New tool for the study of hepatitis C virus genotype 3 and its associated liver disease biology
Authors: Daniel J. Felmlee, Catherine Schuster, Thomas F. Baumert
Journal: Hepatology (2014)

Due to the chronic and typically slowly developing pathogenesis caused by hepatitis C virus (HCV), the population of patients who contracted the virus decades ago portends a coming peak in serious liver disease.1 Since HCV was discovered in 1989, there have been a number of key discoveries that have aided in the development of the current and following generations of direct-acting antivirals (DAAs).2 One of these breakthroughs was the development of the HCV subgenomic replicon that contains the viral proteins that compose the replicase complex and can selectively be sustained in a cultured hepatoma cell line, Huh-7.3 Further optimization of the Huh-7 line for HCV replication resulted in descendant cell lines that are highly capable of sustaining HCV replication.4 Through developing multiple subgenomic replicons, a highly replication-competent isolate was discovered to be capable of sustaining the full HCV life-cycle in cell culture (HCVcc).4-6 These tools were instrumental in advancing the scientific knowledge of HCV and developing the recent and effective panel of specific antiviral agents.2 However, most of the efforts in development were targeted at the particular genotype that was the most prominent in the Western world and most resistant to the traditional standard of care therapies based on interferon alpha and ribavirin, genotype 1 HCV.3, 7 With at least 30% variation between genotypes at the nucleotide level, DAAs discovered by screening genotype 1 HCV subgenomic replicons were less effective against other genotypes.8 This presents a significant problem regarding genotype 3, the second most prevalent genotype in Europe and the most prevalent in the Indian subcontinent. It has long been acknowledged that genotype 3 HCV also has a distinct pathology in associating with hepatic steatosis, an accumulation of lipid droplets in the liver. Furthermore, patients with genotype 3 infection also tend to have hypocholesterolemia and hypobetalipoproteinemia, indicating aberrant lipoprotein metabolism, especially with the cholesterol-laden low-density lipoprotein (LDL).9 While the cause of these lipid abnormalities is not clear, the etiology is almost certainly viral; not only are these pathologies observed associated vastly more prominently with genotype 3 infection, but treatment response also reverses steatosis and hypocholesterolemia. Evidence suggests that some lipid abnormalities may be in part through viral protein interaction with lipoprotein components.10 Importantly, HCV genotype 3 infection is also associated with progression of liver fibrosis to cirrhosis and hepatocellular carcinoma (HCC).11 This is consistent with HCV genotype 3 infection associating with a higher statistical probability of mortality in patients during a patient study period, and at a younger age.12 Furthermore, both the first- and next-generation DAAs appear to be markedly less effective against genotype 3 infection than other genotypes.13 For these reasons HCV genotype 3 has emerged as a priority for future therapy development and understanding of the pathology of fibrosis and steatosis development, and a reliable cell culture model of genotype 3 infection is urgently needed. In this context, Kim et al. in Takaji Wakita's laboratory have published work in this issue of Hepatology detailing their development of a genotype 3 HCV cell-culture system (Fig. 1).14 The work builds on the authors' previous work that identified critical mutations within the HCV genome that facilitated high levels of viral replication in HCV genotype 3 subgenomic replicons after selection in Huh-7 hepatoma cells.15 By engineering these mutations into the full-length genotype 3 HCV isolate S310, the authors discovered that HCV spreading in cell culture was detectable and became even more robust after 12 weeks of further cell culture adaptation. This represents a true breakthrough in having a genuine HCV genotype 3 cell culture model of infection with the final infectivity approximately one-tenth of the classically studied Japanese fulminant hepatitis virus (JFH1).14 The development of the genotype 3 HCVcc system. (A) Schematic of developing a cell culture competent genotype 3 (HCVcc). The consensus sequence from a patient-derived variant with HCV genotype 3 infection was engineered into a subgenomic replicon system with a selectable gentimycin resistant marker (neo). The subgenomic replicon was transfected into Huh-7 cells and selected for G418 resistance. After selection, multiple cell-culture adaptations were identified (*). These mutations were then reengineered into the parental sequence, that after further adaptation formed a robustly infecting virus in cell culture (HCVcc). (B) Genotype 3-infected hepatocytes in vivo have some pathological hallmarks such as microvesicular steatosis. (C) The HCVcc model system appears to mimic some features of the pathology associated with genotype 3 infection. The authors then sought to characterize this newly developed model in comparison to the JFH1 HCVcc. A characteristic hallmark of HCV is its capacity to bind to LDL and other lipoproteins that augment the buoyant density of the virus.9 This results in the peak infectivity of JFH1 being in lower density fractions than the majority of HCV RNA or core protein. In contrast to JFH1, this new genotype 3 model has the infectious peak in sync with HCV RNA and core protein at a density that would suggest limited or no lipoprotein association. This finding suggests that genotype 3 virus may not use lipoproteins in the same way as JFH1, providing a useful tool to probe this interesting aspect of the HCV life cycle. However, CD81 usage appears to be conserved among these genotypes, since antibodies binding this HCV receptor inhibits the infection with both S310 and JFH1 HCVcc potently. Since genotype 3 HCV infection has a well-characterized clinical association with lipid accumulation in the liver, the authors anticipated that some pathological hallmarks might also be observed in the model system. Confocal microscopy analysis revealed that, contrary to the localization of HCV core protein encircling large lipid droplets in JFH1-infected cells, genotype 3-infected cells appeared to have a large proliferation of small lipid droplets with little or no colocalization of core protein. This could reflect the microvesicular steatosis often seen in HCV patients, although the background lipid accumulation of Huh7 cells may make further analysis of this pathological hallmark limited unless using primary cells or less lipogenic cell lines. Finally, the authors tested the current antiviral therapies against the genotype 3 virus in comparison to JFH1 and observed that telaprevir (VX-950) was less effective against genotype 3, consistent with clinical observations.8 The S310 cell culture model developed by Kim et al. represents a significant step forward for a genotype of HCV that has emerged as being both particularly pathogenic and resistant to the current anti-HCV therapies.11 As the HCV research community has gotten progressively closer to a tractable mouse model system,16-18 it will be fascinating to observe how this virus can be used to model genotype specific pathologies in vivo. Since genotype 3 is more robustly associated with HCC than other genotypes, it is possible that this tool may evolve into a model informing general liver cancer research. Furthermore, this technology lays the foundation for screening chemical inhibitors of targeting the complete life cycle of genotype 3 infection, which would effectively close the hole in the armament against HCV infection for a large patient population. We enthusiastically await the next steps against genotype 3 HCV infection that this new model will facilitate. Daniel J. Felmlee, Ph.D.1,2Catherine Schuster, Ph.D.1,2Thomas F. Baumert, M.D.1,2,3 1Inserm, U1110, 67000 Strasbourg, France 2Université de Strasbourg, 67000 Strasbourg, France 3Pôle Hépato-Digestif, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France