Hepatitis C virus particle detected by immunoelectron microscopic study

Kaito, Masahiko; Watanabe, Shozo; Tsukiyama-Kohara, Kyoko; Yamaguchi, K; Kobayashi, Yoshinao; Konishi, Masayoshi; Yokoi, Masato; Ishida, Satoshi; Suzuki, Shiro; Kohara, Michinori

doi:10.1099/0022-1317-75-7-1755

Cited by 225 publications

(146 citation statements)

References 17 publications

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“…In the culture medium from cells transfected with pTHr, a peak of HCV proteins and RNA coincided in fraction 5, which has the density of 1.16 g͞ml. This density is consistent with the published density of free HCV virions (16). Viral particles were visualized by electron microscopy only in fraction 5 (Fig.…”

Section: Hcv Virion Production and Secretionsupporting

confidence: 80%

An in vitro model of hepatitis C virion production

Heller

Saito

Auerbach

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

101

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The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5 and 3 ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive-and negative-strand HCV RNA. Sequence analysis of the 5 and 3 ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g͞ml, the putative density of HCV virions. Electron microscopy showed viral particles of Ϸ50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.assembly ͉ cell culture ͉ infection ͉ ribozyme ͉ viral replication T he hepatitis C virus (HCV) is an important cause of human illness worldwide (1). Although it has proven to be a difficult public health problem, it has been no easier to study in the laboratory. A major impediment has been the lack of robust model systems to study the complete viral life cycle. HCV is a member of the Flaviviridae family of Ϸ9.6 kb, and it has a central ORF flanked by the 5Ј and 3Ј noncoding regions. The ORF is divided into the coding sequences for the structural proteins at the 5Ј end and the nonstructural proteins at the 3Ј end. Study of the biology of hepatitis C at a molecular level focused initially on expression and manipulation of individual viral proteins in tissue culture.The development of the subgenomic and genomic replicons is a major breakthrough to understanding viral replication and viral-cell interactions and provides a means to test therapeutic targets (2, 3). However, as yet, none of these systems produce viral particles, nor do they produce infectious virions. Although some infectious tissue culture systems have been described; in general, these systems have not been robust enough to study the complete viral life cycle (4, 5).Why virion production has been such an elusive goal remains unclear; however, the promise of a system that produces authentic virions is clear. Not only would more of the biology of the virus become accessible for study, but also such a system would provide a means to s...

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Section: Hcv Virion Production and Secretionsupporting

confidence: 80%

An in vitro model of hepatitis C virion production

Heller

Saito

Auerbach

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

101

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“…HCV is an enveloped virus, as indicated by inactivation after chloroform treatment (Feinstone et al, 1983) and the buoyant density of the virus in sucrose has been determined to be between 1.08 and 1.11 g/ml (Bradley et al, 1991;Hijikata et al, 1993;Kanto et al, 1994;Miyamoto et al, 1992). Recently, electron microscopic examination revealed the spherical morphology of HCV particles with a mean diameter of 55-65 nm (Kaito et al, 1994).…”

mentioning

confidence: 99%

Association of hepatitis C virus particles with immunoglobulin: a mechanism for persistent infection

Choo

Cho

et al. 1995

Journal of General Virology

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The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20-60 % isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186-1.213 g/ml) and at low density (1.099-1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation ofimmunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

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“…Via immunoelectron microscopy using an E2-specific monoclonal antibody, the authors succeeded in demonstrating the presence of specifically labeled spherical particles measuring 50-65 nm in diameter in the culture supernatant from JFH-1 RNA-transfected cells, confirming the early filtration and chimpanzee transmission as well as electron microscopy studies mentioned above. [17][18][19] Furthermore, Wakita et al showed that viral particles had a density of approximately 1.17 g/mL and that infection could be blocked by a monoclonal antibody directed against CD81, confirming the importance of this molecule as a component of the HCV entry pathway. The authors also developed a bicistronic luciferase reporter construct with similar capacity to produce infectious particles as a conveniently measurable read-out.…”

Section: Production Of Recombinant Hcv In Tissue Culturementioning

confidence: 96%

“…17 Subsequent electron microscopy studies confirmed that HCV is an enveloped spherical particle that is 40-70 nm in diameter. 18,19 Thus, it is believed that the HCV envelope contains the viral glycoproteins E1 and E2 and encloses a nucleocapsid composed of multiple copies of core protein, which in turn harbors the viral positive-strand RNA genome.…”

Section: Studying the Viral Particle And Its Entry Pathwaymentioning

confidence: 99%