Morphological characterization and fusion properties of triglyceride-rich lipoproteins obtained from cells transduced with hepatitis C virus glycoproteins

Abstract

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of beta-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.

FIGURE 1.

Electron microscopy of TRL hybrid particles. Panels A and B, negative staining of TRL hybrid particles produced from E1-E2-transduced Caco-2 cells or nontransduced cells, respectively, is shown. The bar is 100 nm unless otherwise indicated. Panels C and D, TEM grids were coated with the anti-HCV E2 H48 antibody and rinsed, and particles produced from E1-E2-transduced (C) or untransduced Caco-2 cells (D) were added. Panel E, magnetic beads (black arrow) covered with a layer of protein A coated with H48 were incubated with TRL hybrid particles, rinsed, and observed by cryo-TEM; asterisks, TRL and E2-positive particles retained on the magnetic bead. Panels F and G, immunogold labeling of TRL particles from E1-E2-transduced (F) or untransduced Caco-2 cells (G) are shown; particles deposited on TEM grids were immunoassayed for the presence of HCV E1 with the A4 antibody, and a gold-labeled secondary antibody was added. Panels H and I, TEM grids were coated with H48, rinsed, then incubated with E117-E2-positive particles and immunoassayed for human apoB 48 and 100 with the polyclonal antibody K45253G; a gold-labeled secondary antibody was then added. Images show representative objects at high magnification.

FIGURE 2.

Dose-dependent lipid mixing of eLVP produced from Caco-2 cells (A) or Huh-7.5 cells (B). TRL particles from untransduced (open bars) or E1-E2-transduced cells (solid bars) were added at indicated apoB100 concentrations to a cuvette containing phosphatidylcholine:cholesterol:R₁₈ liposomes (see “Materials and Methods”), and lipid mixing was recorded at pH 5.0 over a 10-min period. Initial rates of lipid mixing were calculated from the tangent at the steepest part of the fusion curves and results normalized, with 1 representing the basal lipid mixing rate measured for the lowest concentration of apoB tested (75 ng/ml).

FIGURE 3.

pH-dependent lipid mixing of eLVP produced from Caco-2 cells (A) or Huh-7.5 cells (B). TRL particles (350 ng/ml apoB concentration) from untransduced (open bars) or E1-E2-transduced cells (solid bars) were added to a cuvette containing phosphatidylcholine:cholesterol:R₁₈ liposomes, and lipid mixing was recorded at the indicated pH values. Initial rates of lipid mixing were then normalized, with 1 corresponding to the rate measured at pH 7.4.

FIGURE 4.

Inhibition of eLVP lipid mixing by Z-fFG. Lipid mixing of TRL particles from Caco-2 cells transduced (solid bars) or not (open bars) with HCV E1-E2 was measured as described above in the presence of indicated concentrations of Z-fFG. Initial rates and final extents of lipid mixing were measured as described under “Materials and Methods” and normalized (1 is the value of the considered parameter obtained in the absence of Z-fFG).

FIGURE 5.

Internal contents mixing of eLVP. Tb-loaded and DPA-loaded liposomes were prepared as described under “Materials and Methods.” A 1:1 mixture of liposomes was added to 10 mm Hepes, 150 mm NaCl, 1 mm EDTA buffer, pH 7.4, at 37 °C, and TRL particles were added (final apoB concentration 350 ng/ml). Formation of the Tb·DPA chelation complex, as a measure of internal contents mixing, leads to a fluorescence increase (λ_exc 276 nm; λ_em 545 nm). Panel A, contents mixing of TRL particles produced from HCV E1-E2-transduced Caco-2 cells is shown; green, pH 7.4; blue, pH 6.5; yellow, pH 5.5; magenta, pH 4.5; black and red curves, Tb- and DPA-loaded liposomes without particles, at pH 7.4 and 5.0, respectively. Panel B, contents mixing of TRL particles devoid of HCV glycoproteins. The color code is as described in panel A. AU, absorbance units.

FIGURE 6.

eLVP lipid mixing in the presence of monoclonal antibodies. TRL particles produced in Caco-2 cells and positive for HCV E1-E2 (panel A) or not (panel B) were incubated for 20 min on ice in buffer at pH 7.4, with the indicated amounts of monoclonal antibodies against apoB 100 and apoB 48 (1D1), HCV E2 (H48), apoB 100 (5E11), or with an irrelevant (but isotype-matched) antibody against measles virus hemagglutinin (anti H). The Ab/particles mixture was then added to a 37 °C-thermostated cuvette containing R₁₈-labeled liposomes, and lipid mixing was recorded after acidification as described above.

FIGURE 7.

Inhibition of eLVP lipid mixing by monoclonal antibodies is dependent on pH. TRL particles produced in Caco-2 cells and positive for HCV E1-E2 were incubated for 20 min on ice at indicated pH without (black bars) or with 25 μg/ml of monoclonal antibodies against HCV E2 (H48, green bars), apoB 100 and apoB 48 (1D1, blue bars), apoB 100 (5E11, yellow bars), or with 60 μg/ml of the irrelevant antibody against measles virus H protein (irrelevant, red bars). The Ab/particles mixture was then added to a 37 °C-thermostated cuvette containing R₁₈-labeled liposomes in a buffer at similar pH, and lipid mixing was recorded. Results shown for one representative set of data, obtained from one batch of eLVP (of three tested).