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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 polyprotein expression induced dysfunction of the mitochondria. A: HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG were labelled in vivo at 24 h p.i with Mitotracker deep red (blue) to detect the mitochondria, with an anti-cytochrome c (red) antibody and with the serum from an HCV-infected patient to detect HCV proteins. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the mitochrondrial membrane potential (ΔΨm) was determined quantifying TMRE fluorescence. C: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the absence or presence of IPTG or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the uninfected and infected cells were stained with dihydroethidium (2-HE) and subjected to flow cytometry. Note: STS: staurosporine.
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Figure 5: HCV polyprotein expression induced dysfunction of the mitochondria. A: HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG were labelled in vivo at 24 h p.i with Mitotracker deep red (blue) to detect the mitochondria, with an anti-cytochrome c (red) antibody and with the serum from an HCV-infected patient to detect HCV proteins. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the mitochrondrial membrane potential (ΔΨm) was determined quantifying TMRE fluorescence. C: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the absence or presence of IPTG or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the uninfected and infected cells were stained with dihydroethidium (2-HE) and subjected to flow cytometry. Note: STS: staurosporine.

Mentions: To determine whether HCV polyprotein expression from the VACV recombinant activates cytochrome c release, HeLa cells were infected with VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine (as a positive control). The cytochrome c release was detected by confocal microscopy. As shown in Fig. 5A, the cytochrome c remained confined to the mitochondria in both uninfected cells and VT7-HCV7.9infected cells in the absence of IPTG. However, in cells infected with VT7-HCV7.9 in the presence of IPTG, there is a diffuse cytosolic pattern of cytochrome c staining, similarly as in cells treated with staurosporine, indicating that cytochrome c was released from the mitochondria.


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 polyprotein expression induced dysfunction of the mitochondria. A: HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG were labelled in vivo at 24 h p.i with Mitotracker deep red (blue) to detect the mitochondria, with an anti-cytochrome c (red) antibody and with the serum from an HCV-infected patient to detect HCV proteins. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the mitochrondrial membrane potential (ΔΨm) was determined quantifying TMRE fluorescence. C: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the absence or presence of IPTG or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the uninfected and infected cells were stained with dihydroethidium (2-HE) and subjected to flow cytometry. Note: STS: staurosporine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: HCV polyprotein expression induced dysfunction of the mitochondria. A: HeLa cells uninfected or infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG were labelled in vivo at 24 h p.i with Mitotracker deep red (blue) to detect the mitochondria, with an anti-cytochrome c (red) antibody and with the serum from an HCV-infected patient to detect HCV proteins. Cells treated with staurosporine at 0.5 μM for 16 h were used as positive control. B: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the mitochrondrial membrane potential (ΔΨm) was determined quantifying TMRE fluorescence. C: HeLa cells were either infected at 5 PFU/cell with the recombinant VT7-HCV7.9 in the absence or presence of IPTG or treated with staurosporine at 0.5 μM for 16 h. At 48 h p.i, the uninfected and infected cells were stained with dihydroethidium (2-HE) and subjected to flow cytometry. Note: STS: staurosporine.
Mentions: To determine whether HCV polyprotein expression from the VACV recombinant activates cytochrome c release, HeLa cells were infected with VT7-HCV7.9 in the presence or absence of IPTG, or treated with staurosporine (as a positive control). The cytochrome c release was detected by confocal microscopy. As shown in Fig. 5A, the cytochrome c remained confined to the mitochondria in both uninfected cells and VT7-HCV7.9infected cells in the absence of IPTG. However, in cells infected with VT7-HCV7.9 in the presence of IPTG, there is a diffuse cytosolic pattern of cytochrome c staining, similarly as in cells treated with staurosporine, indicating that cytochrome c was released from the mitochondria.

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