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), intermediate-density 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.

FIG. 1.

Characterization of HCV RNA-containing particles in very-low- to low-density plasma fractions. (A) Serum and three plasma fractions corresponding to VLDL, IDL, and LDL were prepared from 27 chronically HCV-infected patients. The HCV genotype distribution was as follows: 1 genotype 1a, 15 genotype 1b, 1 genotype 2b, 5 genotype 3a, 5 genotype 4, and 3 ambiguous genotypes. Viremia was calculated as the number of HCV RNA copies per milliliter of serum. Viral loads in fractions were calculated as the number of HCV RNA copies per milligram of protein. The association of HCV RNA with low-density fractions is expressed as an index as described in Materials and Methods. d, density of fraction. (B) Fractions from infected and noninfected plasma samples were analyzed for the presence of immunoglobulins (Ig). Five-microgram quantities of fractions corresponding to VLDL, IDL, and LDL were separated by SDS-10% PAGE and transferred to PVDF membranes for IgG and IgM detection. A positive control experiment was performed by running 200 ng of purified IgG in the gel.

FIG. 2.

Electron microscopy of fractions corresponding to LDL from infected and noninfected patients. (A) LDL fraction depleted of immunoglobulin-positive LVP from an infected donor. (B) LVP purified from the LDL fraction of an infected donor. Protein A-coated magnetic beads are seen as dark granules 10 to 20 nm in diameter. Purified LVP appears as spherical particles whose average diameter is 100 nm (extremes, 50 to 150 nm). (C) Same fraction as in panel B but at a higher magnification (×250,000), showing the internal structures. (D) Ether-butanol-treated purified LVP adsorbed on grids and stained with phosphotungstic acid. Capsid-like particles 25 to 35 nm in diameter can be seen.

FIG. 3.

Immunodetection of HCV core protein in purified and delipidated LVP. (A) Control immunoelectron microscopy with an irrelevant primary monoclonal antibody and a gold-labeled secondary antibody. No significant labeled antibody binding occurred. (B) Immunoelectron microscopy of Tween 80-treated LVP with anti-HCV core protein monoclonal antibody 19D9D6 and a 10-nm-gold-labeled secondary antibody. The binding of core protein-specific antibody on the particles can be seen. (C) Western blot of purified and delipidated LVP. Ether-butanol-treated LVP corresponding to 3 × 10⁶ HCV RNA copies (lane a), HepG2 cells (negative control) (lane b), and HepG2 cells stably transfected with HCV core cDNA (positive control) (lane c) are shown. Detection of HCV core protein was performed with monoclonal antibody 19D9D6.

FIG. 4.

Cell association and uptake of purified LVP. (A) A total of 50,000 copies of HCV RNA from serum, fraction (fract.) 1 (whole fraction containing lipoproteins and LVP), fraction 2 (whole fraction depleted of immunoglobulin-positive LVP but containing lipoproteins and remaining immunoglobulin-negative LVP), and purified (pur.) LVP were incubated with PLC cells (50,000 cells/well). Cell association was quantified after 3 h of incubation and extensive washing and expressed as HCV RNA copy number per well. Data are representative of three independent experiments. (B and C) Purified LVP (50,000 HCV RNA copies) were coincubated with increasing amount of VLDL from a noninfected donor (B) or with increasing ratios of protein A-coated magnetic beads saturated with purified human IgG to purified LVP (C). Quantitation of cell-associated HCV RNA was performed after 3 h of incubation. ip, immunopurified LVP. Error bars indicated standard deviations.

FIG. 5.

Kinetics of internalization of immunoglobulin-positive purified LPV. (A) After 3 h of incubation at 37°C with purified LVP and extensive washing, PLC cells were subjected to suramin treatment at 4°C. The number of HCV RNA copies after suramin treatment is representative of internalized purified LVP. As uptake does not occur at 4°C, the HCV RNA copy number remaining associated with cells following incubation at 4°C and subsequent suramin treatment is indicative of the efficiency of suramin. The efficiency of suramin treatment was greater than 95% (data not shown). Data are expressed as HCV RNA copy number per microgram of protein. (B and C) Purified LVP were incubated with PLC cells for various periods of time before suramin treatment and quantitation of internalized HCV RNA. (B) Incubation with 50,000 copies of HCV RNA from purified LVP. (C) Incubation with 200,000 copies of HCV RNA from purified LVP. Data are representative of three independent experiments. Error bars indicated standard deviations.

FIG. 6.

Endocytosis pathway for purified LVP. (A) A total of 200,000 copies of HCV RNA from purified LVP were incubated for 3 h with 50,000 HepG2 cells grown for 24 h in LPDS or FCS. Internalized HCV RNA was calculated after suramin treatment. (B) A total of 500,000 copies of HCV RNA from purified LVP were incubated with 20,000 N1 or FH fibroblasts for 3 h before suramin treatment and quantitation of internalized HCV RNA. Error bars indicated standard deviations.

FIG. 7.

Characterization of LVP binding to the LDL receptor. Purified LVP (6.6 × 10⁶ HCV RNA copies) were incubated for 1 h with 200 μg of purified human IgG/ml. A total of 300,000 HCV RNA copies were diluted in FCS-free medium supplemented with 0.2% BSA and 50 μg of each anti-ApoB or anti-ApoE monoclonal antibody/ml alone or together. The samples were allowed to stand at room temperature for 1 h with rocking. The samples were then incubated for 45 min on 40,000 HepG2 cells that had been grown for 24 h in medium supplemented with LPDS. Quantitation of cell-associated HCV RNA was then performed. Monoclonal antibodies were 4G3 (anti-ApoB), 5E11 (anti-ApoB), and 1D7 (anti-ApoE). The P value for a comparison of results obtained with the control and the pool of antibodies was <0.02.