Characterization of hepatitis C RNA-containing particles from human liver by density and size

Abstract

Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml−1. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.

Protein Kinase D Negatively Regulates Hepatitis C Virus Secretion through Phosphorylation of Oxysterol-binding Protein and Ceramide Transfer Protein

“…It was shown that silencing apoB and apoE or inhibiting microsomal triglyceride transfer protein activity abrogated HCV secretion. In earlier studies, apoE and apoB were found on the surface of infectious viral particles, suggesting that lipoproteins associate with viral particles to produce lipoviroprotein particles (51)(52)(53)(54). These findings raise the possibilities of viral entry via LDL or SR-B1 receptors, and indeed there have been studies that support this view (51,52,55,56).…”

Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

“…A hallmark of HCV infectious particles is their tight connection with very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), giving rise to a hybrid form of lipoviral particles (LVPs) with heterogeneous buoyant density (5)(6)(7)(8)(9)(10)(11)(12). Nonexchangeable apolipoprotein B (apoB) and several exchangeable apolipoproteins (apoE, apoA-I, and apoC-I) have been found on the LVP surface (13)(14)(15).…”

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

“…E1 could not be detected in the studied samples (data not shown), even though previous reports have described its presence in LVPs. 5,28 Symmetrically, envelope glycoprotein E2 was detected by a genotype 1a-specific monoclonal antibody in two LVP samples from 12 HCV genotype 1 patients (representative blot on Fig. 2C).…”

“…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).…”