Ultrastructural and physicochemical characterization of the hepatitis C virus recovered from the serum of an agammaglobulinemic patient

168

133

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Section: Discussionmentioning

confidence: 96%

Nonenveloped Nucleocapsids of Hepatitis C Virus in the Serum of Infected Patients

Maillard¹,

Krawczyński

Nitkiewicz³

et al. 2001

168

133

“…While the sedimentation coefficient calculated for HCV both in vivo and in vitro falls into the range described for other Flaviviridae (ϳ200S) (32), the broad density range of the particles in the supernatant appears to be a unique feature characteristic of HCV (1,22,25,30,36,40,43,48,49,51,54). This broad range of buoyant densities is thought to be partially dependent on the presence of host lipoprotein components, including apoB and apoE, in the viral particle (1,40,47,48).…”

mentioning

confidence: 84%

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

Gastaminza

Kapadia

Chisari

2006

215

232

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).

“…Previous ultrastructural studies using patient-derived material report particles with heterogeneous diameters ranging from 35 to 100 nm (33,37,42,57,64). Cell culture-derived particles appear to display a diameter within that range (ϳ55 nm) (65,68).…”

mentioning

confidence: 89%

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

Gastaminza

Dryden

Boyd

et al. 2010

181

151

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.