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Worldwide, about 3% of the population is infected with HCV, and chronic infection is associated with a risk of serious progressive liver disease. The recent introduction of direct-acting antiviral agents (DAAs) has considerably improved the treatment of chronic HCV infection, and most patients are now cured without major side effects.1 However, subgroups of patients difficult-to-treat and requiring additional therapeutic approaches will probably emerge. Furthermore, DAAs are very expensive, limiting their use in some countries.1 Thus, in the absence of prophylactic vaccines, improvements to our understanding of the mechanisms underlying virus assembly, maturation and secretion from the cell are required, to facilitate the development of new treatment options.
In patients, HCV particles circulate as hybrid particles, known as lipoviroparticles (LVPs), combining components of both HCV and very low-density lipoprotein (VLDL).2 LVPs have a low density (<1.06 g/mL) and are highly infectious. Their maturation and release are tightly associated with VLDL synthesis, leading to cholesterol and triglyceride export from hepatocytes. Similar characteristics have been described for viruses produced in cell culture (HCVcc) by human Huh7.5 hepatoma cells.2
LVPs are rich in triacylglycerol and total cholesterol. They contain the viral RNA, capsid protein, envelope glycoproteins and apolipoproteins. They carry highly variable amounts of lipids and are therefore highly heterogeneous in size and density. LVPs have been shown to associate with apolipoprotein B and apolipoprotein E (ApoE).3 ApoE is a soluble and exchangeable apolipoprotein. It plays a critical role in VLDL assembly and is also involved in cellular lipid transport.2
The importance of ApoE for the life cycle of HCV has been confirmed by studies showing ApoE to be required for LVP production4 ,5 HCV infectivity being associated with the presence of ApoE in sucrose gradients. ApoE knockdown greatly impairs the assembly and release of infectious HCV, whereas ectopic ApoE expression restores virus production. Furthermore, HCV RNA is efficiently immunoprecipitated by an anti-ApoE antibody (Ab), and this same Ab neutralises HCV infection.4 ,5
Following an unknown triggering event, the nucleocapsid is translocated to the endoplasmic reticulum (ER), where it associates with glycoproteins, forming HCV RNA-containing particles. The initial viral structure associates with apolipoproteins along the VLDL maturation pathway, generating highly infectious low-density viral particles, which are secreted via the secretory pathway of the host cell. However, the mechanisms by which ApoE acts in HCV morphogenesis remain poorly understood. Two simultaneously published studies have shed light on the initial contact between HCV components and ApoE.6 ,7 They demonstrated that HCV E1E2 protein and ApoE associate early in the ER. The resulting complex is then detected on the surface of infectious particles. This complex, which forms through protein-protein interactions, initiates the formation of hybrid particles of viral and cellular proteins. The nascent LVP then associates with triacylglycerols/cholesteryl esters along the VLDL pathway in the ER, generating highly infectious, low-density viral particles (figure 1).
Several studies have highlighted the direct role of ApoE in mediating HCV entry.8 ,9 They showed that an anti-ApoE Ab efficiently blocked viral attachment to the target cells. A similar inhibitory effect was obtained with a human ApoE synthetic peptide derived from the ApoE receptor-binding region. In this context, several molecules have been reported to mediate HCV entry. One study from Gale's laboratory described the LDL-receptor (LDL-R) as a cooperative HCV coreceptor mediating viral entry through interaction with ApoE.10 Jiang et al demonstrated that ApoE mediates HCV attachment through specific interactions with cell surface heparan sulfates8 (figure 1).
An understanding of the mechanisms involved in the hijacking of cellular lipid metabolism by HCV is crucial to decipher the life cycle of this pathogen and its ability to evade host immune responses. The group of Catherine Schuster and Thomas Baumert in Strasbourg (France) recently showed that ApoE mediates evasion from HCV-neutralising Ab.11 Functional studies with human monoclonal antiviral antibodies have shown that conformational epitopes of envelope glycoprotein E2 domains B and C are exposed after ApoE deletion from infectious cell-culture HCV. The virion-associated ApoE enables the virus to escape Ab-mediated neutralisation. Interestingly, another study implicated LVP-associated ApoE in interferon sensitivity.12 It showed that a complete early virological response in HCV+ patients was associated with a lower serum ApoE concentration, suggesting that lipid modulation could be used to limit HCV propagation (figure 1).
These studies clearly indicate that ApoE is a direct-acting molecule and is essential for completion of the HCV life cycle. This molecule appears to have multiple functions: facilitating HCV entry by interacting with several potential receptors (LDL-R, heparan sulfate proteoglycan (HSPG)), participating in or promoting HCV morphogenesis and helping the virus escape innate (interferon) and adaptive (neutralising Ab) immune responses. However, the role of extracellular lipid-free ApoE in the HCV life cycle remained unclear. In this issue, the group from Strasbourg uses the HCVcc system in Huh7.5 cells and primary human hepatocytes to determine the role of extracellular soluble ApoE in the early steps of the HCV life cycle and in the lipid metabolism of hepatic cells.13 They show that increasing amounts of ApoE3 result in a dose-dependent decrease in HCV RNA replication in both the subgenomic replicon and HCVcc models. This effect was not dependent on the potential receptors of ApoE described above, LDL-R and HSPG. Nevertheless, ApoE seemed to be taken up by cells, and this uptake was essential for the efficient inhibition of HCV replication. According to Crouchet et al, the cellular membrane lipid rafts may be involved in apolipoprotein uptake. The inhibition of HCV replication also seems to be related to cholesterol efflux under the control of ATP-binding cassette subfamily G member 1 (ABCG1), which has been reported to load cholesterol onto high-density lipoproteins (HDL). This finding led the authors to investigate the production of apolipoprotein A1 (ApoA1), a key molecule for HDL production. They observed a marked increase in intracellular ApoA1 production and extracellular ApoA1-HDL formation. Together, these findings indicate that ApoE3 promotes the formation of ApoA1-HDL, which is then available for cholesterol loading by ABCG1. This mechanism accounts for the inhibition of HCV replication as a consequence of cholesterol depletion13 (figure 1).
Simultaneously, other functions of free extracellular ApoE in the HCV life cycle have been described by Yang et al,14 who demonstrated the importance of ApoE levels for permissiveness to HCV infection. Secreted ApoE-associated lipoprotein specifically enhances infection with HCV LVP. Exchanges of ApoE between LVP and lipoproteins play an important role in maintaining sufficiently high levels of ApoE on LVPs for attachment to the cell surface (figure 1).
HCV is an uncommon virus remarkably well adapted to its host. It subverts most of the components of the VLDL morphogenesis pathway for its own benefit: LVP morphogenesis, entry into target cells and evasion from the immune system. ApoE appears to play a key role in these mechanisms, with all ApoE forms apparently involved. Nascent ApoE in the ER is involved in the initiation of LVP formation, extracellular lipid-free ApoE is involved in viral entry and HCV-associated ApoE facilitates entry and immune system escape. Finally, as demonstrated by Crouchet et al, lipid-free ApoE can modulate HCV replication by regulating hepatic lipid metabolism. Much remains to be done if we are to use ApoE against the virus itself, but deciphering HCV-ApoE interactions might reveal new targets for anti-HCV treatment. Furthermore, ApoE has been reported to affect the life cycles of at least two other hepatic viruses (HBV and HEV). The knowledge gleaned from studies of ApoE-HCV interactions may therefore benefit research on other pathogens.
Contributors J-CM wrote the commentary, JD draw figure 1, PR provided the laboratory infrastructure and edited the commentary.
Competing interests None declared.
Patient consent Obtained.
Provenance and peer review Commissioned; internally peer reviewed.
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