Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

Abstract

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (ϳ40-to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ϳ60 and ϳ45 nm in diameter. The ϳ60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ϳ45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-lowdensity, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ϳ60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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.…”

“…Because the number of E2 proteins per particle is most likely not affected by the tagging, we conclude that either the FLAG beads have an avidity much higher than any of the used E2 antibodies or that the tag is exposed on the surface of the particles and therefore more accessible than the epitope for any of the used E2-specific antibodies. Whatever the reason, we note that similarly low labeling efficiency with an E2-specific antibody (5-20% of density gradient-purified HCV particles) has been reported very recently (76).…”

Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

“…Viruses from the two genera have similar morphologies in electron micrographs and contain only two envelope glycoproteins, E1 and E2 (3)(4)(5). Moreover, formation of E1-E2 heterodimers through interactions involving the transmembrane segments of the two proteins is required for cell entry of both pesti-and hepaciviruses (22,23).…”

“…Pesti-and hepaciviruses have a similar genomic organization and they both form mostly spherical particles 40-60 nm in diameter with relatively smooth surfaces. Due to variations in particle size and shape, electron microscopy studies have provided few additional structural insights and it is not yet clear whether the virions have regular icosahedral symmetry (3)(4)(5). Inhibition of cell entry by endocytic inhibitors suggests that pesti-and hepaciviruses enter the cell by clathrin-mediated endocytosis (6,7).…”

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).…”