Limits...
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.

Show MeSH

Related in: MedlinePlus

Hepatocyte cell alterations following infection of HepG2 with VT7-HCV7.9. HepG2 cells uninfected or infected with the recombinant VT7-HCV7.9 in the presence or absence of the inducer IPTG were chemically fixed at 16 h p.i and then processed for conventional embedding in an epoxy resin.A: Cellular architecture of uninfected HepG2 cells. B: A general view of a cell infected with VT7-HCV7.9 in the absence of IPTG, showing the VACV forms IVs and IMVs.C, D and E: A general view of a cell infected with VT7-HCV7.9 in the presence of IPTG, showing large EDS surrounded by vacuoles and the presence of "virus-like particles" surrounded with membranes (*). Note: Vacuole (V) and electron dense structures in membranous webs (EDS). Bar: 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553408&req=5

Figure 3: Hepatocyte cell alterations following infection of HepG2 with VT7-HCV7.9. HepG2 cells uninfected or infected with the recombinant VT7-HCV7.9 in the presence or absence of the inducer IPTG were chemically fixed at 16 h p.i and then processed for conventional embedding in an epoxy resin.A: Cellular architecture of uninfected HepG2 cells. B: A general view of a cell infected with VT7-HCV7.9 in the absence of IPTG, showing the VACV forms IVs and IMVs.C, D and E: A general view of a cell infected with VT7-HCV7.9 in the presence of IPTG, showing large EDS surrounded by vacuoles and the presence of "virus-like particles" surrounded with membranes (*). Note: Vacuole (V) and electron dense structures in membranous webs (EDS). Bar: 200 nm.

Mentions: Since hepatocytes are the main targets of HCV virus, we next analyzed if expression of HCV polyprotein from the VT7-HCV7.9 infected cells also affected the ultra-structure of hepatic cells. Thus, monolayers of a human hepatoblast cell line (HepG2) were infected with VT7-HCV7.9 under the same conditions as for HeLa cells and processed at 16 h p.i for EM analysis. In this cell line, similarly as in HeLa cells, the inducible expression of HCV proteins blocks VACV morphogenesis and induces the same alterations described above. In contrast to uninfected (Fig 3A) and infected hepG2 cells in absence of IPTG (Fig. 3B), in cells expressing HCV proteins we distinguish EDS in membranous webs (Fig. 3C and 3E), formation of large vacuoles (Fig. 3C and 3D), and also identified "virus like-particles" structures surrounded by membranes and dispersed in several areas of the cell cytoplasm (Fig. 3D).


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)

Hepatocyte cell alterations following infection of HepG2 with VT7-HCV7.9. HepG2 cells uninfected or infected with the recombinant VT7-HCV7.9 in the presence or absence of the inducer IPTG were chemically fixed at 16 h p.i and then processed for conventional embedding in an epoxy resin.A: Cellular architecture of uninfected HepG2 cells. B: A general view of a cell infected with VT7-HCV7.9 in the absence of IPTG, showing the VACV forms IVs and IMVs.C, D and E: A general view of a cell infected with VT7-HCV7.9 in the presence of IPTG, showing large EDS surrounded by vacuoles and the presence of "virus-like particles" surrounded with membranes (*). Note: Vacuole (V) and electron dense structures in membranous webs (EDS). Bar: 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Hepatocyte cell alterations following infection of HepG2 with VT7-HCV7.9. HepG2 cells uninfected or infected with the recombinant VT7-HCV7.9 in the presence or absence of the inducer IPTG were chemically fixed at 16 h p.i and then processed for conventional embedding in an epoxy resin.A: Cellular architecture of uninfected HepG2 cells. B: A general view of a cell infected with VT7-HCV7.9 in the absence of IPTG, showing the VACV forms IVs and IMVs.C, D and E: A general view of a cell infected with VT7-HCV7.9 in the presence of IPTG, showing large EDS surrounded by vacuoles and the presence of "virus-like particles" surrounded with membranes (*). Note: Vacuole (V) and electron dense structures in membranous webs (EDS). Bar: 200 nm.
Mentions: Since hepatocytes are the main targets of HCV virus, we next analyzed if expression of HCV polyprotein from the VT7-HCV7.9 infected cells also affected the ultra-structure of hepatic cells. Thus, monolayers of a human hepatoblast cell line (HepG2) were infected with VT7-HCV7.9 under the same conditions as for HeLa cells and processed at 16 h p.i for EM analysis. In this cell line, similarly as in HeLa cells, the inducible expression of HCV proteins blocks VACV morphogenesis and induces the same alterations described above. In contrast to uninfected (Fig 3A) and infected hepG2 cells in absence of IPTG (Fig. 3B), in cells expressing HCV proteins we distinguish EDS in membranous webs (Fig. 3C and 3E), formation of large vacuoles (Fig. 3C and 3D), and also identified "virus like-particles" structures surrounded by membranes and dispersed in several areas of the cell cytoplasm (Fig. 3D).

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