Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

Abstract

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediatedensity lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

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

“…Quantification of HCV RNA in LVPs indicated that on average, 34% 6 26% of HCV RNA associated with low-density particles circulate as LVP immune complexes as reported. 4 ApoB was detected and quantified via ELISA in all but four patient samples (Fig. 1B).…”

“…Electron microscopy of LVPs revealed large and single particles. 4 Similarly, in vitro-produced apoB and E1E2-positive, nucleocapsid-free particles have two-to three-fold larger diameters than E1E2-negative lipoproteins. 32 In addition, the higher molar ratios of neutral lipids on apoB in LVPs compared with TRLs indicates that such particles are not agglomerates of standard lipoproteins with LVPs.…”

“…LVP purification from normal lipoproteins contained in LDF was performed via protein A immunoprecipitation of the immune complexes that are only found in the LDFs of infected patients as described. 4 Briefly, protein Acoated magnetic beads (Miltenyi Biotec, Paris, France) were incubated with LDFs in phosphate-buffered saline (PBS) with gentle rocking for 30 minutes (20 lL of beads per 1 mL LDF). A total of 2 mL sample were then passed through one magnetic column (Miltenyi Biotec), washed with PBS, and collected in 500 lL PBS.…”

“…Index calculations were determined with apoB included as an internal standard for the lipoprotein compartment as follow: (RNA copy number per milligram of ApoB in LDFs/RNA copy number per milligram of apoB in serum) Â100. 4 Lipid Extraction, Phospholipid, and Triacylglyceride Analyses. Lipid extracts obtained via the Folch procedure 23 from 3 mL of LVPs or from 500 lL of each lipoprotein fraction were separated by thin-layer chromatography (TLC) on Silica Gel G60 plates (Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid (60/40/1, vol/vol) solvents.…”

“…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).…”