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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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Immunogold electron microscopy analysis of the localization of HCV proteins in VT7-HCV7.9 infected HeLa cells. HeLa cells infected with VT7-HCV7.9 in the presence or absence of IPTG were chemically fixed, quickly frozen in liquid propane and then processed at low temperature in Lowicryl K4M resin. Immunogold labelling was performed with different antibodies. A: Cells infected with VT7-HCV7.9 in the absence of IPTG reacted with a serum from an HCV-infected patient.B: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a serum from an HCV-infected patient C: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS4B. D: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS5A. E: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a mouse monoclonal antibody anti-PDI. Note: Electron dense structures in membranous webs (EDS); mitochondria (m), immature virus (IV), intracellular mature virus (IMV), nucleus (N) and Vacuole (V). Bar: 250 nm.
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Figure 4: Immunogold electron microscopy analysis of the localization of HCV proteins in VT7-HCV7.9 infected HeLa cells. HeLa cells infected with VT7-HCV7.9 in the presence or absence of IPTG were chemically fixed, quickly frozen in liquid propane and then processed at low temperature in Lowicryl K4M resin. Immunogold labelling was performed with different antibodies. A: Cells infected with VT7-HCV7.9 in the absence of IPTG reacted with a serum from an HCV-infected patient.B: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a serum from an HCV-infected patient C: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS4B. D: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS5A. E: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a mouse monoclonal antibody anti-PDI. Note: Electron dense structures in membranous webs (EDS); mitochondria (m), immature virus (IV), intracellular mature virus (IMV), nucleus (N) and Vacuole (V). Bar: 250 nm.

Mentions: To assure that the electron dense structures appearing in the cytoplasm of infected cells are the result of HCV polyprotein expression, we performed immunogold electron microscopy analysis with antibodies against HCV structural and nonstructural proteins (Fig. 4). Thus, HeLa cells were infected with VT7-HCV7.9 in the presence or absence of IPTG and at 16 h p.i, infected and uninfected cells were processed for immunogold labelling on ultrathin sections. Due to the fixation and embedding procedures used in immunostaining, the cell structures are less visible than by embedding in an epoxy resin. While in cells infected with VT7-HCV7.9 in absence of IPTG there was no specific labelling detected with the serum from an HCV-infected patient (Fig. 4A), in contrast, in antibody-reacted cells expressing HCV proteins most gold particles were concentrated into electron dense and membranous structures (Fig. 4B). Discrete labelling was observed in other parts of the cell cytoplasm. The localization of some of the nonstructural HCV proteins was determined using rabbit polyclonal antibodies against NS4B or NS5A proteins. The membranous and electron dense structures were also specifically recognized by antibodies against NS4B (Fig. 4C) and NS5A (Fig. 4D), indicating that both proteins are part of electron dense membrane-associated cytoplasmic complexes.


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)

Immunogold electron microscopy analysis of the localization of HCV proteins in VT7-HCV7.9 infected HeLa cells. HeLa cells infected with VT7-HCV7.9 in the presence or absence of IPTG were chemically fixed, quickly frozen in liquid propane and then processed at low temperature in Lowicryl K4M resin. Immunogold labelling was performed with different antibodies. A: Cells infected with VT7-HCV7.9 in the absence of IPTG reacted with a serum from an HCV-infected patient.B: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a serum from an HCV-infected patient C: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS4B. D: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS5A. E: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a mouse monoclonal antibody anti-PDI. Note: Electron dense structures in membranous webs (EDS); mitochondria (m), immature virus (IV), intracellular mature virus (IMV), nucleus (N) and Vacuole (V). Bar: 250 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Immunogold electron microscopy analysis of the localization of HCV proteins in VT7-HCV7.9 infected HeLa cells. HeLa cells infected with VT7-HCV7.9 in the presence or absence of IPTG were chemically fixed, quickly frozen in liquid propane and then processed at low temperature in Lowicryl K4M resin. Immunogold labelling was performed with different antibodies. A: Cells infected with VT7-HCV7.9 in the absence of IPTG reacted with a serum from an HCV-infected patient.B: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a serum from an HCV-infected patient C: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS4B. D: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a rabbit polyclonal anti-NS5A. E: Cells infected with VT7-HCV7.9 in the presence of IPTG reacted with a mouse monoclonal antibody anti-PDI. Note: Electron dense structures in membranous webs (EDS); mitochondria (m), immature virus (IV), intracellular mature virus (IMV), nucleus (N) and Vacuole (V). Bar: 250 nm.
Mentions: To assure that the electron dense structures appearing in the cytoplasm of infected cells are the result of HCV polyprotein expression, we performed immunogold electron microscopy analysis with antibodies against HCV structural and nonstructural proteins (Fig. 4). Thus, HeLa cells were infected with VT7-HCV7.9 in the presence or absence of IPTG and at 16 h p.i, infected and uninfected cells were processed for immunogold labelling on ultrathin sections. Due to the fixation and embedding procedures used in immunostaining, the cell structures are less visible than by embedding in an epoxy resin. While in cells infected with VT7-HCV7.9 in absence of IPTG there was no specific labelling detected with the serum from an HCV-infected patient (Fig. 4A), in contrast, in antibody-reacted cells expressing HCV proteins most gold particles were concentrated into electron dense and membranous structures (Fig. 4B). Discrete labelling was observed in other parts of the cell cytoplasm. The localization of some of the nonstructural HCV proteins was determined using rabbit polyclonal antibodies against NS4B or NS5A proteins. The membranous and electron dense structures were also specifically recognized by antibodies against NS4B (Fig. 4C) and NS5A (Fig. 4D), indicating that both proteins are part of electron dense membrane-associated cytoplasmic complexes.

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