High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

Abstract

Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture-produced HCV (HCVcc) and ex vivo-characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid-free subviral LVPs are assembled and secreted by apoB-producing cell lines. To determine whether such subviral particles circulate in HCV-infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low-density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A-purified LVPs contained at least the envelope glycoprotein E2 and E2-specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride-rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 3 10 6 apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid-free, envelope glycoprotein-containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. Conclusion: LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family. (HEPATOLOGY 2012;56:39-48) H epatitis C virus (HCV) is a member of the Flaviviridae family and a major cause of chronic hepatitis often leading to liver cirrhosis and hepatocellular carcinoma. 1 Chronic hepatitis C is a complex disease associated with host metabolic modifications resulting in a unique metabolic syndrome including insulin resistance, liver steatosis, and hypobetalipoproteinemia. 2,3 The most striking link between HCV and lipids resides in the association of HCV particles with lipoproteins. 4,5 Indeed, HCV virions with a density <1.06 g/mL are associated with lipoproteins, thus forming hybrid particles known as lipoviral particles (LVPs). These low-density viral particles are globular, rich in triacylglycerol and total cholesterol (TChol) and contain the viral envelope glycoproteins and nucleocapsid (composed of HCV RNA and core protein). In addition, LVPs contain all the apolipoproteins (apo) that define the triacylglycerolrich lipoproteins (TRLs). Indeed, apolipoprotein (apo) B, apoE, apoCI, apoCII, and apoCIII, all of which characterize very low-density, intermediate-density, and low-density lipoproteins (VLDL, IDL, and LDL,

Ultrastructural organisation of HCV from the bloodstream of infected patients revealed by electron microscopy after specific immunocapture

“…A mixed population composed of two types of particles was also previously observed by Scholtes et al 24 in the low-density fractions of HCV+ patient plasmas. However, the authors purified LVPs from the low density fractions by protein A-mediated precipitation of viral particles naturally coated with patients’ antibodies.…”

“…This result is consistent with the findings of a study in which the group of André23 characterised hybrid particles produced by Huh-7.5 cells transduced with HCV envelope glycoproteins. Transduction with the genes encoding E1 and E2, in the absence of additional viral genes, is sufficient to induce the assembly and secretion of these defective low-density hybrid particles resembling TG-rich lipoproteins (VLDL/LDL) and bearing the viral glycoproteins on their surface 23 24. As suggested by these authors, many defective particles containing only E1 and E2 are likely to circulate in the bloodstream of chronically infected patients.…”

“…Thus, we expected an enrichment of our IC populations with genuine and infectious LVPs and the selection of well folded E1E2 heterodimer carrying lipoproteins. The association of lipoproteins with HCV E1E223 24 may make specific studies of the putative LVP population separately from lipoproteins difficult. Since we captured these lipoproteins using an anti-E2 Ab, they may be similar to low density hybrid particles resembling TG-rich lipoproteins (VLDL, LDL) and bearing the viral glycoproteins on their surface as described by Icard et al 23 In our IC experiments, genuine lipoproteins were visualised using anti-ApoE and anti-ApoB.…”

Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

“…In this respect, in vivo data suggesting a strong association between apoB and HCV have been recently revisited. Scholtes and collaborators demonstrated that LVP could mainly be apoB/gpE1-gpE2 positive, but nucleocapsid-free, particles (85). This new subclass of particles could then be defined as defective subviral particles (named eLVP).…”

“…In fact, it is not excluded that infectious HCV particles could harbor only apoE and not apoB. It remains to be determined whether other types of viral or subviral particles, such as eLVP (85), could be produced by HCV-replicating cells. In this respect the HCV-replicating HepG2 line developed here will be instrumental.…”