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Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines.
A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (ApoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant.
Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. ApoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with ApoE. CONCLUSIONS: Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.
Hepatitis C virus (HCV) is a major causative agent of liver fibrosis, cirrhosis and heptocellular carcinoma. Recently, the first direct-acting antivirals (DAAs) have been approved for use along side the existing standard of care, pegylated interferon (IFN) α and ribavirin. HCV treatment, however, continues to be associated with adverse side effects and variable success rates. Studies of the HCV life cycle and the rational design of DAAs were delayed for many years by difficulties in culturing the virus in the laboratory. The advent of pseudotyped lentiviral particles bearing HCV glycoproteins (HCVpp) and the replicon system allowed initial investigation of entry and replication, respectively, and also provided platforms for screening potential drug compounds. It was not until 2005, however, that the discovery of a unique HCV isolate, termed JFH-1, allowed the complete viral life cycle - from entry to particle assembly - to be recapitulated in cultured cells (reviewed in (1). Since this time, mounting evidence has pointed to a link between HCV entry, replication, and assembly and the biogenesis of host very low-density lipoproteins (VLDL) (2-4). The interplay between HCV and VLDL is emphasized by the existence of very low-density viral particles that can be immunoprecipitated from patient sera with antibodies targeting lipoprotein-associated proteins, notably apolipoproteins (apo) B and E (5). ApoE may promote HCV uptake via its interaction with the low-density lipoprotein receptor (LDLR). The role of specific apolipoproteins in assembly is currently a topic of intense investigation and knockdown approaches have implicated apoA1 (6), apoB (2), and apoE (reviewed in (7)) as potential players in this process.
As the host components required for HCV assembly in human liver cells are discerned, the ability of other cell types and species to produce infectious particles remains an open question. Mouse cells, which are of particular interest to animal model developers, show restrictions in HCV entry and replication; the ability of these cells to support assembly is not known. This question was addressed by Long and colleagues in a recent issue of Gastroenterology (8). Using murine hepatic cell lines, the authors first sought to bypass known roadblocks to HCV life cycle steps preceding assembly. To avoid limited HCV production yielded by transient genome transfection and unwanted structural protein deletions found in selectable genomes, they devised a trans-complementation system to exogenously express HCV core, E1, E2, p7 and NS2 proteins in murine cells harboring subgenomic HCV replicons, which replicate autonomously under antibiotic selection. Limited particle production prompted a comparative transcriptome analysis between naïve mouse cells and those containing HCV replicons that revealed low levels of apoE in the replicon-containing cells. Remarkably, ectopic expression of human or mouse apoE was sufficient to rescue infectious HCV production from mouse cells, yielding infectious titers similar to those observed in the widely used human hepatoma cell line, Huh-7.5. Notably, Long et al. achieved comparable levels of infectious particle production in cells expressing individual human apoE isoforms (apoE2, E3 and E4). This corroborates a recent study by Cun et al. (9) suggesting all isoforms are competent to promote HCV assembly, but contradicts Hishiki et al., who correlated HCV infectivity with isoform affinity for LDLR binding (10). While the reason for this discrepancy is unclear, this emphasizes the difficulty in separating the role of apoE in particle assembly from its role in entry. It is still unclear whether noninfectious particles can be produced by cells lacking apoE or expressing only a single isoform of the protein. The mechanism of apoE function during HCV assembly in mouse or human cells also remains to be determined.
Coaxing HCV assembly in mouse cells adds a new piece to the puzzle in the development of a fully functional rodent model for the virus. HCV has a narrow host range, infecting only humans and chimpanzees, and the lack of a suitable small animal model has limited preclinical testing of drugs and candidate vaccines, as well as hampered mechanistic studies of virus-host interactions. Advances have been made, including murine xenorecipient strains that can be engrafted with human hepatocytes and rendered susceptible to HCV challenge. Liver chimeric mice are, however, a relatively low throughput system with high costs and logistical challenges. Developing an inbred mouse model, by understanding and overcoming each block to interspecies tropism, has been championed as a desirable alternative. At the level of entry, four cellular entry factors are required for HCV uptake - CD81, scavenger type B class I (SCARB1), claudin 1 (CLDN1) and occludin (OCLN) - but only CD81 and OCLN have to be of human origin for entry into murine cell lines (11). This discovery was recently translated into the first inbred mouse model for the early stages of HCV infection (12). RNA replication in mouse hepatoma cells does not seem to be restricted by dominant negative factors (13). In fact, stable, albeit low-level, HCV RNA replication can be established in hepatic and non-hepatic murine cell lines harboring subgenomic or full-length drug selectable replicons (14, 15). These data suggest that all essential cellular factors required for HCV replication are present in mouse cells, but that the viral proteins may not optimally interact with the murine orthologs. An additional limitation to HCV replication in murine cells may relate to antiviral defense mechanisms. For example, the viral protease cleaves critical immune signaling intermediates TRIF and MAVS in humans, but it is not known if this evasion mechanism occurs in mouse cells. Indeed, HCV RNA replication is more efficient in mouse cells lacking immune sensors, such as PKR (16), or transcription factors, such as IRF3 (17).
Long and colleagues provide first evidence that mouse cells can support the late stages for the HCV life cycle, if critical components of the VLDL pathway are present. Expression of either human or mouse apoE dramatically increases packaging efficiency, indicating that apoE is not a species-specific restriction factor. Furthermore, although the murine hepatic cell line reported here was deficient in apoE, primary murine hepatocytes assayed in parallel boasted high expression, suggesting that this host factor will not be limiting in mouse models in vivo. Still, the authors note that additional host factors may be lacking, inhibitory, or incompatible with HCV assembly in primary murine hepatocytes that are not evident in murine hepatic cell lines. This highlights the impact of the cell culture system chosen for analysis and emphasizes that cell lines or in vitro models often do not recapitulate primary cell or in vivo phenotypes. Nonetheless, these important findings by Long et al. shed light on HCV assembly and further raise the hope that an inbred mouse model for HCV infection can be achieved.