Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients

Abstract

Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, < or =1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.

FIG. 1.

Density distribution of HCV RNA in serum from immunodeficient patient S6 on iodixanol and sucrose gradients. Two identical serum samples with 6 × 10⁶ IU of HCV RNA were applied to either an iodixanol (A) or a sucrose (B) density gradient. The two gradients were centrifuged and harvested under identical conditions. Fractions are numbered from the bottom, dense end of the gradient. HCV RNA in each fraction was detected by RT-PCR and analyzed by agarose gel electrophoresis. The PCR product from each fraction was semiquantified by densitometry, and values are shown as bar graphs. The linearity of each density gradient is shown as a line graph. The peak fraction of HCV RNA in the iodixanol gradient had a density below 1.04 g/ml. Two peak fractions of HCV RNA in the sucrose gradient were in the range below 1.08 g/ml and 1.12 to 16 g/ml.

FIG. 2.

Density distribution of HCV RNA in plasma from three immunocompetent patients using iodixanol gradients. All three patients, S7, S8, and S9, had been diagnosed with chronic HCV infection, and antiviral treatment had not been initiated. (A) Patient S7 had moderate HCV infection, and 100 μl plasma with 2.6 × 10⁵ IU of HCV RNA was applied to the gradient. (B) Patient S8 had mild HCV infection, and 100 μl plasma with 1.6 × 10⁴ IU of HCV RNA was applied to the gradient. (C) Patient S9 had been diagnosed with cirrhosis due to HCV infection, and 100 μl plasma with 1.3 × 10⁴ IU of HCV RNA was applied to the gradient. RNA in each fraction from the gradients was detected by RT-PCR and analyzed by agarose gel electrophoresis. The PCR product was semiquantified by densitometry, and values are shown as bar graphs. The linearity of each density gradient is shown as a line graph.

FIG. 3.

Effect of low-pH treatment on the density distribution of HCV in an iodixanol density gradient. A serum sample from patient S6 with 6 × 10⁶ IU of HCV RNA was incubated with PIPPS buffer, pH 4.0, and applied to an iodixanol density gradient prepared with the same buffer. The distribution of HCV RNA in the gradient was determined by RT-PCR and semiquantitated by densitometry from an agarose gel. A peak of HCV RNA was observed with a density between 1.10 and 1.12 g/ml, and HCV RNA was also observed toward the top of the gradient with a density below 1.08 g/ml.

FIG. 4.

Effects of detergents on the density distribution of HCV RNA in iodixanol gradients. A serum sample from patient S6 with 15 × 10⁶ IU of HCV RNA was applied to an iodixanol density gradient either before treatment (A) or after treatment with 0.1% deoxycholic acid with 400 mM KBr (B), 0.5% deoxycholic acid without NaCl or KBr (C), or 0.18% NP-40 (D). Changes in the density profile of HCV were assessed by the amount of HCV RNA in fractions from the gradient as determined by quantitative real-time PCR. The linearity of the density gradient is shown as a line graph, and the bar graphs show the amount of HCV RNA in each density gradient fraction. Each bar is the mean from two determinations.

FIG. 5.

SDS-PAGE analysis after precipitation of LDL/VLDL from patient S6 serum, using either a polyclonal antibody against human ApoB or heparin/Mn²⁺. Protein samples were applied to a gradient polyacrylamide gel (5) and stained with Coomassie brilliant blue. Molecular mass markers (lane M). Immunoprecipitation of LDL/VLDL from 20 μl serum with a polyclonal antibody against human ApoB (lanes 1 to 4). Serum before immunoprecipitation (lane 1), supernatant (lane 2), and pellet (lane 3) after immunoprecipitation. Lane 4 received only the ApoB antibody, and protein bands at 50 kDa and 25 kDa correspond to the heavy and light chains of rabbit IgG. Precipitation of LDL/VLDL from 20 μl serum with heparin/Mn²⁺ (lanes 5 and 6). The supernatant (lane 5) and pellet (lane 6) after the precipitation were applied to the gel. Purified LDL from normal human plasma collected in the presence of EDTA and protease inhibitors (lanes 7 and 8). The density range of the LDL is between 1.019 and 1.063 g/ml, and two samples with either 2 μg (lane 7) or 10 μg (lane 8) protein were applied to the gel. The position of ApoB-100 with a molecular mass of 540 kDa, as well as two 410-kDa and 140-kDa fragmentation products of ApoB, are indicated with arrows.

FIG. 6.

Western blot analysis of HCV glycoprotein E2 from transplant liver S6b. Samples from the low-density fraction (ρ ≤ 1.06 g/ml) of liver macerate were analyzed either undiluted (lane 1), diluted 1:2 (lane 2), or diluted 1:4 (lane 3). The Western blot was immunostained using mouse MAb AP33 against HCV E2. The positions of E2 and the IgG light chain (L) are indicated with arrows. Immunoprecipitations of E2 from undiluted samples of liver macerate were performed using monoclonal and polyclonal antibodies. Normal goat IgG (lane 4), B65581G goat anti-E2 (lane 5), MAb 2G12-2 (negative control; lane 6), MAb AP33 anti-E2 (lane 7), human MAb CBH-2 anti-E2 (lane 8), and human MAb CBH-5 anti-E2 (lane 9). Biotinylated molecular mass markers (lane M).