using the 2a genome (JFH1) without any adaptive mutations, and a J6/JFH1 chimeric clone significantly improved the virus titer. Adaptive mutations are usually required for the construction of infectious clones for other genotypes. The innate immune-deficient hepatoma cell line Huh7.5 is highly permissive for HCV replication and is widely used for HCV culturing [Figure 1B]. Currently, infectious clones of limited genotypes are available [Figure 3], and direct culturing of clinical isolates remains extremely challenging. Overexpression of SEC14L2 allows several clinical isolates to replicate in cultured cells, probably through modulation of the host lipid environment and viral replicase assembly.[14,15] Similarly, modification of cellular phosphatidylinositol 4-phosphate favors the replication of unadapted clinical isolates. The cell-cultured virus (HCVcc) is capable of infecting chimpanzees, the only natural animal model for HCV. HCVcc also infects innate immune-deficient humanized mice and immune-competent ICR mice with transgenic overexpression of human receptors CD81 and occludin (OCLN) [Figure 1B].
Direct antiviral agents (DAAs) against HCV
By using a subgenomic replicon system, DAAs are developed, and antiviral efficacy is evaluated. Telaprevir (VX-950) was designed as a covalent and reversible inhibitor of NS3-4A serine protease based on the NS3 crystal structure. Using a subgenomic replicon system, daclatasvir (BMS-790052) was identified by screening as a potent inhibitor of viral replication. Drug resistance mutation analysis revealed that daclatasvir was an inhibitor of non-enzymatic NS5A [Figure 2]. Daclatasvir impairs the formation of double-membrane vesicles, probably by impairing protein-protein interactions within the viral replicase and disrupting the replicase quaternary structure. Sofosbuvir (GS-7977) was developed as a prodrug of 2’-deoxy-2’-fluoro-2’-C-methyluridine monophosphate, which is metabolized to an active uridine triphosphate form, acting as a nucleotide analog and promoting non-obligate chain termination. Sofosbuvir shows pan-genotype in vitro activity. Treatment with combinations of DAA cocktails for 12 to 24weeks cures more than 90% of chronically infected patients. Three scientists, Ralf F.W. Bartenschlager, Charles M. Rice, and Michael J. Sofia, were awarded the 2016 Lasker-DeBakey Clinical Medical Research Award for the HCV replicon system and drug development (http://www.laskerfoundation.org/awards/year/2016/).
Vaccine development for HCV
Despite the tremendous efficacy of DAAs, eradication of HCV infection is unlikely to be achieved without a vaccine. The envelope glycoprotein E2 engages host receptors to mediate virus entry and serves as a main target for neutralizing antibodies [Figure 3]. Vaccine candidates include inactivated virion, E1E2 complex, and E2. An inactivated virion derived from cell-cultured J6/JFH-1 elicits neutralizing antibodies in BALB/c mice. The E1E2746 of HCV 1a complex elicits broad cross-genotype neutralizing antibodies in human volunteers. A trivalent vaccine that is composed of insect cell-produced truncated E2 (soluble E2) from genotype 1a, 1b, and 3a elicits pangenotypic neutralizing antibodies in mice and rhesus macaques. Because of the diversity of HCV genotypes, a successful vaccine candidate should elicit broadly neutralizing antibodies against most genotypes [Figure 3]. Robust HCV infection animal models are still needed for vaccine development.
The complexity and heterogeneity of HCV virions, due to the mixing with lipoprotein and host factors, such as syntenin, hinder the activity of neutralizing antibodies in cell culture models,[32,33] which may also affect neutralizing efficacy in vivo. T-cell immunity against viral non-structural proteins may bypass the obstacles of inefficient antibody neutralization. However, testing of a T-cell vaccine showed no protective effect in a clinical trial.
Conclusion and prospects
Despite the great triumph of HCV cure by DAAs, HCV still remains a challenge for public health because of poor access to DAA treatment, a relatively low rate of diagnosis, the emergence of drug-resistant mutations, etc. However, looking back at the HCV journey, from discovery to cure, and learning the methodology used in HCV studies and the knowledge of developing DAAs against HCV may inspire studies of other difficult-to-culture viruses, such as hepatitis E virus and norovirus, as well as the development of DAAs for other single-stranded, positive-sense RNA viruses, including the pandemic-causing SARS-CoV-2 virus, which shares the common replication strategy of forming a membrane-bound viral replicase.
This study is supported in part by the National Natural Science Foundation of China (No. 81772181, No. 81971926).
Conflicts of Interest
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