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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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HCV proteins induced apoptosis in a caspase-dependent manner. A: Extent of apoptosis. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG. At 24 h p.i, uninfected and infected cells where fixed with an EtOH 70%-PBS solution, washed and stained with propidium iodide (PI) as explained in Material and Methods. The cell cycle was measure by flow cytometry. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: Activation of effector caspases. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of IPTG individually or in combination with a general caspase inhibitor, zVAD-fmk at 50 μM. Cell lysates from uninfected and infected cells were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes full-length (116 kDa) and cleavage-PARP (89 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). C: Caspase-8 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually or in combination with a caspase-8 inhibitor, zIEDT-fmk at 50 μM, in the presence (+) or absence (-) of IPTG. Uninfected and infected cell lysates were collected at 48 h p.i. and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes procaspase- (57 kDa) and active-caspase-8 (43 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). D: Caspase-9 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually in the presence (+) or absence (-) of IPTG or in combination with a caspase-9 inhibitor, zLEHD-fmk at 50 μM. Uninfected and infected cell lysates were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes the active-caspase 9 (37 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). Cells infected with the inducible VV-PKR were used as positive control.
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Figure 6: HCV proteins induced apoptosis in a caspase-dependent manner. A: Extent of apoptosis. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG. At 24 h p.i, uninfected and infected cells where fixed with an EtOH 70%-PBS solution, washed and stained with propidium iodide (PI) as explained in Material and Methods. The cell cycle was measure by flow cytometry. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: Activation of effector caspases. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of IPTG individually or in combination with a general caspase inhibitor, zVAD-fmk at 50 μM. Cell lysates from uninfected and infected cells were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes full-length (116 kDa) and cleavage-PARP (89 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). C: Caspase-8 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually or in combination with a caspase-8 inhibitor, zIEDT-fmk at 50 μM, in the presence (+) or absence (-) of IPTG. Uninfected and infected cell lysates were collected at 48 h p.i. and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes procaspase- (57 kDa) and active-caspase-8 (43 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). D: Caspase-9 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually in the presence (+) or absence (-) of IPTG or in combination with a caspase-9 inhibitor, zLEHD-fmk at 50 μM. Uninfected and infected cell lysates were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes the active-caspase 9 (37 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). Cells infected with the inducible VV-PKR were used as positive control.

Mentions: To quantify the extent of apoptosis, DNA content was analyzed by flow cytometry after permeabilization and labelling with the DNA-specific fluorochrome propidium iodide. As shown by flow cytometry, 78% of HeLa cells infected with VT7-HCV7.9 in the presence of IPTG were apoptotic in contrast with the 26% determined when cells were infected with VT7-HCV7.9 in the absence of the inducer (Fig. 6A).


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)

HCV proteins induced apoptosis in a caspase-dependent manner. A: Extent of apoptosis. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG. At 24 h p.i, uninfected and infected cells where fixed with an EtOH 70%-PBS solution, washed and stained with propidium iodide (PI) as explained in Material and Methods. The cell cycle was measure by flow cytometry. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: Activation of effector caspases. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of IPTG individually or in combination with a general caspase inhibitor, zVAD-fmk at 50 μM. Cell lysates from uninfected and infected cells were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes full-length (116 kDa) and cleavage-PARP (89 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). C: Caspase-8 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually or in combination with a caspase-8 inhibitor, zIEDT-fmk at 50 μM, in the presence (+) or absence (-) of IPTG. Uninfected and infected cell lysates were collected at 48 h p.i. and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes procaspase- (57 kDa) and active-caspase-8 (43 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). D: Caspase-9 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually in the presence (+) or absence (-) of IPTG or in combination with a caspase-9 inhibitor, zLEHD-fmk at 50 μM. Uninfected and infected cell lysates were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes the active-caspase 9 (37 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). Cells infected with the inducible VV-PKR were used as positive control.
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Related In: Results  -  Collection

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Figure 6: HCV proteins induced apoptosis in a caspase-dependent manner. A: Extent of apoptosis. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG. At 24 h p.i, uninfected and infected cells where fixed with an EtOH 70%-PBS solution, washed and stained with propidium iodide (PI) as explained in Material and Methods. The cell cycle was measure by flow cytometry. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: Activation of effector caspases. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence (+) or absence (-) of IPTG individually or in combination with a general caspase inhibitor, zVAD-fmk at 50 μM. Cell lysates from uninfected and infected cells were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes full-length (116 kDa) and cleavage-PARP (89 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). C: Caspase-8 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually or in combination with a caspase-8 inhibitor, zIEDT-fmk at 50 μM, in the presence (+) or absence (-) of IPTG. Uninfected and infected cell lysates were collected at 48 h p.i. and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes procaspase- (57 kDa) and active-caspase-8 (43 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). D: Caspase-9 activation. HeLa cells were infected at 5 PFU/cell with the recombinant VT7-HCV7.9 individually in the presence (+) or absence (-) of IPTG or in combination with a caspase-9 inhibitor, zLEHD-fmk at 50 μM. Uninfected and infected cell lysates were collected at 48 h p.i and separated on a 12% SDS-PAGE for immunoblot analysis using an antibody that recognizes the active-caspase 9 (37 kDa) (left panel) or used for the quantification of the apoptotic levels by ELISA (right panel). Cells infected with the inducible VV-PKR were used as positive control.
Mentions: To quantify the extent of apoptosis, DNA content was analyzed by flow cytometry after permeabilization and labelling with the DNA-specific fluorochrome propidium iodide. As shown by flow cytometry, 78% of HeLa cells infected with VT7-HCV7.9 in the presence of IPTG were apoptotic in contrast with the 26% determined when cells were infected with VT7-HCV7.9 in the absence of the inducer (Fig. 6A).

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