A notable difference between hepatitis C virus (HCV) and the other members of the Flaviviridae family is the absence of a conventional model of free HCV particle in vivo, whereas the structure of the flavivirus virion has been described in detail. HCV has indeed been detected in many heterogeneous forms of diverse buoyant density (d <1.06 to >1.25 g/mL). In the blood of infected patients, HCV presents itself either in “free” forms, complexed with immunogobulins, associated with high-, low- or very low-density lipoproteins, or “encrusted” in β-lipoproteins as so called lipo-viro-particles (LVPs).1–5 Indirect evidence suggests that low-density HCV (i.e., LVPs or other lipoprotein-associated HCV forms) are more infectious than the lipoprotein-free viral forms of higher densities.2, 3 This could be due to neutralization of the viral forms of high densities by immunoglobulins, to protection of low-density HCV against neutralizing antibodies, and/or to lipoprotein-association that stimulates HCV infection. Remarkably, in contrast to the positive modulation of lipoproteins on HCV infection, oxidized forms of lipoproteins may specifically inhibit HCV infection. As reported in this issue, von Hahn and collaborators demonstrate a significant and specific inhibition of HCV (IC50 at ca. 1μg/mL) in infection assays in vitro by oxLDL or oxHDL, most likely induced via their oxidized lipid moieties. The inhibitory effect is conserved across diverse HCV genotypes (genotypes 1a, 1b, 2a) and for all target cells tested. The authors further examined the mechanisms of inhibition and they propose that the oxidized lipoproteins inhibit cell entry by interfering with the scavenger receptor BI (SR-BI), one of the putative receptors of the virus.
The investigation of HCV, its many different forms, and its receptors is a particularly complex task since ex vivo characterization of HCV derived from plasma has proven to be extremely difficult. First, the efficacy of the virus to establish productive infection of primary hepatocytes or hepatoma cell lines is very low; second, the availability of primary hepatocytes and of well-characterized native HCV populations is significantly restricted; third, purification of plasma-derived HCV may alter the properties of the viral particles and/or their association with lipoproteins.4 A significant number of model systems of HCV particles have been developed in an attempt to overcome these severe limitations associated with the study of HCV structure and cell entry.6 They include (1) non-infectious HCV-like particles (HCV-LP),7 (2) “infectious” HCV pseudo-particles derived from vesicular stomatitis virus (VSV)8 or from retroviruses (HCVpp),9, 10 (3) cell culture–grown authentic HCV (HCVcc) that produces infectious virus particles.11–13 HCVpp and HCVcc allow a robust investigation of most functions mediated by the HCV glycoproteins and, hence, may reproduce some cell entry features of native HCV.11–18 Of note, these different HCV model systems are generated in vitro from transformed cell lines, which could preclude some feature of HCV association with lipoprotein metabolism. However, recent results suggest that there may be more similarities than anticipated between current in vitro HCV models and virus purified from patients' plasma. HCV E1E2-targeted antibodies have been detected in patients' blood, or induced in immunized animals, that prevented contamination of patients, or infection of chimpanzees, with plasma-derived HCV19, 20 and that readily neutralized both HCVpp and/or HCVcc,20, 21 (Dreux et al., unpublished observations, 2006), indicating some shared serological and antigenic properties. Moreover, HCVcc with the highest specific infectivity was shown to have a low buoyant density,11 similar to clinical HCV isolates with high infectivity in animals,2 indicating that they may associate with lipoproteins. Finally, several recent reports,15, 17, 18 including that of von Hahn et al.,22 have addressed the fascinating complexity of the interrelation between HCV, its receptors and serum components as reconstituted in vitro, using either HCV infection models.
Cell attachment of flaviviruses generally leads to endocytosis of bound virions, an event required to induce fusion between viral and cell membranes in acidic intracellular vesicles and subsequent initiation of viral replication. As the liver is the major organ known to replicate HCV, the expression pattern of the cellular HCV receptor(s) has been suggested to be liver specific, or dominant within the liver; but this issue will only resolve with the identification of the receptor(s) used by HCV in vivo. A considerable number of receptors have been proposed for HCV, and most of them have already been isolated based on binding studies with soluble recombinant E2 (sE2) or HCV-LPs. Potential receptors include several “capture” molecules that induce concentration of viral particles at the cell surface,6 the human tetraspanin CD81,23 the low-density lipoprotein receptor (LDLr),24 and SR-BI,25 that binds HDL, native or modified LDL and vLDL. Experimental data using either HCVpp or HCVcc have confirmed functional roles for CD81 and SR-BI in HCV entry10, 12, 14 (Dreux et al., unpublished observations, 2006), (Fig. 1A), substantiating previous results that indicated that HCV may directly interact with either receptor via the E1E2 glycoproteins. Furthermore, cell entry of HCV has been shown to occur via endocytosis and membrane fusion to be dependent on acidic pH and cholesterol.10, 14, 16, 26
How do oxidized lipoproteins interfere with HCV entry and what is the significance of this event in vivo? First, it is important to point out that lipoproteins are completely dispensable in infection assays in vitro with HCVpp or HCVcc. Nevertheless, modulation of their infectivity has been reported via their functional interplays with lipoproteins.15, 17, 18 Second, it is highly interesting that redundant and/or overlapping pathways involving direct or indirect interactions with lipoproteins and their receptors have been adopted by HCV to mediate its cellular uptake (Fig. 1). This leads to the stimulating hypothesis that such interactions may facilitate virus uptake and/or infection, as a “Trojan horse” (Fig. 1B-C).
Evidence suggests that the LDL receptor, which recognizes ApoB and ApoE apolipoproteins exposed on lipoproteins, mediates the binding and endocytosis of native HCV particles isolated from patients' blood, most likely via their association with LDL or vLDL.1, 24 Interestingly, the implication of LDLr in HCV entry was corroborated using HCVpp, since apoE-antibodies partially neutralize their infectivity.9 How LDLr subsequently mediates membrane fusion of β-lipoprotein-associated HCV, leading to cytosolic release of HCV core, remains to be clarified since interactions between β-lipoproteins and their receptors are not likely to induce such fusion events by themselves. Furthermore, LDLr directs the lipoproteins to lysosomes where they are disrupted, allowing liberation of their cholesterol content. However, an initial interaction of lipoprotein-associated HCV to receptor does not preclude an involvement of E1E2 and other receptors at a later stage of the cell entry process; i.e., at the level of membrane fusion processes. Thus, cell entry of β-lipoprotein-associated HCV via this route would require endosomal escape before the viral particles are degraded. This may require the engagement of the HCV glycoproteins with their own receptors and their subsequent activation by the low pH environment of endosomes (Fig. 1B). Likewise, cellular uptake of native HCV particles isolated from infected patients seems to be induced by SR-BI27 via an interaction with β-lipoproteins (Fig. 1B). Again, the outcome of this cellular uptake in HCV penetration and replication has not yet been characterized.
As mentioned above, recent evidence using HCVpp, HCV-LP and sE2 suggest that HCV entry exploits SR-BI via a direct interaction with HCV E2.14, 15, 18, 25 This interaction leads to infection and requires both CD81 and HCV E1E2 glycoproteins. It is stimulated by HDL at a post-binding stage but is not in competition with β-lipoproteins; these interact with a different binding site on SR-BI (Fig. 1C). The available data indicate that ApoC1, exposed on HDL, and the hypervariable region 1 (HVR1) of HCV E2 are key components of this infection enhancement pathway, through a mechanism that involves the lipid transfer function of SR-BI.15, 17, 18 As for HCV inhibition by oxidized lipoproteins, which are other high-affinity ligands of SR-BI, their effect may also proceed through interaction with SR-BI. Yet, the results of von Hahn et al. indicate that such oxidized lipoproteins do not inhibit HCV via competition for a cellular binding site, but rather, act at a post-binding step. Due to their similar binding capacities to SR-BI, oxLDL and HCV particles may be concentrated at the same cellular site. Alternatively, the data in this article suggest the possibility that oxLDL may directly interact with the viral particle. Altogether, this may change the biophysical properties of the HCV particle, particularly its lipid membranes, or of neighboring cell membranes, which may have possible negative implications during subsequent membrane fusion steps and merging of the lipid bilayers (Fig. 1D). Additional investigation will be required to clarify through which mechanism oxLDL inhibits HCV infectivity; yet, the striking potency and specificity of the effect will stimulate further studies aiming at therapeutically exploiting this Achilles' heel of HCV and lipoprotein interplay.