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Falcon Viviana,
Menéndez Ivon,
Acosta-Rivero Nelson,
Shibayama Mineko,
Marıa-C de la Rosa, Jose Luna-Munoz,
Miranda-Sanchez Magdalena,
Jorge V Gavilondo,
Lopez Deliana,
Duenas-Carrera Santiago, [......],
Vidal Eduardo,
Arus-Soler Enrique,
Silva Jose,
Alvarez Felix,
Emilio F. Acosta,
Seoane Jesus,
Morales-Grillo Juan,
Penton Eduardo,
Kouri Juan,
Tsutsumi Victor
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ABSTRACT: Despite of recent advances on acknowledge of hepatitis B virus (HBV) structure and biology, little is known about the morphogenesis and release of virions. In this study, the ultrastructural analysis of HBV components in liver biopsies from chronically HBV-infected patients disclosed complex assembly and morphogenesis pathways for HBV. HBV core (HBcAg) and surface (HBsAg) antigens were specifically identified in all liver biopsies from HVB-infected patients. HBcAgcontaining core-like particles 24-28 nm in diameter were observed both in nucleus and cytoplasm of hepatocytes. Dane’s-like particles were detected either budding to or into the lumen of ER close toHBsAg containing tubular structures. Besides, Dane’s-like particles were detected in different vesicular compartments resembling multivesicular endosomes. Other kind of enveloped HBV-like particles similar to the previously described cobra-shaped and horn-shaped particles were also observed in hepatocytes. Some of these particles were connected to the vesicle membrane through a stalk 20-22 nm in diameter. On the other hand, spherical subviral particles (SVP) were frequently observed in cytoplasmic vesicles. Moreover, a minor proportion of enveloped HBV-like particles budding through the plasma membrane to the extracellular space and bile canaliculi were detected.Interestingly, Dane’s-like particles in the bile canaliculi and entering into ductular-like cells were shown. Strikingly, Dane’s-like particles close to tubular structures and specific immunolabeling forHBcAg both in cytoplasm and nuclei of stellate-like cells were detected. Remarkably, HVB-like particles appeared entering hepatocytes from large cytoplasmic processes of fibrocytes raising theinteresting possibility of a cell to cell passage of HBV through direct transcellular migration.
American Journal of Infectious Diseases. 01/2008;
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Viviana Falcón,
Nelson Acosta-Rivero,
Mineko Shibayama,
Glay Chinea,
Jorge V Gavilondo,
María C de la Rosa,
Ivón Menéndez,
Bienvenido Gra,
Santiago Dueñas-Carrera,
Ariel Viña,
Waldo García,
Maritza González-Bravo, Jose Luna-Munoz,
Magdalena Miranda-Sanchez,
Juan Morales-Grillo,
Juan Kouri,
Victor Tsutsumi
[show abstract]
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ABSTRACT: Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.
Biochemical and Biophysical Research Communications 05/2005; 329(4):1320-8. · 2.48 Impact Factor
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Viviana Falcón,
Nelson Acosta-Rivero,
María-C de la Rosa,
Ivón Menéndez,
Santiago Dueñas-Carrera,
Deliana Lopez,
Celia Fernández-Ortega,
Dionne Casillas,
Juan Morales,
Bienvenido Gra,
Waldo García,
Eduardo Vilar,
González-Bravo,
Maritza,
Mineko Shibayama, Jose Luna-Munoz,
Magdalena Miranda-Sanchez,
Juan Kouri and Victor Tsutsumi
[show abstract]
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ABSTRACT: In this study, we examined the expression of hepatitis C virus (HCV) components in hepatocytes and peripheral blood mononuclear cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA. The samples obtained from 25 randomly selected patients (13 of 25 patients showed no histological evidences of chronic hepatitis and had normal serum ALAT and GGTP levels) were studied by in situ Hybridization and Immunofluorescence assays. The findings show that HCV-RNA of both positive and negative polarity was carried in the hepatocytes of more than 80% of cases. The proportion of cells expressing the negative strand (mean ± SD, 10.25% ± 5.56%) was lower than those expressing positive strand (mean ± SD, 19.88% ± 9.19%) (p=0.0076; Student’s t test). In addition, reaction products suggestive of HCV core, E1 and E2 antigens within hepatocytes were also observed. Both hybridization and immunofluorescence signals were localized in the cytoplasm suggesting that this is the place of active HCV replication. On the other hand, the HCV-RNA of positive polarity was detected in PBMC from 16 out of 17 samples analyzed (94%) while the HCV-RNA of negative polarity was detected in 82% of samples investigated. Positive hybridization signals were localized in the cytoplasm of PBMC. Interestingly, 12 out of 13 patients with clinical and histological resolution of hepatitis, contain HCV-RNA in either liver or PBMC. These results provide further evidences that the intermediate replicative form of the HCV genome can persist in hepatocytes and PBMC after apparently complete resolution of chronic hepatitis C.
American Journal of Infectious Diseases. 01/2005;
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Viviana Falcón,
Nelson Acosta-Rivero,
María-C de la Rosa,
Ivón Menéndez,
Santiago Dueñas-Carrera,
Deliana Lopez,
Celia Fernández-Ortega,
Dionne Casillas,
Juan Morales,
Bienvenido Gra,
Waldo García,
Maritza González Bravo,
Mineko Shibayama, Jose Luna-Munoz,
Magdalena Miranda-Sanchez,
Juan Kouri,
Victor Tsutsumi
[show abstract]
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ABSTRACT: Understanding the mechanism of Hepatitis C Virus (HCV) pathogenesis is an important part of HCV research. In this study, the presence of apoptosis in HCV-infected liver and Peripheral Blood Mononuclear Cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA was investigated. The samples obtained from 21 patients were studied by in situ Hybridization (ISH), Immunofluorescence, TUNEL reaction and caspase 3 activation assays. The findings show that both DNA fragmentation by TUNEL assay and activation of caspase 3 were detected in hepatocytes from patients histologically confirmed as bearing chronic hepatitis or with abnormal ALAT or GGTP as well as in patients with no histological evidences of chronic hepatitis and normalization of transaminases. Apoptotic cells were also detected in PBMC samples by the TUNEL assay. ISH analysis of liver biopsies and PBMC samples showed both positive and negative strands of the HCV genome localized in some cells showing nuclear characteristics of apoptosis such as chromatin margination, condensation and fragmentation. These typical morphological changes of apoptotic cell death were also observed in some hepatocytes showing reaction products suggestive of HCcAg. Data suggest that under certain conditions HCV induces apoptosis in the absence of liver injury. Induction of apoptosis in HCV-infected cells may interfere with viral replication, which may lead to undetectable levels of HCV-RNA in serum.
American Journal of Infectious Diseases. 01/2005;
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Nelson Acosta-Rivero,
Viviana Falcón,
Catalina Alvarez,
Alexis Musacchio,
Glay Chinea,
María Cristina de la Rosa,
Armando Rodriguez,
Santiago Dueñas-Carrera,
Victor Tsutsumi,
Mineko Shibayama,
Ivón Menéndez, Jose Luna-Munoz,
Maria M Miranda-Sanchez,
Juan Kouri,
Juan Morales-Grillo
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ABSTRACT: The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.
Biochemical and Biophysical Research Communications 11/2003; 310(1):48-53. · 2.48 Impact Factor
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Viviana Falcón,
Nelson Acosta-Rivero,
Glay Chinea,
María Cristina de la Rosa,
Ivón Menéndez,
Santiago Dueñas-Carrera,
Bienvenido Gra,
Armando Rodriguez,
Victor Tsutsumi,
Mineko Shibayama, Jose Luna-Munoz,
Maria M Miranda-Sanchez,
Juan Morales-Grillo,
Juan Kouri
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ABSTRACT: Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.
Biochemical and Biophysical Research Communications 11/2003; 310(1):54-8. · 2.48 Impact Factor
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Viviana Falc,
On,
Nelson Acosta-Rivero,
Glay Chinea,
Mar I Ia,
Cristina De La Rosa,
Iv O On Men E Endez,
Santiago Due~,
Nas-Carrera,
Bienvenido Gra,
Armando Rodriguez,
Victor Tsutsumi,
Mineko Shibayama, Jose Luna-Munoz,
Maria M Miranda-Sanchez,
Juan Morales-Grillo,
Juan Kouri
[show abstract]
[hide abstract]
ABSTRACT: Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.
