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

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
Journal of Virology (Impact Factor: 4.44). 11/2010; 84(21):10999-1009. DOI: 10.1128/JVI.00526-10
Source: PubMed

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 (∼40- 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 ∼60 and ∼45 nm in diameter. The ∼60-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 ∼45-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-low-density, >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 ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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Available from: Pablo Gastaminza, Jun 26, 2014
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    • "HCV is an enveloped virus of the Flaviviridae family, measuring 40–80 nm in diameter with a heterogeneous morphology and no clear form of symmetry [17] [18] [19]. For these reasons, a high-resolution model of particle structure is not available, in contrast to the well-defined particle structures of the related flaviviruses. "
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    ABSTRACT: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. Acute infection often progresses to chronicity resulting frequently in fibrosis, cirrhosis, and in rare cases, in the development of hepatocellular carcinoma (HCC). Although HCV has proven to be an arduous object of research and has raised important technical challenges, several experimental models have been developed all over the last two decades in order to improve our understanding of the virus life cycle, pathogenesis and virus-host interactions. The recent development of direct acting-agents (DAAs), leading to considerable progress in treatment of patients, represents the direct outcomes of these achievements. Proteomic approaches have been of critical help to shed light on several aspect of the HCV biology such as virion composition, viral replication, and virus assembly and to unveil diagnostic or prognostic markers of HCV-induced liver disease. Here, we review how proteomic approaches have led to improve our understanding of HCV life cycle and liver disease, thus highlighting the relevance of these approaches for studying the complex interactions between other challenging human viral pathogens and their host. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Proteomics 03/2015; 15(12). DOI:10.1002/pmic.201500009 · 3.81 Impact Factor
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    • "Previous studies have indicated that cell culture- [20]–[24] and patients' serum-derived [25]–[29] HCV particles display heterogeneous diameters (from 35 to 145 nm) and have a broad range of buoyant density (between 1.01 g/ml and 1.17 g/ml). The main peak of both viral Core protein and RNA exhibited at a density of 1.15 to 1.17 g/ml in the cell culture derived-HCV (HCVcc) [30], [31], and the highest specific infectivity of extracellular virion was observed at a density of 1.14 g/ml [20]. Notably, the low-density fraction (density of <1.1 g/ml) displays exosome-like structures and also contains infectivity [20], but the nature and origin of their properties are still unknown. "
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    ABSTRACT: Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.
    PLoS ONE 06/2014; 9(6):e99022. DOI:10.1371/journal.pone.0099022 · 3.23 Impact Factor
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    • "We believe that the density profile of sf-HCVcc might allow more effective density-based purification and concentration using ultracentrifugation and gel chromatography, since a single fraction, containing the majority of infectious virus, could be collected. Density changes were previously observed for HCVcc without hypervariable region 1 (HVR1) (Bankwitz et al., 2010; Prentoe et al., 2011), HCVcc with a specific E2 mutation (Zhong et al., 2006; Grove et al., 2008; Gastaminza et al., 2010) and for HCV recovered from HCVcc-infected chimpanzees and uPA- SCID mice engrafted with human liver cells (Lindenbach et al., 2006). These density changes were suggested to be due to differences in lipoprotein association (Lindenbach et al., 2006; Zhong et al., 2006; Grove et al., 2008; Bankwitz et al., 2010; Prentoe et al., 2011). "
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    ABSTRACT: Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1log10 higher infectivity titers (4.7-6.2log10Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.
    Virology 06/2014; 458-459C(1):190-208. DOI:10.1016/j.virol.2014.03.021 · 3.32 Impact Factor
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