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Subcellular forms and biochemical events triggered in human cells by HCV polyprotein expression from a viral vector.

Vandermeeren AM, Gómez CE, Patiño C, Domingo-Gil E, Guerra S, González JM, Esteban M - Virol. J. (2008)

Bottom Line: Biochemical analysis demonstrate that HCV proteins bring about the activation of initiator and effector caspases followed by severe apoptosis and mitochondria dysfunction, hallmarks of HCV cell injury.Microarray analysis revealed that HCV polyprotein expression modulated transcription of genes associated with lipid metabolism, oxidative stress, apoptosis, and cellular proliferation.Our findings demonstrate the uniqueness of the VT7-HCV7.9 system to characterize morphological and biochemical events related to HCV pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Madrid, Spain. avandermeeren@pharmamar.com

ABSTRACT
To identify the subcellular forms and biochemical events induced in human cells after HCV polyprotein expression, we have used a robust cell culture system based on vaccinia virus (VACV) that efficiently expresses in infected cells the structural and nonstructural proteins of HCV from genotype 1b (VT7-HCV7.9). As determined by confocal microscopy, HCV proteins expressed from VT7-HCV7.9 localize largely in a globular-like distribution pattern in the cytoplasm, with some proteins co-localizing with the endoplasmic reticulum (ER) and mitochondria. As examined by electron microscopy, HCV proteins induced formation of large electron-dense cytoplasmic structures derived from the ER and containing HCV proteins. In the course of HCV protein production, there is disruption of the Golgi apparatus, loss of spatial organization of the ER, appearance of some "virus-like" structures and swelling of mitochondria. Biochemical analysis demonstrate that HCV proteins bring about the activation of initiator and effector caspases followed by severe apoptosis and mitochondria dysfunction, hallmarks of HCV cell injury. Microarray analysis revealed that HCV polyprotein expression modulated transcription of genes associated with lipid metabolism, oxidative stress, apoptosis, and cellular proliferation. Our findings demonstrate the uniqueness of the VT7-HCV7.9 system to characterize morphological and biochemical events related to HCV pathogenesis.

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Compartmentalization of HCV proteins produced in HeLa cells infected with VT7-HCV7.9. Subconfluent HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of the inducer IPTG, were fixed at 24 h p.i, labelled with the corresponding primary antibody followed by the appropriate fluorescent secondary antibody and visualized by confocal microscopy. The antibodies or reagents used were Hα HCV to detect HCV proteins; Topro-3 to detect DNA; Rα Giantin to detect the Golgi complex (A); Rα Calnexine to detect the endoplasmatic reticulum (B) and Mitotracker Deep Red 633 to detect mitochondria (C). The co-localization is shown with a higher resolution in the white square.
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Figure 1: Compartmentalization of HCV proteins produced in HeLa cells infected with VT7-HCV7.9. Subconfluent HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of the inducer IPTG, were fixed at 24 h p.i, labelled with the corresponding primary antibody followed by the appropriate fluorescent secondary antibody and visualized by confocal microscopy. The antibodies or reagents used were Hα HCV to detect HCV proteins; Topro-3 to detect DNA; Rα Giantin to detect the Golgi complex (A); Rα Calnexine to detect the endoplasmatic reticulum (B) and Mitotracker Deep Red 633 to detect mitochondria (C). The co-localization is shown with a higher resolution in the white square.

Mentions: To identify the cytoplasmic compartment(s) in which viral HCV proteins accumulated during infection of HeLa cells with VT7-HCV7.9, we performed immunofluorescence analysis using serum from an HCV-infected patient to recognize HCV proteins and antibodies specific for the Golgi apparatus (anti-gigantin), the endoplasmic reticulum (anti-calnexin) or the mitochondria (mitotracher) (Fig. 1). Inducible expression of HCV proteins caused severe disruption of the Golgi apparatus as revealed by labelling this organelle with a specific antibody (Fig. 1A). This effect was not observed in cells infected with VT7-HCV7.9 in the absence of IPTG. There is no co-localization of HVC proteins with the disrupted Golgi, whereas in the labelling of the endoplasmic reticulum, a clear co-localization between HCV proteins expressed from VT7-HCV7.9 and ER proteins was observed (Fig. 1B). With an in vivo mitochondrial marker (Fig. 1C), we detected partial co-localization between HCV proteins expressed from VT7-HCV7.9 and mitochondria organelles. Moreover, the mitochondria appeared more rounded in cells infected with VT7-HCV7.9 + IPTG, in comparison with uninfected cells or with cells infected in the absence of IPTG.


Subcellular forms and biochemical events triggered in human cells by HCV polyprotein expression from a viral vector.

Vandermeeren AM, Gómez CE, Patiño C, Domingo-Gil E, Guerra S, González JM, Esteban M - Virol. J. (2008)

Compartmentalization of HCV proteins produced in HeLa cells infected with VT7-HCV7.9. Subconfluent HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of the inducer IPTG, were fixed at 24 h p.i, labelled with the corresponding primary antibody followed by the appropriate fluorescent secondary antibody and visualized by confocal microscopy. The antibodies or reagents used were Hα HCV to detect HCV proteins; Topro-3 to detect DNA; Rα Giantin to detect the Golgi complex (A); Rα Calnexine to detect the endoplasmatic reticulum (B) and Mitotracker Deep Red 633 to detect mitochondria (C). The co-localization is shown with a higher resolution in the white square.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553408&req=5

Figure 1: Compartmentalization of HCV proteins produced in HeLa cells infected with VT7-HCV7.9. Subconfluent HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of the inducer IPTG, were fixed at 24 h p.i, labelled with the corresponding primary antibody followed by the appropriate fluorescent secondary antibody and visualized by confocal microscopy. The antibodies or reagents used were Hα HCV to detect HCV proteins; Topro-3 to detect DNA; Rα Giantin to detect the Golgi complex (A); Rα Calnexine to detect the endoplasmatic reticulum (B) and Mitotracker Deep Red 633 to detect mitochondria (C). The co-localization is shown with a higher resolution in the white square.
Mentions: To identify the cytoplasmic compartment(s) in which viral HCV proteins accumulated during infection of HeLa cells with VT7-HCV7.9, we performed immunofluorescence analysis using serum from an HCV-infected patient to recognize HCV proteins and antibodies specific for the Golgi apparatus (anti-gigantin), the endoplasmic reticulum (anti-calnexin) or the mitochondria (mitotracher) (Fig. 1). Inducible expression of HCV proteins caused severe disruption of the Golgi apparatus as revealed by labelling this organelle with a specific antibody (Fig. 1A). This effect was not observed in cells infected with VT7-HCV7.9 in the absence of IPTG. There is no co-localization of HVC proteins with the disrupted Golgi, whereas in the labelling of the endoplasmic reticulum, a clear co-localization between HCV proteins expressed from VT7-HCV7.9 and ER proteins was observed (Fig. 1B). With an in vivo mitochondrial marker (Fig. 1C), we detected partial co-localization between HCV proteins expressed from VT7-HCV7.9 and mitochondria organelles. Moreover, the mitochondria appeared more rounded in cells infected with VT7-HCV7.9 + IPTG, in comparison with uninfected cells or with cells infected in the absence of IPTG.

Bottom Line: Biochemical analysis demonstrate that HCV proteins bring about the activation of initiator and effector caspases followed by severe apoptosis and mitochondria dysfunction, hallmarks of HCV cell injury.Microarray analysis revealed that HCV polyprotein expression modulated transcription of genes associated with lipid metabolism, oxidative stress, apoptosis, and cellular proliferation.Our findings demonstrate the uniqueness of the VT7-HCV7.9 system to characterize morphological and biochemical events related to HCV pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Madrid, Spain. avandermeeren@pharmamar.com

ABSTRACT
To identify the subcellular forms and biochemical events induced in human cells after HCV polyprotein expression, we have used a robust cell culture system based on vaccinia virus (VACV) that efficiently expresses in infected cells the structural and nonstructural proteins of HCV from genotype 1b (VT7-HCV7.9). As determined by confocal microscopy, HCV proteins expressed from VT7-HCV7.9 localize largely in a globular-like distribution pattern in the cytoplasm, with some proteins co-localizing with the endoplasmic reticulum (ER) and mitochondria. As examined by electron microscopy, HCV proteins induced formation of large electron-dense cytoplasmic structures derived from the ER and containing HCV proteins. In the course of HCV protein production, there is disruption of the Golgi apparatus, loss of spatial organization of the ER, appearance of some "virus-like" structures and swelling of mitochondria. Biochemical analysis demonstrate that HCV proteins bring about the activation of initiator and effector caspases followed by severe apoptosis and mitochondria dysfunction, hallmarks of HCV cell injury. Microarray analysis revealed that HCV polyprotein expression modulated transcription of genes associated with lipid metabolism, oxidative stress, apoptosis, and cellular proliferation. Our findings demonstrate the uniqueness of the VT7-HCV7.9 system to characterize morphological and biochemical events related to HCV pathogenesis.

Show MeSH
Related in: MedlinePlus