Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells

Roingeard, Philippe; Lu, Shilun; Sureau, Camille; Freschlin, M.; Arbeille, B.; Essex, M.; Romet-Lemonne, Jean-Loup

doi:10.1002/hep.1840110219

Cited by 37 publications

(26 citation statements)

References 32 publications

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“…1), as previously described. 11,12 However, an extensive and more accurate analysis revealed an unusual feature of the HBV particles when more than one nucleocapsid is surrounded by a single envelope (Fig. 1A, E, and F).…”

Section: Resultsmentioning

confidence: 99%

“…The HepG2-T14 clone has been described elsewhere. 11,12 This clone was selected after a stable transfection of the HepG2 cell line with cloned HBV DNA for its ability to produce HBV particles, 11 which were shown to be infectious in the chimpanzee. 13 By electron microscopy, HBV virions and subviral envelope filaments were found in intracellular cisternae, 12 in contrast to the absence of detectable intracellular particles in other HBV-producer-transfected cells such as the HepG2-T2.2.15 clone.…”

Section: Methodsmentioning

confidence: 99%

“…11,12 This clone was selected after a stable transfection of the HepG2 cell line with cloned HBV DNA for its ability to produce HBV particles, 11 which were shown to be infectious in the chimpanzee. 13 By electron microscopy, HBV virions and subviral envelope filaments were found in intracellular cisternae, 12 in contrast to the absence of detectable intracellular particles in other HBV-producer-transfected cells such as the HepG2-T2.2.15 clone. 14,15 This was the consequence of the particularly high expression level of the L protein in the HepG2-T14 cells, as seen by a strong immunocytochemical staining for the preS1 antigen in these cells.…”

Section: Methodsmentioning

confidence: 99%

“…14,15 This was the consequence of the particularly high expression level of the L protein in the HepG2-T14 cells, as seen by a strong immunocytochemical staining for the preS1 antigen in these cells. 12 The HepG2-T14 clone as well as the HepG2 parent cell line were grown at 37°C with 5% CO 2 in RPMI 1640 medium (Gibco, Grand Island, NY) supple-mented with 20% inactivated fetal calf serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…14,15 This was the consequence of the particularly high expression level of the L protein in the HepG2-T14 cells, as seen by a strong immunocytochemical staining for the preS1 antigen in these cells. 12 The HepG2-T14 clone as well as the HepG2 parent cell line were grown at 37°C with 5% CO 2 in RPMI 1640 medium (Gibco, Grand Island, NY) suppleAbbreviations: HBV, hepatitis B virus; L protein, large protein; M protein, middle protein; S protein, small protein; ER, endoplasmic reticulum; TNE, 150 mmol/L NaCl and 20 mmol/L Tris HCl, pH 7.4; FCH, fibrosing cholestatic hepatitis.From the …”

mentioning

confidence: 99%

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Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

Roingeard

Sureau

1998

Hepatology

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The intracellular accumulation of empty hepatitis B virus (HBV) particles of filamentous shape leads to a direct cytopathic effect in so-called ground-glass hepatocytes. The aim of this study was to investigate how these filaments can be structurally formed at the cellular level. By electron microscopy, we reexamined the HBV-producer HepG2T-14 cells, which have been described as producing a substantial amount of empty HBV filaments compared with the other forms of HBV particles. Examination of ultrathin sections of HepG2T14 cells revealed the presence of HBV virions and filaments at the periphery of extremely large intracellular cisternae, probably related to a pre-Golgi compartment. Very long filaments appeared to be formed by a tubular budding of a long portion of the cisterna membrane. This phenomenon may be identical to that observed in the hepatocytes of HBV chronic carriers, in which the inability of the infected cell to export long HBV filamentous particles through the cellular secretion pathway seems to be at the origin of a direct cytopathic effect. (HEPATOLOGY 1998;28: 1128-1133.)Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus that infects a large number of people worldwide. 1 Its genome is encapsulated with a virus-encoded polymerase in a nucleocapsid surrounded by a host-derived lipid envelope bearing three viral surface proteins. 2 The large (L) protein, containing the preS1, preS2, and S domains, is translated from the first start codon of the surface open reading frame. The middle (M) protein, containing the preS2 and S domain, and the small (S) protein are translated from in-frame start codons further downstream. 2 During synthesis, all three proteins are cotranslationally inserted into the endoplasmic reticulum (ER) as transmembrane proteins that can form disulfide bridges with each other. 3 The S protein traverses the ER membrane at least twice to form a cytosolic loop and a luminal domain. 4 The M protein has a similar topology with the additional N-terminal preS2 domain located in the ER lumen. 5 The L protein can adopt two different transmembrane topologies, allowing the exposure of the N-terminal preS domain at either the luminal or the cytosolic side of the ER membrane. 6 This latter form is essential during HBV morphogenesis to establish a protein-protein interaction between the cytoplasmic core particle and a specific sequence within the preS domain of the L protein. 7,8 One peculiarity of HBV morphogenesis compared with that of other viruses is that the surface proteins not only envelop the nucleocapsid to form a mature virion, they are capable of forming subviral empty spherical or filamentous particles in the absence of nucleocapsid. 2 Although the envelopes of all types of HBV particles contain the M and S proteins, filamentous and virion envelopes are richer in the L protein. 2 It is thought that during morphogenesis, the M and S proteins, in the absence of any other viral proteins, can bud into a post-ER/pre-Golgi compartment to form 20-nm spherical particles that ca...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…14,15 This was the consequence of the particularly high expression level of the L protein in the HepG2-T14 cells, as seen by a strong immunocytochemical staining for the preS1 antigen in these cells. 12 The HepG2-T14 clone as well as the HepG2 parent cell line were grown at 37°C with 5% CO 2 in RPMI 1640 medium (Gibco, Grand Island, NY) suppleAbbreviations: HBV, hepatitis B virus; L protein, large protein; M protein, middle protein; S protein, small protein; ER, endoplasmic reticulum; TNE, 150 mmol/L NaCl and 20 mmol/L Tris HCl, pH 7.4; FCH, fibrosing cholestatic hepatitis.From the …”

mentioning

confidence: 99%

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Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

Roingeard

Sureau

1998

Hepatology

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Expression of X protein and hepatitis B virus replication in chronic hepatitis

et al. 1991

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The X protein can act on the enhancer of hepatitis B virus in an in vitro system and elevate the transcriptional level of hepatitis B virus. However, because no relationship had been reported between X protein expression and hepatitis B virus replication in patients with chronic hepatitis B, we focused on its expression in the liver in comparison with markers of hepatitis B virus replication. Liver biopsy samples and sera from 59 carriers with HBsAg were examined immunohistochemically for X protein using rabbit IgG against recombinant X protein. There was a significant difference in the serum hepatitis B virus DNA level between X protein-positive and -negative patients (p less than 0.001). Serum pre-S1 and pre-S2 antigens were also measured quantitatively by enzyme immunoassay using monoclonal antibodies specific against each antigen. The titers of pre-S1 antigen in patients positive for X protein were significantly higher (p less than 0.001) than those of the X protein-negative patients (3.02 +/- 0.99 vs. 2.00 +/- 0.59, respectively). Similarly, the titers of pre-S2 antigen were 2.98 +/- 0.91 vs. 1.94 +/- 0.54, respectively (p less than 0.001). The rate of positivity of the X protein was higher (38 of 49; 77.6%) in the replicative group (serum HBeAg, serum hepatitis B virus DNA or HBcAg in liver positive) compared with that observed in the nonreplicative group (3 of 10; 30%--serum HBeAg, serum hepatitis B virus DNA and HBcAg in liver negative) (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

et al. 2007

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Formation of enveloped viruses involves assembly and budding at cellular membranes. In this study, we elucidated the morphogenesis of hepadnaviruses on the ultrastructural and biochemical level using duck hepatitis B virus (DHBV) as a model system. Formation of virus progeny initiates at the endoplasmic reticulum (ER) and is conserved both in vitro and in vivo. The morphogenesis proceeds via membrane-surrounded vesicles containing both virions and subviral particles, indicating a common morphogenetic pathway. The virus particle-containing vesicles (VCVs) are generated and maintained by reorganization of endomembranes accompanied by a striking disorganization of the rough ER (rER). DHBV-infected hepatocytes produce not only complete virions, but also large quantities of subviral particles (SVPs). SVPs are devoid of the nucleocapsid and viral DNA, 4,5 but contain the envelope proteins. The extracellular DHB virion has a spherical structure of approximately 45 to 65 nm and contains an electron-dense nucleocapsid of about 27 nm. 6 Little is known about the morphogenesis of this virus family, but several of the following features indicate that this process is likely to be unique and complex: the DHB viral envelope proteins are multispanning transmembrane proteins, encoded by a single open reading frame, whose differential transcription results in the large (L) and the small (S) surface protein. 7 Both proteins share an identical carboxyterminal region, representing the S protein, with an additional Nterminal extension forming the preS-region of L. The ratio between the L and S proteins in the viral particles is about 1:4. 8 As the major structural component of the envelope, the S protein determines envelope curvature and is indispensable for both budding and secretion of viral particles. 9 Both surface proteins are synthesized at the rough endoplasmic reticulum (ER), or rER, and then bud into the ER lumen, forming SVPs. This budding event is autonomous and independent of all other viral components (e.g., nucleocapsid). 10,11 The intrinsic membrane-deforming activity of hepadnaviral envelope proteins is host cell-independent and conserved from yeast to

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Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells

Cited by 37 publications

References 32 publications

Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

Expression of X protein and hepatitis B virus replication in chronic hepatitis

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

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