Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

Abstract

The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (ϳ65 to 70 nm) but different in buoyant density (ϳ1.15 to 1.20 g/ml) from extracellular particles (ϳ1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Approximately 3% of the human population is infected, and more than 80% of all HCV infections progress to chronicity, ultimately leading to fibrosis, cirrhosis, and hepatocellular carcinoma (24). There is no vaccine against HCV, and the most widely used therapy involves the administration of type I interferon (␣2A) combined with ribavirin. However, this treatment strategy is toxic and has been shown to be ineffective in a significant proportion of the cases (41).HCV is a member of the Flaviviridae family and the sole member of the genus Hepacivirus (34). HCV is an enveloped virus with a single-strand positive RNA genome that codes for a unique polyprotein of approximatively 3,000 amino acids (11,12). A single open reading frame is flanked by 5Ј and 3Ј untranslated regions that contain RNA sequences essential for RNA translation and replication, respectively (17,18,23). The translation of the single open reading frame is driven by an internal ribosomal entry site sequence present within the 5Ј untranslated region (23), and the resulting polyprotein is processed by cellular and viral proteases into its individual components (reviewed in reference 42). The E1, E2, and core structural proteins are assembled into particles (3, 4), but are not essential for viral RNA replication or translation. The NS2, NS3, NS4A, NS4B, NS5A, and NS5B nonstructural proteins constitute the viral components necessary for efficient viral RNA replication, although NS2 is dispensable for this function (5, 33). In the linear sequence of the polyprotein, the structural proteins are separated from the nonstructural proteins by a small hydrophobic protein, p7 (29), whose function remains unknown but that has the potential to form ion channels (19). Viral proteins are localized in the cytoplasm, and it is assumed, by analogy with other members of the Flaviviridae family, that the entire life cycle of the virus is exclusively cytoplasmic (32).

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

“…E2-labeled particles had an average diameter of 73 Ϯ 18 nm, which is in agreement with previous predictions (9,15) and observations (9,16,54,63,76). Particles were predominantly singular (Fig.…”

“…Up to now, characterization of HCV particles is based on studies using partially purified virions from patient sera, experimentally inoculated chimpanzees, or rather low titer JFH1 particle preparations generated in cell cultures (9,(13)(14)(15)(16)(17). It is well established that infectious HCV particles have a low den-sity (Յ1.1 g/ml) that appears to be determined by the host cell, in which the virus is produced (18).…”

“…It is well established that infectious HCV particles have a low den-sity (Յ1.1 g/ml) that appears to be determined by the host cell, in which the virus is produced (18). This is probably due to the tight association of virions with host cell components, most notably lipoproteins (15,16,19). The association appears to occur during assembly, because HCV formation and secretion depend on components of the very low density lipoprotein (VLDL) assembly and secretion pathways, including apolipoprotein E (apoE) and microsomal triglyceride transfer protein (19 -23).…”

Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

“…2, A and B). In line with studies of others (10,11,14), viral particles could also be detected at higher densities (to up to 1.26 g/ml, data not shown) and at lower densities (to up to 1.02 g/ml; Fig. 2, A and B).…”

Hepatitis c virus and host cell lipids: An intimate connection

“…167 This result is in keeping with the notion that assembly and release of HCV particles are tightly linked to host cell lipoproteins and lipids. 162,[168][169][170][171][172] Moreover, the nature of the lipidic constituents of HCV particles appears to depend on the host cell in which the virus is produced. 173 This is best illustrated by the observation that cell culture grown HCV (HCVcc) produced in the human hepatoma cell line Huh7 differs from HCV circulating in patient serum as so-called lipoviro particles (LVPs).…”