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.
Highlights
hepatitis C virus (HCV); TRL, triglyceride-rich lipoproteins; LVP, lipo-viral particle; empty lipo-viral particles (eLVP), empty LVP; DPA, dipicolinic acid; R18, octadecylrhodamine B chloride; TEM, transmission electron microscopy; Z-fFG, carbobenzoxy-D-phenyl-L-phenylglycine; Ab, antibody
Taken together these data demonstrate the concomitant presence of HCV E1 and E2 and of apoB 100 or 48 at the surface of the eLVP produced from E1-E2-transduced Caco-2 cells
Our present study examines the structural details of eLVP and dissects for the first time one of their function, namely membrane fusion. eLVP observed by cryo-TEM appeared as very poorly contrasted spherical objects not delimited by a bilayer and devoid of internal density, closely resembling -lipoproteins visualized by-TEM (38 – 40)
Summary
HCV; TRL, triglyceride-rich lipoproteins; LVP, lipo-viral particle; eLVP, empty LVP; DPA, dipicolinic acid; R18, octadecylrhodamine B chloride; TEM, transmission electron microscopy; Z-fFG, carbobenzoxy-D-phenyl-L-phenylglycine; Ab, antibody. Serum viral particles in density fractions below 1.06 g/ml are associated with triglyceride-rich lipoproteins (TRLs) bearing apolipoprotein B (apoB), i.e. the low, intermediate and very low density lipoproteins (low, intermediate, and very low density lipoproteins, respectively) and chylomicrons [9, 11, 17, 18] Taken together, these data suggest a key role of lipids and/or lipoprotein-associated lipids for productive infection by HCV, which may be related to facilitated virus binding, entry, and/or fusion. These data suggest a key role of lipids and/or lipoprotein-associated lipids for productive infection by HCV, which may be related to facilitated virus binding, entry, and/or fusion These highly infectious low density HCV particles were termed lipo-viral particles (LVPs). Because they contain HCV glycoproteins together with both apoB isoforms (apoB 100 and apoB 48), our results, point to a crucial role of the E1-E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of the triglyceride-rich lipoprotein hybrid particle
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