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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.

SUBMITTER: Nielsen SU

PROVIDER: S-EPMC2557069 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Metabolic diseases such as obesity and diabetes are associated with changes in high-density lipoprotein (HDL) particles, including changes in particle size and protein composition, often resulting in abnormal function. Recent studies suggested that patients with non-alcoholic fatty liver disease (NAFLD), including individuals with non-alcoholic steatohepatitis (NASH), have smaller HDL particles when compared to individuals without liver pathologies. However, no studies have investigated potential changes in HDL particle protein composition in patients with NAFLD, in addition to changes related to obesity, to explore putative functional changes of HDL which may increase the risk of cardiovascular complications. Methods:From a cohort of morbidly obese females who were diagnosed with simple steatosis (SS), NASH, or normal liver histology, we selected five matched individuals from each condition for a preliminary pilot HDL proteome analysis. HDL particles were enriched using size-exclusion chromatography, and the proteome of the resulting fraction was analyzed by liquid chromatography tandem mass spectrometry. Differences in the proteomes between the three conditions (normal, SS, NASH) were assessed using label-free quantitative analysis. Gene ontology term analysis was performed to assess the potential impact of proteomic changes on specific functions of HDL particles. Results:Of the 95 proteins identified, 12 proteins showed nominally significant differences between the three conditions. Gene ontology term analysis revealed that severity of the liver pathology may significantly impact the anti-thrombotic functions of HDL particles, as suggested by changes in the abundance of HDL-associated proteins such as antithrombin III and plasminogen. Conclusions:The pilot data from this study suggest that changes in the HDL proteome may impact the functionality of HDL particles in NAFLD and NASH patients. These proteome changes may alter cardio-protective properties of HDL, potentially contributing to the increased cardiovascular disease risk in affected individuals. Further validation of these protein changes by orthogonal approaches is key to confirming the role of alterations in the HDL proteome in NAFLD and NASH. This will help elucidate the mechanistic effects of the altered HDL proteome on cardioprotective properties of HDL particles.

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

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